Novel human 26649, 3259, 57809, 57798, 33358, and 32529 molecules and uses therefor

[0006] The entire contents of each of the above-referenced patent applications are incorporated herein by this reference.

1INDEXChapterPageTitleI.226649, A NOVEL HUMAN GTPASE ACTIVATINGMOLECULE AND USES THEREFORII.7832591, A NOVEL HUMAN GTPASE ACTIVATINGMOLECULE AND USES THEREFORIII.15257809 AND 57798, NOVEL HUMAN CADHERINMOLECULES AND USES THEREFORIV.23333358, A NOVEL HUMAN ANKYRIN FAMILYMEMBER AND USES THEREOFV.30832529, A NOVEL HUMAN GUANINE NUCLEOTIDEEXCHANGE FACTOR FAMILY MEMBER ANDUSES THEREOF

[0007] I. 26649, A Novel Human GTPase Activating Molecule and Uses Therefor

BACKGROUND OF THE INVENTION

[0008] The family of G proteins encompasses a diverse array of proteins which regulate a complex range of biological processes, including the regulation of protein synthesis, vesicular and nuclear transport, regulation of the cell cycle, differentiation, and cytoskeletal rearrangements. The common motif among this important family of proteins is the presence of a GTP-binding domain (Alberts et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., New York, N.Y. pp. 206-207, 641). These proteins act as molecular switches that can cycle between active (GTP-bound) and inactive (GDP-bound) states (Bourne et al. (1990) Nature, 348:125-132). In the active state, G proteins are able to interact with a broad range of effector molecules. These effector molecules constitute components of a variety of signaling cascades. The lifetime of the active state of a G protein is determined by the rate at which the bound GTP is converted to GDP by the GTP-hydrolytic activity (GTPase activity) that is intrinsic to most G proteins. Upon hydrolysis of the bound GTP, the G protein reverts to the inactive state. This intrinsic enzymatic activity is accelerated by orders of magnitude in the presence of a family of molecules which interact with G proteins called “GTPase-activating proteins” (GAPs) (Scheffzek et al. (1998) Trends Biochem Sci., 23:257-262; Gamblin and Smerdon (1998) Curr. Opinion in Struct. Biol. 8:195-201). The members of this family of molecules appear to interact with domains of a given G protein, causing conformational changes which activate GTPase activity. The opposing transition from GDP-bound inactive state to GTP-bound active state appears to be facilitated by another class of molecules known as guanine-nucleotide-exchange factors (GEFs).

[0009] It is the regulated cycling between active and inactive states of G proteins that allows for proper transduction of many vital cellular signals. Indeed, the regulation of GTP/GDP levels in the cell by G proteins, and their accessory GAP molecules, has been implicated in a number of diseases, including atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, type 1 neurofibromatosis, Wiskott-Aldrich syndrome, cystic fibrosis, Microphthalmia with linear skin defects syndrome, and viral infection (Meijt, (1996) Mol. Cell. Biochem. 157:31-38; Olson, (1996) J. Mol. Med. 74:563-571; Wilson et al. (1988) J. Cell Biol. 107:69-77; Gutmann and Collins, (993) Neuron 10:335-343; Kolluri et al. (1996), PNAS 93:5615-5618; Schaefer et al., (1997) Genomics 46:268-277; Tan et al., (1993) Biol. Chem. 268:27291-27298).

[0010] Several GAP family members have been identified to date, including C. elegans gap-1 and gap-2 (Hajnal et al. (1997) Genes Dev, 11:2715-2728; Hayashizaki et al. (1998) Genes Cells 3:189-202), bovine GAP-1 and GAP-3 (Nice et al. (1992) J Biol. Chem. 267:1546-1553), and Drosophila Gap1 (Gaul et al. (1992) Cell 68:1007-1019).

SUMMARY OF THE INVENTION

[0011] The present invention is based, at least in part, on the discovery of a novel family of GTPase activating proteins, referred to herein interchangeably as “GTPase Activating Protein-4,” “G Protein Activating Protein-4,” or “GAP-4” nucleic acid and protein molecules. The GAP-4 molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes which are influenced by the regulated hydrolysis of GTP to GDP and the resulting GTP/GDP ratios. These processes include transduction of intracellular signaling, structuring of the cytoskeleton, vesicular trafficking, and progression through the cell cycle. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding GAP-4 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of GAP-4-encoding nucleic acids.

[0012] In one embodiment, a GAP-4 nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:1 or 3 or the nucleotiden sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, or a complement thereof.

[0013] In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:1 or 3, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 1-126 of SEQ ID NO:1. In another embodiment, the nucleic acid molecule includes SEQ ID NO:3 and nucleotides 2773-3536 of SEQ ID NO:1. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:1 or 3. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, or more nucleotides (e.g., contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.

[0014] In another embodiment, a GAP-4 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2 or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851. In a preferred embodiment, a GAP-4 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the entire length of the amino acid sequence of SEQ ID .NO:2, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851.

[0015] In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human GAP-4. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851.

[0016] Another embodiment of the invention features nucleic acid molecules, preferably GAP-4 nucleic acid molecules, which specifically detect GAP-4 nucleic acid molecules relative to nucleic acid molecules encoding non-GAP-4 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 50-100, 100-500, 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:1, the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, or a complement thereof.

[0017] In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:1 or 3 under stringent conditions.

[0018] Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a GAP-4 nucleic acid molecule, e.g., the coding strand of a GAP-4 nucleic acid molecule.

[0019] Another aspect of the invention provides a vector comprising a GAP-4 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably a GAP-4 protein family member, by culturing a host cell in a suitable medium, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.

[0020] Another aspect of this invention features isolated or recombinant GAP-4 proteins and polypeptides. In preferred embodiments, the isolated GAP-4 protein family member includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0021] In a preferred embodiment, the GAP-4 protein family member has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the amino acid sequence of SEQ ID NO:2, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, and includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0022] In another preferred embodiment, the GAP-4 protein family member modulates GTPase activity, and includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0023] In yet another preferred embodiment, the GAP-4 protein family member is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, and includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0024] In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:2, wherein the fragment comprises at least 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:2, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number PTA-1851. In another embodiment, the protein, preferably a GAP-4 protein, has the amino acid sequence of SEQ ID NO:2.

[0025] In another embodiment, the invention features an isolated GAP-4 protein family member which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof. This invention further features an isolated protein, preferably a GAP-4 protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or a complement thereof.

[0026] The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-GAP-4 polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably GAP-4 proteins. In addition, the GAP-4 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

[0027] In another aspect, the present invention provides a method for detecting the presence of a GAP-4 nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a GAP-4 nucleic acid molecule, protein or polypeptide such that the presence of a GAP-4 nucleic acid molecule, protein or polypeptide is detected in the biological sample.

[0028] In another aspect, the present invention provides a method for detecting the presence of GAP-4 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of GAP-4 activity such that the presence of GAP-4 activity is detected in the biological sample.

[0029] In another aspect, the invention provides a method for modulating GAP-4 activity comprising contacting a cell capable of expressing GAP-4 with an agent that modulates GAP-4 activity such that GAP-4 activity in the cell is modulated. In one embodiment, the agent inhibits GAP-4 activity. In another embodiment, the agent stimulates GAP-4 activity. In one embodiment, the agent is an antibody that specifically binds to a GAP-4 protein. In another embodiment, the agent modulates expression of GAP-4 by modulating transcription of a GAP-4 gene or translation of a GAP-4 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a GAP-4 mRNA or a GAP-4 gene.

[0030] In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted GAP-4 protein or nucleic acid expression or activity by administering an agent which is a GAP-4 modulator to the subject. In one embodiment, the GAP-4 modulator is a GAP-4 protein. In another embodiment the GAP-4 modulator is a GAP-4 nucleic acid molecule. In yet another embodiment, the GAP-4 modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant or unwanted GAP-4 protein or nucleic acid expression is a GTP hydrolysis-related disorder, such as atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, cystic fibrosis and viral infection.

[0031] The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a GAP-4 protein; (ii) mis-regulation of the GAP-4 gene; and (iii) aberrant post-translational modification of a GAP-4 protein, wherein a wild-type form of the gene encodes a protein with a GAP-4 activity.

[0032] In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of a GAP-4 protein, by providing an indicator composition comprising a GAP-4 protein having GAP-4 activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on GAP-4 activity in the indicator composition to identify a compound that modulates the activity of a GAP-4 protein.

[0033] Other features and advantages of the invention will be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIGS. 1A-E depict the cDNA sequence and predicted amino acid sequence of the human GAP-4. The nucleotide sequence corresponds to nucleic acids 1 to 3536 of SEQ ID NO:1. The amino acid sequence corresponds to amino acids 1 to 881 of SEQ ID NO:2. The coding region of the human GAP-4 corresponds to SEQ ID NO:3.

[0035]
FIG. 2 depicts a structural, hydrophobicity, and antigenicity analysis of the human GAP-4 protein.

[0036]
FIG. 3 depicts the results of a search which was performed against the HMM database using the amino acid sequence of human GAP-4. This search resulted in the identification of a “RhoGAP” domain in the human GAP-4 protein.

[0037] FIGS. 4A-D depict the results of a search performed against the ProDom database using the amino acid sequence of human GAP-4.

[0038] FIGS. 5A-D depict the cDNA sequence and predicted amino acid sequence of the human GAP-5. The nucleotide sequence corresponds to nucleic acids 1 to 4431 of SEQ ID NO:4. The amino acid sequence corresponds to amino acids 1 to 1101 of SEQ ID NO:5.

[0039] The coding region of the human GAP-5 corresponds to SEQ ID NO:6.

[0040] FIGS. 6A-B depict the results of a search which was performed against the HMM database using the amino acid sequence of human GAP-5. This search resulted in the identification of a “RhoGAP” domain in the human GAP-5 protein.

[0041]
FIG. 7 depicts the results of a search performed against the ProDom database using the amino acid sequence of human GAP-5.

[0042]
FIG. 8 depicts a domain analysis of the amino acid sequence of human GAP-5.

[0043] FIGS. 9A-C depict the cDNA sequence and predicted amino acid sequence of human CDHN-1 (clone Fbh57798). The nucleotide sequence corresponds to nucleic acids 1 to 3181 of SEQ ID NO:7. The amino acid sequence corresponds to amino acids 1 to 924 of SEQ ID NO:8. The coding region without the 5′ and 3′ untranslated regions of the human CDHN-1 gene is shown in SEQ ID NO:9.

[0044]
FIG. 10 depicts a structural, hydrophobicity, and antigenicity analysis of the human CDHN-1 protein (SEQ ID NO:8).

[0045]
FIG. 11 depicts the results of a search which was performed against the MEMSAT database and which resulted in the identification of “transmembrane domains” in the human CDHN-1 protein (SEQ ID NO:8).

[0046] FIGS. 12A-B depict the results of a search which was performed against the HMM (PFAM) database and which resulted in the identification of “cadherin domains” in the human CDHN-1 protein (SEQ ID NO:8).

[0047] FIGS. 13A-B depict the results of a search which was performed against the HMM (SMART) database and which resulted in the identification of “CA” domains in the human CDHN-1 protein (SEQ ID NO:8). FIGS. 14A-H depict the results of a search which was performed against the ProDom database and which resulted in the local alignment of the human CDHN-1 protein with p99.2 (671) FAT(32) Q14517(28) O88277(27); p99.2 (1) P81137_MANSE; p99.2 (1) O01909_CAEEL; p99.2 (1) O93508_BRARE; and p99.2 (1) Q19319_CAEEL.

[0048] FIGS. 15A-C depict the cDNA sequence and predicted amino acid sequence of human CDHN-2 (clone Fbh57809). The nucleotide sequence corresponds to nucleic acids 1 to 2938 of SEQ ID NO:10. The amino acid sequence corresponds to amino acids 1 to 830 of SEQ ID NO:11. The coding region without the 5′ and 3′ untranslated regions of the human CDHN-2 gene is shown in SEQ ID NO:12.

[0049]
FIG. 16 depicts a structural, hydrophobicity, and antigenicity analysis of the human CDHN-2 protein (SEQ ID NO:11).

[0050]
FIG. 17 depicts the results of a search which was performed against the MEMSAT database and which resulted in the identification of “transmembrane domains” in the human CDHN-2 protein (SEQ ID NO:11).

[0051] FIGS. 18A-B depict the results of a search which was performed against the HMM (PFAM) database and which resulted in the identification of “cadherin domains” in the human CDHN-2 protein (SEQ ID NO:11).

[0052] FIGS. 19A-B depict the results of a search which was performed against the HMM (SMART) database and which resulted in the identification of “CA” domains in the human CDHN-2 protein (SEQ ID NO:11).

[0053] FIGS. 20A-I depict the results of a search which was performed against the ProDom database and which resulted in the local alignment of the human CDHN-2 protein with p99.2 (3) O75309(1) Q28634(1) O88338(1); p99.2 (1) O76356_CAEEL; p99.2 (1) Q19319_CAEEL; p99.2 (671) FAT (32) Q14517(28) O88277(27); p99.2 (1) P81137_MANSE; p99.2 (3) O75309(1) O88338(1) Q28634(1); p99.2 (1) ENDR_BOVIN; p99.2 (38) CAD1(4) DSC1(3) CAD2(3); p99.2 (3) CADL(1) Q12864(1) Q15336(1); and p99.2 (3) O75309(1) O88338(1) Q28634(1).

[0054] FIGS. 21A-B depict a cDNA sequence (SEQ ID NO:14) and predicted amino acid sequence (SEQ ID NO:15) of human C/SKARP-1. The methionine-initiated open reading frame of human C/SKARP-1 (without the 5′ and 3′ untranslated regions) starts at nucleotide 75 until the termination codon (shown also as coding sequence SEQ ID NO:16).

[0055] FIGS. 22A-D depict C/SKARP-1 mRNA expression by probing a library array using RT-PCR.

[0056]
FIG. 23 depicts a structural, hydrophobicity, and antigenicity analysis of the human C/SKARP-1 protein.

[0057] FIGS. 24A-E depict the cDNA sequence and predicted amino acid sequence of human GEF32529. The nucleotide sequence corresponds to nucleic acids 1 to 3075 of SEQ ID NO:17. The amino acid sequence corresponds to amino acids 1 to 802 of SEQ ID NO:18. The coding region without the 5′ and 3′ untranslated regions of the human GEF32529 gene is shown in SEQ ID NO:19.

[0058]
FIG. 25 depicts a structural, hydrophobicity, and antigenicity analysis of the human GEF32529 polypeptide.

[0059] FIGS. 26A-C depict the results of a search which was performed against the HMM database in PFAM and SMART and which resulted in the identification of a “GEF domain,” a “PH domain,” and a “SH3 domain” in the human GEF32529 polypeptide (SEQ ID NO:18).

DETAILED DESCRIPTION OF THE INVENTION

[0060] The present invention is based, at least in part, on the discovery of a novel family of GTPase activating proteins, referred to herein interchangeably as “GTPase Activating Protein-4,” “G Protein Activating Protein-4,” or “GAP-4.” GAP-4 is a GTPase-associating protein which resembles members of the GAP (GTPase activating protein) family of proteins (described in, for example, Scheffzek et al. (1998) Trends Biochem Sci., 23:257-262) that normally activate the hydrolysis of GTP into GDP by GTPases.

[0061] The GAP-4 molecules of the present invention play a role in GTP hydrolysis and regulation of GTP/GDP levels. As used herein, the term “GTP hydrolysis” includes the dephosphorylation of GTP, resulting in the formation of GDP or other forms of guanine. GTP hydrolysis is mediated by GTPases, e.g., Rho-GTPases, ras-GTPases, rac-GTPases, and rab-GTPases. As used herein, the term “regulation of GTP/GDP levels” includes cellular mechanisms involved in regulating and influencing the levels, e.g., intracellular levels, of GTP and GDP. Such mechanisms include the hydrolysis of GTP to GDP (GTP hydrolysis) in response to biological cues, e.g., by a GTPase. The maintenance of GTP/GDP levels is particularly important for a cell's signaling needs. Thus, the GAP-4 molecules, by participating in GTP hydrolysis and regulation of GTP/GDP levels, may modulate GTP hydrolysis and GTP/GDP levels and provide novel diagnostic targets and therapeutic agents to control GTP hydrolysis-related disorders.

[0062] As used herein, the term “GTP hydrolysis-related disorders” includes disorders, diseases, or conditions which are characterized by aberrant, e.g., upregulated or downregulated, GTP hydrolysis and/or aberrant, e.g., unregulated or downregulated, GTP and/or GDP levels. Examples of such disorders may include cardiovascular disorders, e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, or arrhythmia.

[0063] Other examples of GTP hydrolysis-related disorders include disorders of the central nervous system, e.g., cystic fibrosis, type 1 neurofibromatosis, cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[0064] Still other examples of GTP hydrolysis-related disorders include cellular proliferation, growth, differentiation, or migration disorders. Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes. As used herein, a “cellular proliferation, growth, differentiation, or migration process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.

[0065] Still other examples of GTP hydrolysis-related disorders include disorders of the immune system, such as Wiskott-Aldrich syndrome, viral infection, autoimmune disorders or immune deficiency disorders, e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency. Other examples of GTP hydrolysis-related disorders include congenital malformalities, including facio-genital dysplasia; and skin disorders, including microphthalmia with linear skin defects syndrome.

[0066] The term “family” when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin. Members of a family may also have common functional characteristics.

[0067] For example, the family of GAP-4 proteins comprise at least one, and preferably two to three “transmembrane domains.” As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 10, 15, 20, 25, 30, 35, 40, 45 or more amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have a helical structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acid residues of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al., (1996) Annual Rev. Neurosci. 19:235-63, the contents of which are incorporated herein by reference. Amino acid residues 265-281, 394-410, and 419-435 of the human GAP-4 polypeptide (SEQ ID NO:2) comprise transmembrane domains (FIG. 2).

[0068] In another embodiment, a GAP-4 molecule of the present invention is identified based on the presence of a “RhoGAP domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “RhoGAP domain” includes a protein domain having an amino acid sequence of about 150 amino acid residues and having a bit score for the alignment of the sequence to the RhoGAP domain (HMM) of at least 193. Preferably, a RhoGAP domain includes at least about 130-200, more preferably about 140-175 amino acid residues, or about 145-155 amino acids and has a bit score for the alignment of the sequence to the RhoGAP domain (HMM) of at least 100, 150, 160, 170,180, 190, 200, or greater. The ankyrin repeat domain RhoGAP domain has been assigned the PFAM Accession PF00620 (http://genome.wustl.edu/Pfam/.html). RhoGAP domains are involved in protein-protein interactions and are described in, for example, Musacchio et al., (1996) PNAS, 93:14373-14378, the contents of which are incorporated herein by reference.

[0069] To identify the presence of an RhoGAP domain in a GAP-4 protein and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. MoL Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a RhoGAP domain in the amino acid sequence of SEQ ID NO:2 (at about residues 266-415). The results of this search are set forth in FIG. 3.

[0070] Isolated GAP-4 proteins of the present invention, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:2, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:1 or 3. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.

[0071] As used interchangeably herein, a “GAP-4 activity”, “biological activity of GAP-4,” or “functional activity of GAP-4,” includes an activity exerted by a GAP-4 protein, polypeptide or nucleic acid molecule on a GAP-4-responsive cell or tissue, or on a GAP-4 protein substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a GAP-4 activity is a direct activity, such as an association with a GAP-4-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which a GAP-4 protein binds or interacts in nature, such that GAP-4- mediated function is achieved. A GAP-4 target molecule can be a non-GAP-4 molecule or a GAP-4 protein or polypeptide of the present invention. In an exemplary embodiment, a GAP-4 target molecule is a GAP-4 ligand, e.g., a GTPase. Alternatively, a GAP-4 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the GAP-4 protein with a GAP-4 ligand, e.g., a GTPase. Preferably, a GAP-4 activity is the ability to modulate the hydrolysis of GTP via, e.g., interactions with GTPase molecules.

[0072] Accordingly, another embodiment of the invention features isolated GAP-4 polypeptides having a GAP-4 activity. Preferred proteins are GAP-4 proteins having at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain, and, preferably, a GAP-4 activity. Additional preferred GAP-4 proteins have at least one RhoGAP domain, and/or at least one transmembrane domain and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3.

[0073] The nucleotide sequence of the isolated human GAP-4 cDNA and the predicted amino acid sequence of the human GAP-4 polypeptide are shown in FIGS. 1A-E and in SEQ ID NO:1 and SEQ ID NO:2, respectively. A plasmid containing the nucleotide sequence encoding human GAP-4 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on May 9, 2000 and assigned Accession Number PTA-1851.

[0074] These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112.

[0075] The human GAP-4 gene, which is approximately 3536 nucleotides in length, encodes a protein having a molecular weight of approximately 97 kD and which is approximately 881 amino acid residues in length.

[0076] Various aspects of the invention are described in further detail in the following subsections:

[0077] I. Isolated Nucleic Acid Molecules

[0078] One aspect of the invention pertains to isolated nucleic acid molecules that encode GAP-4 proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify GAP-4-encoding nucleic acid molecules (e.g., GAP-4 mRNA) and fragments for use as PCR primers for the amplification or mutation of GAP-4 nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0079] The term “isolated nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated GAP-4 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0080] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, as a hybridization probe, GAP-4 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0081] Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851.

[0082] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GAP-4 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0083] In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:1. The sequence of SEQ ID NO:1 corresponds to the human GAP-4 cDNA. This cDNA comprises sequences encoding the human GAP-4 protein (i.e., “the coding region”, from nucleotides 127-2772), as well as 5′ untranslated sequences (nucleotides 1-126) and 3′ untranslated sequences (nucleotides 2773-3536). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:1 (e.g., nucleotides 127-2772, corresponding to SEQ ID NO:3).

[0084] In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, thereby forming a stable duplex.

[0085] In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:1 or 3, or the entire length of the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, or a portion of any of these nucleotide sequences.

[0086] Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a GAP-4 protein, e.g., a biologically active portion of a GAP-4 protein. The nucleotide sequence determined from the cloning of the GAP-4 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other GAP-4 family members, as well as GAP-4 homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, of an anti-sense sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, or of a naturally occurring allelic variant or mutant of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851. In one embodiment, a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is greater than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851.

[0087] Probes based on the GAP-4 nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a GAP-4 protein, such as by measuring a level of a GAP-4-encoding nucleic acid in a sample of cells from a subject, e.g., detecting GAP-4 mRNA levels or determining whether a genomic GAP-4 gene has been mutated or deleted.

[0088] A nucleic acid fragment encoding a “biologically active portion of a GAP-4 protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, which encodes a polypeptide having a GAP-4 biological activity (the biological activities of the GAP-4 proteins are described herein), expressing the encoded portion of the GAP-4 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the GAP-4 protein.

[0089] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, due to degeneracy of the genetic code and, thus, encode the same GAP-4 proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number PTA-1851. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2.

[0090] In addition to the GAP-4 nucleotide sequences shown in SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the GAP-4 proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the GAP-4 genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a GAP-4 protein, preferably a mammalian GAP-4 protein, and can further include non-coding regulatory sequences, and introns.

[0091] Allelic variants of human GAP-4 include both functional and non-functional GAP-4 proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the human GAP-4 protein that maintain the ability to bind a GAP-4 ligand or substrate (e.g., a GTPase) and/or modulate GTP hydrolysis and/or GTPase signaling mechanisms, and/or disorders related to regulation of levels of GTP/GDP. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:2, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.

[0092] Non-functional allelic variants are naturally occurring amino acid sequence variants of the human GAP-4 proteins that do not have the ability to either bind a GAP-4 ligand or substrate (e.g., a GTPase) and/or modulate GTP hydrolysis and/or GTPase signaling mechanisms, and/or disorders related to regulation of levels of GTP/GDP. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:2, or a substitution, insertion or deletion in critical residues or critical regions. The present invention further provides non-human orthologues of the human GAP-4 protein. Orthologues of the human GAP-4 protein are proteins that are isolated from non-human organisms and possess the same GAP-4 ligand binding and/or modulation of GTPase activity and/or GTPase related signaling mechanisms and/or modulation of GTP/GDP levels. Orthologues of the human GAP-4 protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:2.

[0093] Moreover, nucleic acid molecules encoding other GAP-4 family members and, thus, which have a nucleotide sequence which differs from the GAP-4 sequences of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851 are intended to be within the scope of the invention. For example, another GAP-4 cDNA can be identified based on the nucleotide sequence of human GAP-4. Moreover, nucleic acid molecules encoding GAP-4 proteins from different species, and which, thus, have a nucleotide sequence which differs from the GAP-4 sequences of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851 are intended to be within the scope of the invention. For example, a mouse GAP-4 cDNA can be identified based on the nucleotide sequence of a human GAP-4.

[0094] Nucleic acid molecules corresponding to natural allelic variants and homologues of the GAP-4 cDNAs of the invention can be isolated based on their homology to the GAP-4 nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the GAP-4 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the GAP-4 gene.

[0095] Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851. In other embodiment, the nucleic acid is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500 or more nucleotides in length.

[0096] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4×sodium chloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in 1×SSC, at about 65-70° C. (or hybridization in 1×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 0.3×SSC, at about 65-70° C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide at about 40-45° C.) followed by one or more washes in 2×SSC, at about 50-60° C. Ranges intermediate to the above-recited values, e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassed by the present invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log10[Na+])+0.41(% G+C)-(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH2PO4, 7% SDS at about 65° C., followed by one or more washes at 0.02M NaH2PO4, 1% SDS at 65° C., see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or alternatively 0.2×SSC, 1% SDS).

[0097] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1 or 3 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0098] In addition to naturally-occurring allelic variants of the GAP-4 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, thereby leading to changes in the amino acid sequence of the encoded GAP-4 proteins, without altering the functional ability of the GAP-4 proteins. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of GAP-4 (e.g., the sequence of SEQ ID NO:2) without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the GAP-4 proteins of the present invention, e.g., those present in the ankyrin repeat domain(s) or the ion transport protein domain(s) or the transmembrane domain(s), are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the GAP-4 proteins of the present invention and other members of the vanilloid receptor family are not likely to be amenable to alteration.

[0099] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding GAP-4 proteins that contain changes in amino acid residues that are not essential for activity. Such GAP-4 proteins differ in amino acid sequence from SEQ ID NO:2, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to SEQ ID NO:2.

[0100] An isolated nucleic acid molecule encoding a GAP-4 protein identical to the protein of SEQ ID NO:2, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a GAP-4 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a GAP-4 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GAP-4 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0101] In another preferred embodiment, a mutant GAP-4 protein can be assayed for the ability to (1) interact with a non-GAP-4 protein molecule, e.g., a GTPase or a GAP-4 ligand or substrate; (2) modulate a GAP-4-dependent signal transduction pathway; (3) modulate GTPase-dependant signal transduction; (4) modulate GTP hydrolysis activity; (5) modulate levels of GTP/GDP.

[0102] In addition to the nucleic acid molecules encoding GAP-4 proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire GAP-4 coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding GAP-4. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human GAP-4 corresponds to SEQ ID NO:3). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding GAP-4. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0103] Given the coding strand sequences encoding GAP-4 disclosed herein (e.g., SEQ ID NO:3), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of GAP-4 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of GAP-4 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GAP-4 mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0104] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a GAP-4 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0105] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0106] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave GAP-4 mRNA transcripts to thereby inhibit translation of GAP-4 mRNA. A ribozyme having specificity for a GAP-4-encoding nucleic acid can be designed based upon the nucleotide sequence of a GAP-4 cDNA disclosed herein (i.e., SEQ ID NO:1 or 3, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a GAP-4-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, GAP-4 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0107] Alternatively, GAP-4 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory and/or 5′ untranslated region of the GAP-4 nucleotides (e.g., the GAP-4 promoter and/or enhancers; e.g., nucleotides 1-126 of SEQ ID NO:1) to form triple helical structures that prevent transcription of the GAP-4 gene in target cells. Seegenerally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

[0108] In yet another embodiment, the GAP-4 nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0109] PNAs of GAP-4 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of GAP-4 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0110] In another embodiment, PNAs of GAP-4 can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of GAP-4 nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

[0111] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0112] II. Isolated GAP-4 Proteins and Anti-GAP-4 Antibodies One aspect of the invention pertains to isolated GAP-4 proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-GAP-4 antibodies. In one embodiment, native GAP-4 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, GAP-4 proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a GAP-4 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[0113] An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GAP-4 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of GAP-4 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of GAP-4 protein having less than about 30% (by dry weight) of non-GAP-4 protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-GAP-4 protein, still more preferably less than about 10% of non-GAP-4 protein, and most preferably less than about 5% non-GAP-4 protein. When the GAP-4 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0114] The language “substantially free of chemical precursors or other chemicals” includes preparations of GAP-4 protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of GAP-4 protein having less than about 30% (by dry weight) of chemical precursors or non-GAP-4 chemicals, more preferably less than about 20% chemical precursors or non-GAP-4 chemicals, still more preferably less than about 10% chemical precursors or non-GAP-4 chemicals, and most preferably less than about 5% chemical precursors or non-GAP-4 chemicals.

[0115] As used herein, a “biologically active portion” of a GAP-4 protein includes a fragment of a GAP-4 protein which participates in an interaction between a GAP-4 molecule and a non-GAP-4 molecule, e.g., a GTPase. Biologically active portions of a GAP-4 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the GAP-4 protein, e.g., the amino acid sequence shown in SEQ ID NO:2, which include less amino acids than the full length GAP-4 proteins, and exhibit at least one activity of a GAP-4 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the GAP-4 protein, e.g., interacting with GTPase molecules, modulating GTPase activity, and/or modulating GTP/GDP levels. A biologically active portion of a GAP-4 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200, 500, or more amino acids in length. Biologically active portions of a GAP-4 protein can be used as targets for developing agents which modulate a GAP-4 mediated activity, e.g., modulation of GTP hydrolysis or modulation of GTP/GDP levels.

[0116] In one embodiment, a biologically active portion of a GAP-4 protein comprises at least one RhoGAP domain, and/or at least one transmembrane domain. It is to be understood that a preferred biologically active portion of a GAP-4 protein of the present invention may contain at least one RhoGAP domain. Another preferred biologically active portion of a GAP-4 protein may contain at least one transmembrane domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native GAP-4 protein.

[0117] In a preferred embodiment, the GAP-4 protein has an amino acid sequence shown in SEQ ID NO:2. In other embodiments, the GAP-4 protein is substantially identical to SEQ ID NO:2, and retains the functional activity of the protein of SEQ ID NO:2, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the GAP-4 protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to SEQ ID NO:2.

[0118] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the GAP-4 amino acid sequence of SEQ ID NO:2 having 725 amino acid residues, at least 264, preferably at least 352, more preferably at least 441, even more preferably at least 529, and even more preferably at least 617, 705 or 794 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0119] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Myers and Miller, 1988, Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0120] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to GAP-4 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=100, wordlength=3 to obtain amino acid sequences homologous to GAP-4 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0121] The invention also provides GAP-4 chimeric or fusion proteins. As used herein, a GAP-4 “chimeric protein” or “fusion protein” comprises a GAP-4 polypeptide operatively linked to a non-GAP-4 polypeptide. A “GAP-4 polypeptide” includes a polypeptide having an amino acid sequence corresponding to GAP-4, whereas a “non-GAP-4 peptide” includes a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to a GAP-4 protein, e.g., a protein which is different from the GAP-4 protein and which is derived from the same or a different organism. Within a GAP-4 fusion protein the GAP-4 polypeptide can correspond to all or a portion of a GAP-4 protein. In a preferred embodiment, a GAP-4 fusion protein comprises at least one biologically active portion of a GAP-4 protein. In another preferred embodiment, a GAP-4 fusion protein comprises at least two biologically active portions of a GAP-4 protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the GAP-4 polypeptide and the non-GAP-4 polypeptide are fused in-frame to each other. The non-GAP-4 polypeptide can be fused to the N-terminus or C-terminus of the GAP-4 polypeptide.

[0122] For example, in one embodiment, the fusion protein is a GST-GAP-4 fusion protein in which the GAP-4 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant GAP-4.

[0123] In another embodiment, the fusion protein is a GAP-4 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of GAP-4 can be increased through use of a heterologous signal sequence.

[0124] The GAP-4 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The GAP-4 fusion proteins can be used to affect the bioavailability of a GAP-4 ligand or substrate. Use of GAP-4 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a GAP-4 protein; (ii) mis-regulation of the GAP-4 gene; and (iii) aberrant post-translational modification of a GAP-4 protein.

[0125] Moreover, the GAP-4-fusion proteins of the invention can be used as immunogens to produce anti-GAP-4 antibodies in a subject, to purify GAP-4 ligands and in screening assays to identify molecules which inhibit the interaction of GAP-4 with a GAP-4 ligand or substrate.

[0126] Preferably, a GAP-4 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A GAP-4-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GAP-4 protein.

[0127] The present invention also pertains to variants of the GAP-4 proteins which function as either GAP-4 agonists (mimetics) or as GAP-4 antagonists. Variants of the GAP-4 proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a GAP-4 protein. An agonist of the GAP-4 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a GAP-4 protein. An antagonist of a GAP-4 protein can inhibit one or more of the activities of the naturally occurring form of the GAP-4 protein by, for example, competitively modulating a GAP-4-mediated activity of a GAP-4 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the GAP-4 protein.

[0128] In one embodiment, variants of a GAP-4 protein which function as either GAP-4 agonists (mimetics) or as GAP-4 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a GAP-4 protein for GAP-4 protein agonist or antagonist activity. In one embodiment, a variegated library of GAP-4 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated,library of GAP-4 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GAP-4 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GAP-4 sequences therein. There are a variety of methods which can be used to produce libraries of potential GAP-4 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential GAP-4 sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.(1983) Nucleic Acid Res. 11:477.

[0129] In addition, libraries of fragments of a GAP-4 protein coding sequence can be used to generate a variegated population of GAP-4 fragments for screening and subsequent selection of variants of a GAP-4 protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a GAP-4 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the GAP-4 protein.

[0130] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of GAP-4 proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GAP-4 variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering 6(3):327-331).

[0131] In one embodiment, cell based assays can be exploited to analyze a variegated GAP-4 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a neuronal cell line, which ordinarily responds to GAP-4 in a particular GAP-4 ligand-dependent manner. The transfected cells are then contacted with a GAP-4 ligand and the effect of expression of the mutant on signaling by the GAP-4 ligand can be detected, e.g., by monitoring GTPase activity, GTPase-related signaling mechanisms, or the activity of a GAP-4-regulated transcription factor. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the GAP-4 ligand, and the individual clones further characterized. In related cell-based assays, changes in GTP/GDP levels (i.e., signal transduction) can be measured in live cells which express GAP-4 molecules of the invention. Such an assay can be used for screening compound libraries for useful ligands which interact with GAP-4, or can be used to identify variants of GAP-4 which have useful properties. Other cell based assay include those which can monitor fluxes in intracellular calcium levels which result from GTPase-mediated signaling, e.g., flow cytometry (Valet and Raffael, 1985, Naturwiss., 72:600-602). Also within the scope of the invention are assays and models which utilize GAP-4 nucleic acids to create transgenic organisms for identifying useful pharmaceutical compounds or variants of the GAP-4 molecules.

[0132] An isolated GAP-4 protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind GAP-4 using standard techniques for polyclonal and monoclonal antibody preparation. A full-length GAP-4 protein can be used or, alternatively, the invention provides antigenic peptide fragments of GAP-4 for use as immunogens. The antigenic peptide of GAP-4 comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 and encompasses an epitope of GAP-4 such that an antibody raised against the peptide forms a specific immune complex with GAP-4. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0133] Preferred epitopes encompassed by the antigenic peptide are regions of GAP-4 that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity (see, for example, FIG. 2).

[0134] An GAP-4 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed GAP-4 protein or a chemically synthesized GAP-4 polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic GAP-4 preparation induces a polyclonal anti-GAP-4 antibody response.

[0135] Accordingly, another aspect of the invention pertains to anti-GAP-4 antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as GAP-4. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind GAP-4. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GAP-4. A monoclonal antibody composition thus typically displays a single binding affinity for a particular GAP-4 protein with which it immunoreacts.

[0136] Polyclonal anti-GAP-4 antibodies can be prepared as described above by immunizing a suitable subject with a GAP-4 immunogen. The anti-GAP-4 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized GAP-4. If desired, the antibody molecules directed against GAP-4 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-GAP-4 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lemer (198 1) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a GAP-4 immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds GAP-4.

[0137] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-GAP-4 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lemer, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind GAP-4, e.g., using a standard ELISA assay.

[0138] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-GAP-4 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with GAP-4 to thereby isolate immunoglobulin library members that bind GAP-4. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0139] Additionally, recombinant anti-GAP-4 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0140] An anti-GAP-4 antibody (e.g., monoclonal antibody) can be used to isolate GAP-4 by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-GAP-4 antibody can facilitate the purification of natural GAP-4 from cells and of recombinantly produced GAP-4 expressed in host cells. Moreover, an anti-GAP-4 antibody can be used to detect GAP-4 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the GAP-4 protein. Anti-GAP-4 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, P-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0141] III. Recombinant Expression Vectors and Host Cells

[0142] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a GAP-4 protein (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0143] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GAP-4 proteins, mutant forms of GAP-4 proteins, fusion proteins, and the like).

[0144] The recombinant expression vectors of the invention can be designed for expression of GAP-4 proteins in prokaryotic or eukaryotic cells. For example, GAP-4 proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0145] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0146] Purified fusion proteins can be utilized in GAP-4 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for GAP-4 proteins, for example. In a preferred embodiment, a GAP-4 fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

[0147] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0148] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0149] In another embodiment, the GAP-4 expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec 1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corporation, San Diego, Calif.).

[0150] Alternatively, GAP-4 proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0151] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufinan et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0152] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0153] The expression characteristics of an endogenous GAP-4 gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous GAP-4 gene. For example, an endogenous GAP-4 gene which is normally “transcriptionally silent”, i.e., a GAP-4 gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism. Alternatively, a transcriptionally silent, endogenous GAP-4 gene may be activated by insertion of a promiscuous regulatory element that works across cell types.

[0154] A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous GAP-4 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

[0155] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to GAP-4 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1)1986.

[0156] Another aspect of the invention pertains to host cells into which a GAP-4 nucleic acid molecule of the invention is introduced, e.g., a GAP-4 nucleic acid molecule within a recombinant expression vector or a GAP-4 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0157] A host cell can be any prokaryotic or eukaryotic cell. For example, a GAP-4 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0158] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0159] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a GAP-4 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0160] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a GAP-4 protein. Accordingly, the invention further provides methods for producing a GAP-4 protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a GAP-4 protein has been introduced) in a suitable medium such that a GAP-4 protein is produced. In another embodiment, the method further comprises isolating a GAP-4 protein from the medium or the host cell.

[0161] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GAP-4-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous GAP-4 sequences have been introduced into their genome or homologous recombinant animals in which endogenous GAP-4 sequences have been altered. Such animals are useful for studying the function and/or activity of a GAP-4 and for identifying and/or evaluating modulators of GAP-4 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GAP-4 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0162] A transgenic animal of the invention can be created by introducing a GAP-4-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The GAP-4 cDNA sequence of SEQ ID NO:1 or 3 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human GAP-4 gene, such as a mouse or rat GAP-4 gene, can be used as a transgene. Alternatively, a GAP-4 gene homologue, such as another GAP-4 family member, can be isolated based on hybridization to the GAP-4 cDNA sequences of SEQ ID NO:1 or 3, or the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-1851 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a GAP-4 transgene to direct expression of a GAP-4 protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a GAP-4 transgene in its genome and/or expression of GAP-4 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a GAP-4 protein can further be bred to other transgenic animals carrying other transgenes.

[0163] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a GAP-4 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GAP-4 gene. The GAP-4 gene can be a human gene (e.g., the cDNA of SEQ ID NO:1,3, 4, or 6), but more preferably, is a non-human homologue of a human GAP-4 gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:1,3, 4, or 6). For example, a mouse GAP-4 gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous GAP-4 gene in the mouse genome.

[0164] In a preferred embodiment, the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous GAP-4 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous GAP-4 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GAP-4 protein). In the homologous recombination nucleic acid molecule, the altered portion of the GAP-4 gene is flanked at its 5′ and 3′ ends by additional nucleic acid sequence of the GAP-4 gene to allow for homologous recombination to occur between the exogenous GAP-4 gene carried by the homologous recombination nucleic acid molecule and an endogenous GAP-4 gene in a cell, e.g., an embryonic stem cell. The additional flanking GAP-4 nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GAP-4 gene has homologously recombined with the endogenous GAP-4 gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

[0165] In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase. Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter GO phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0166] IV. Pharmaceutical Compositions

[0167] The GAP-4 nucleic acid molecules, fragments of GAP-4 proteins, and anti-GAP-4 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0168] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0169] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0170] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a GAP-4 protein or an anti-GAP-4 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0171] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0172] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0173] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0174] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0175] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0176] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0177] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0178] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0179] In a preferred example, a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

[0180] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.

[0181] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0182] Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0183] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0184] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0185] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmnaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0186] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration. V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, a GAP-4 protein of the invention has one or more of the following activities: (1) it interacts with a non-GAP-4 protein molecule, e.g., a GTPase or a GAP-4 ligand; (2) it modulated a GAP-4-dependent signal transduction pathway; (3) it modulates GTP/GDP levels; and (4) it modulates GTPase signaling mechanisms, and, thus, can be used to, for example, (1) modulate the interaction with a non-GAP-4 protein molecule, e.g., a GTPase; (2) activate a GAP-4-dependent signal transduction pathway; (3) modulate GTP/GDP levels; and (4) modulate GTPase signaling mechanisms.

[0187] The isolated nucleic acid molecules of the invention can be used, for example, to express GAP-4 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GAP-4 mRNA (e.g., in a biological sample) or a genetic alteration in a GAP-4 gene, and to modulate GAP-4 activity, as described further below. The GAP-4 proteins can be used to treat disorders characterized by insufficient or excessive production of a GAP-4 ligand or substrate or production of GAP-4 inhibitors. In addition, the GAP-4 proteins can be used to screen for naturally occurring GAP-4 ligands or substrates to screen for drugs or compounds which modulate GAP-4 activity, as well as to treat disorders characterized by insufficient or excessive production of GAP-4 protein or production of GAP-4 protein forms which have decreased, aberrant or unwanted activity compared to GAP-4 wild type protein (e.g., GTP hydrolysis-related disorders and/or disorders related to GTP/GDP levels). Moreover, the anti-GAP-4 antibodies of the invention can be used to detect and isolate GAP-4 proteins, regulate the bioavailability of GAP-4 proteins, and modulate GAP-4 activity.

[0188] A. Screening Assays:

[0189] The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to GAP-4 proteins, have a stimulatory or inhibitory effect on, for example, GAP-4 expression or GAP-4 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a GAP-4 ligand or substrate.

[0190] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates or ligands of a GAP-4 protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a GAP-4 protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

[0191] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0192] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner USP 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

[0193] In one embodiment, an assay is a cell-based assay in which a cell which expresses a GAP-4 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate GAP-4 activity is determined. Determining the ability of the test compound to modulate GAP-4 activity can be accomplished by monitoring, for example, changes in intracellular calcium concentration by, e.g., flow cytometry, or by the activity of a GAP-4-regulated transcription factor. The cell, for example, can be of mammalian origin, e.g., a neuronal cell.

[0194] The ability of the test compound to modulate GAP-4 binding to a ligand or substrate or to bind to GAP-4 can also be determined. Determnining the ability of the test compound to modulate GAP-4 binding to a ligand or substrate can be accomplished, for example, by coupling the GAP-4 ligand or substrate with a radioisotope or enzymatic label such that binding of the GAP-4 ligand or substrate to GAP-4 can be determined by detecting the labeled GAP-4 ligand or substrate in a complex. Determining the ability of the test compound to bind GAP-4 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to GAP-4 can be determined by detecting the labeled GAP-4 compound in a complex. For example, compounds (e.g., GAP-4 ligands or substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0195] It is also within the scope of this invention to determine the ability of a compound (e.g., a GAP-4 ligand or substrate) to interact with GAP-4 without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with GAP-4 without the labeling of either the compound or the GAP-4. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and GAP-4.

[0196] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a GAP-4 target molecule (e.g., a GAP-4 ligand or substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GAP-4 target molecule. Determining the ability of the test compound to modulate the activity of a GAP-4 target molecule can be accomplished, for example, by determining the ability of the GAP-4 protein to bind to or interact with the GAP-4 target molecule.

[0197] Determining the ability of the GAP-4 protein or a biologically active fragment thereof, to bind to or interact with a GAP-4 target molecule (e.g., a GTPase) can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the GAP-4 protein to bind to or interact with a GAP-4 target molecule or GTPase can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting the ability of the GTPase to hydrolyze GTP, or by detecting induction of a cellular second messenger of the target (i.e., intracellular Ca2+, diacylglycerol, lP3, and the like), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter-gene (comprising a target responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response such as changes in cytoskeletal structure or nuclear transport.

[0198] In yet another embodiment, an assay of the present invention is a cell-free assay in which a GAP-4 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the GAP-4 protein or biologically active portion thereof is determined. Preferred biologically active portions of the GAP-4 proteins to be used in assays of the present invention include fragments which participate in interactions with non-GAP-4 molecules, e.g., fragments with high surface probability scores (see, for example, FIG. 2). Binding of the test compound to the GAP-4 protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the GAP-4 protein or biologically active portion thereof with a known compound which binds GAP-4 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GAP-4 protein, wherein determining the ability of the test compound to interact with a GAP-4 protein comprises determining the ability of the test compound to preferentially bind to GAP-4 or biologically active portion thereof as compared to the known compound.

[0199] In another embodiment, the assay is a cell-free assay in which a GAP-4 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GAP-4 protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a GAP-4 protein can be accomplished, for example, by determining the ability of the GAP-4 protein to bind to a GAP-4 target molecule by one of the methods described above for determining direct binding. Determining the ability of the GAP-4 protein to bind to a GAP-4 target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[0200] In an alternative embodiment, determining the ability of the test compound to modulate the activity of a GAP-4 protein can be accomplished by determining the ability of the GAP-4 protein to further modulate the activity of a downstream effector of a GAP-4 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.

[0201] In yet another embodiment, the cell-free assay involves contacting a GAP-4 protein or biologically active portion thereof with a known compound which binds the GAP-4 protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the GAP-4 protein, wherein determining the ability of the test compound to interact with the GAP-4 protein comprises determining the ability of the GAP-4 protein to preferentially bind to or modulate the activity of a GAP-4 target molecule.

[0202] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either GAP-4 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a GAP-4 protein, or interaction of a GAP-4 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/GAP-4 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or GAP-4 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of GAP-4 binding or activity determined using standard techniques.

[0203] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a GAP-4 protein or a GAP-4 target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated GAP-4 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with GAP-4 protein or target molecules but which do not interfere with binding of the GAP-4 protein to its target molecule can be derivatized to the wells of the plate, and unbound target or GAP-4 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GAP-4 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the GAP-4 protein or target molecule.

[0204] In another embodiment, modulators of GAP-4 expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of GAP-4 mRNA or protein in the cell is determined. The level of expression of GAP-4 mRNA or protein in the presence of the candidate compound is compared to the level of expression of GAP-4 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of GAP-4 expression based on this comparison. For example, when expression of GAP-4 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GAP-4 mRNA or protein expression. Alternatively, when expression of GAP-4 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GAP-4 mRNA or protein expression. The level of GAP-4 mRNA or protein expression in the cells can be determined by methods described herein for detecting GAP-4 mRNA or protein.

[0205] In yet another aspect of the invention, the GAP-4 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with GAP-4 (“GAP-4-binding proteins” or “GAP-4-bp”) and are involved in GAP-4 activity. Such GAP-4-binding proteins are also likely to be involved in the propagation of signals by the GAP-4 proteins or GAP-4 targets as, for example, downstream elements of a GAP-4-mediated signaling pathway. Alternatively, such GAP-4-binding proteins are likely to be GAP-4 inhibitors.

[0206] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a GAP-4 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a GAP-4- or VR5-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the GAP-4 protein.

[0207] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a GAP-4 protein can be confirmed in vivo, e.g., in an animal such as an animal model for cancer or cardiovascular disease.

[0208] Examples of animal models of cancer include transplantable models (e.g., xenografts of colon tumors such as Co-3, AC3603 or WiDr or into immunocompromised mice such as SCID or nude mice); transgenic models (e.g., B66-Min/+mouse); chemical induction models, e.g., carcinogen (e.g., azoxymethane, 2-dimethylhydrazine, or N-nitrosodimethylamine) treated rats or mice; models of liver metastasis from colon cancer such as that described by Rashidi et al. (2000) Anticancer Res. 20(2A):715; and cancer cell implantation or inoculation models as described in, for example, Fingert, et al. (1987) Cancer Res. 46(14):3824-9 and Teraoka, et aL (1995) Jpn. J. Cancer Res. 86(5):419-23.

[0209] Examples of animal models for cardiovascular disease include mouse models for renal ischemic reperfusion injury (IRI) such as that described in Burne et al. (2000) Transplantation 69(5):1023-5; animal models of congestive heart failure (CHF) such as that described in Smith, et al. (2000) J. Pharmacol. Toxicol. Methods 43(2):125; animal models of restenosis such as that described in Hehrlein et al. (2000) Eur. Heart 21(24):2056-62; and animal models of heart failure such as that described in Arnolda et al. (1999) Aust. N. Z. J Med. 29(3):403-9.

[0210] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a GAP-4 modulating agent, an antisense GAP-4 nucleic acid molecule, a GAP-4-specific antibody, or a GAP-4-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0211] B. Detection Assays Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[0212] 1. Chromosome Mapping

[0213] Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the GAP-4 nucleotide sequences, described herein, can be used to map the location of the GAP-4 genes on a chromosome. The mapping of the GAP-4 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

[0214] Briefly, GAP-4 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GAP-4 nucleotide sequences. Computer analysis of the GAP-4 sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GAP-4 sequences will yield an amplified fragment.

[0215] Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

[0216] PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the GAP-4 nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a GAP-4 sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

[0217] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

[0218] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[0219] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[0220] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GAP-4 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[0221] 2. Tissue Typing

[0222] The GAP-4 sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0223] Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the GAP-4 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[0224] Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The GAP-4 nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:1 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 75-100 bases. If predicted coding sequences, such as those in SEQ ID NO:3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0225] If a panel of reagents from GAP-4 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[0226] 3. Use of Partial GAP-4 Sequences in Forensic Biology

[0227] DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[0228] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:1 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the GAP-4 nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:1, having a length of at least 20 bases, preferably at least 30 bases.

[0229] The GAP-4 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such GAP-4 probes can be used to identify tissue by species and/or by organ type.

[0230] In a similar fashion, these reagents, e.g., GAP-4 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[0231] C. Predictive Medicine:

[0232] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining GAP-4 protein and/or nucleic acid expression as well as GAP-4 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted GAP-4 expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GAP-4 protein, nucleic acid expression or activity. For example, mutations in a GAP-4 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GAP-4 protein, nucleic acid expression or activity.

[0233] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GAP-4 in clinical trials.

[0234] These and other agents are described in further detail in the following sections.

[0235] 1. Diagnostic Assays

[0236] An exemplary method for detecting the presence or absence of GAP-4 protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting GAP-4 protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes GAP-4 protein such that the presence of GAP-4 protein or nucleic acid is detected in the biological sample. A preferred agent for detecting GAP-4 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GAP-4 mRNA or genomic DNA. The nucleic acid probe can be, for example, the GAP-4 nucleic acid set forth in SEQ ID NO:1 or 3, or the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-185 1, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GAP-4 mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[0237] A preferred agent for detecting GAP-4 protein is an antibody capable of binding to GAP-4 protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect GAP-4 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of GAP-4 mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of GAP-4 protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of GAP-4 genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of GAP-4 protein include introducing into a subject a labeled anti-GAP-4 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0238] In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.

[0239] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting GAP-4 protein, mRNA, or genomic DNA, such that the presence of GAP-4 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GAP-4 protein, mRNA or genomic DNA in the control sample with the presence of GAP-4 protein, mRNA or genomic DNA in the test sample.

[0240] The invention also encompasses kits for detecting the presence of GAP-4 in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting GAP-4 protein or mRNA in a biological sample; means for determining the amount of GAP-4 in the sample; and means for comparing the amount of GAP-4 in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GAP-4 protein or nucleic acid.

[0241] 2. Prognostic Assays

[0242] The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted GAP-4 expression or activity. As used herein, the term “aberrant” includes a GAP-4 expression or activity which deviates from the wild type GAP-4 expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant GAP-4 expression or activity is intended to include the cases in which a mutation in the GAP-4 gene causes the GAP-4 gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional GAP-4 protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a GAP-4 ligand, e.g., a GTPase, or one which interacts with a non-GAP-4 ligand, e.g. a non-GTPase molecule. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as aberrant hydrolysis of GTP or aberrant levels of GTP/GDP or aberrant GTPase-related signaling. For example, the term unwanted includes a GAP-4 expression or activity which is undesirable in a subject.

[0243] The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in GAP-4 protein activity or nucleic acid expression, such as disorders related to GTP/GDP levels, e.g., atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, type 1 neurofibromatosis, Wiskott-Aldrich syndrome, cystic fibrosis, microphthalmia with linear skin defects syndrome, and viral infection. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in GAP-4 protein activity or nucleic acid expression, such as GTP hydrolysis-related disorders, e.g., cardiovascular disorders, such as atherosclerosis, hypertension, and heart disease; disorders of the central nervous system, such as cystic fibrosis, type 1 neurofibromatosis, Alzheimer's disease; cell growth disorders such as cancers (e.g., carcinoma, sarcoma, or leukemia), tumor angiogenesis and metastasis, skeletal dysplasia, hepatic disorders, hematopoietic and/or myeloproliferative disorders; immune disorders such as Wiskott-Aldrich syndrome, viral infection, autoimmune disorders, immune deficiency disorders (e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency); skin disorders such as microphthalmia with linear skin defects syndrome; and congenital and/or developmental abnormalities such as facio-genital dysplasia. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted GAP-4 expression or activity in which a test sample is obtained from a subject and GAP-4 protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of GAP-4 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted GAP-4 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

[0244] Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted GAP-4 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a GTP hydrolysis-related disorder or a disorder related to GTP/GDP levels. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted GAP-4 expression or activity in which a test sample is obtained and GAP-4 protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of GAP-4 protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted GAP-4 expression or activity).

[0245] The methods of the invention can also be used to detect genetic alterations in a GAP-4 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in GAP-4 protein activity or nucleic acid expression, such as disorders related to GTP/GDP levels, e.g. atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, type 1 neurofibromatosis, Wiskott-Aldrich syndrome, cystic fibrosis, microphthalmia with linear skin defects syndrome, and viral infection. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a GAP-4- or VR5-protein, or the mis-expression of the GAP-4 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a GAP-4 gene; 2) an addition of one or more nucleotides to a GAP-4 gene; 3) a substitution of one or more nucleotides of a GAP-4 gene, 4) a chromosomal rearrangement of a GAP-4 gene; 5) an alteration in the level of a messenger RNA transcript of a GAP-4 gene, 6) aberrant modification of a GAP-4 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a GAP-4 gene, 8) a non-wild type level of a GAP-4 protein, 9) allelic loss of a GAP-4 gene, and 10) inappropriate post-translational modification of a GAP-4 protein. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in a GAP-4 gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.

[0246] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in the GAP-4- or VR5-gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a GAP-4 gene under conditions such that hybridization and amplification of the GAP-4- or VR5-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[0247] Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0248] In an alternative embodiment, mutations in a GAP-4 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0249] In other embodiments, genetic mutations in GAP-4 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2:753-759). For example, genetic mutations in GAP-4 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0250] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the GAP-4 gene and detect mutations by comparing the sequence of the sample GAP-4 with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

[0251] Other methods for detecting mutations in the GAP-4 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type GAP-4 sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

[0252] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in GAP-4 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a GAP-4 sequence, e.g., a wild-type GAP-4 sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[0253] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in GAP-4 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control GAP-4 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0254] In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[0255] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[0256] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0257] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a GAP-4 gene.

[0258] Furthermore, any cell type or tissue in which GAP-4 is expressed may be utilized in the prognostic assays described herein.

[0259] 3. Monitoring of Effects during Clinical Trials

[0260] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a GAP-4 protein (e.g., the modulation of GTPase activity, GTP hydrolysis, the modulation of GTPase-related signaling mechanisms, the regulation of GTP/GDP levels) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase GAP-4 gene expression, protein levels, or upregulate GAP-4 activity, can be monitored in clinical trials of subjects exhibiting decreased GAP-4 gene expression, protein levels, or downregulated GAP-4 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease GAP-4 gene expression, protein levels, or suppress GAP-4 activity, can be monitored in clinical trials of subjects exhibiting increased GAP-4 gene expression, protein levels, or upregulated GAP-4 activity. In such clinical trials, the expression or activity of a GAP-4 gene, and preferably, other genes that have been implicated in, for example, a GAP-4-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[0261] For example, and not by way of limitation, genes, including GAP-4, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates GAP-4 activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on GAP-4-associated disorders (e.g., GTP hydrolysis-related disorder, disorders related to GTP/GDP levels), for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of GAP-4 and other genes implicated in the GAP-4-associated disorder, respectively. The levels of gene expression (e.g., a gene expression pattern) can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of GAP-4 or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.

[0262] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a GAP-4 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the GAP-4 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the GAP-4 protein, mRNA, or genomic DNA in the pre-administration sample with the GAP-4 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of GAP-4 to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of GAP-4 to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, GAP-4 expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.

[0263] D. Methods of Treatment:

[0264] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted GAP-4 expression or activity, e.g., a GTP hydrolysis-related disorder. “Treatment”, as used herein, is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of disease or disorder or the predisposition toward a disease or disorder. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the GAP-4 molecules of the present invention or GAP-4 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0265] 1. Prophylactic Methods

[0266] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted GAP-4 expression or activity, by administering to the subject a GAP-4 or an agent which modulates GAP-4 expression or at least one GAP-4 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted GAP-4 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the GAP-4 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of GAP-4 aberrancy, for example, a GAP-4, GAP-4 agonist or GAP-4 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[0267] 2. Therapeutic Methods

[0268] Another aspect of the invention pertains to methods of modulating GAP-4 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a GAP-4 or agent that modulates one or more of the activities of GAP-4 protein activity associated with the cell. An agent that modulates GAP-4 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a GAP-4 protein (e.g., a GAP-4 ligand or substrate), a GAP-4 antibody, a GAP-4 agonist or antagonist, a peptidomimetic of a GAP-4 agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more GAP-4 activities. Examples of such stimulatory agents include active GAP-4 protein and a nucleic acid molecule encoding GAP-4 that has been introduced into the cell. In another embodiment, the agent inhibits one or more GAP-4 activities. Examples of such inhibitory agents include antisense GAP-4 nucleic acid molecules, anti-GAP-4 antibodies, and GAP-4 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a GAP-4 protein or nucleic acid molecule such as a GTP hydrolysis-related disorder. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) GAP-4 expression or activity. In another embodiment, the method involves administering a GAP-4 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted GAP-4 expression or activity.

[0269] Stimulation of GAP-4 activity is desirable in situations in which GAP-4 is abnormally downregulated and/or in which increased GAP-4 activity is likely to have a beneficial effect. Likewise, inhibition of GAP-4 activity is desirable in situations in which GAP-4 is abnormally upregulated and/or in which decreased GAP-4 activity is likely to have a beneficial effect.

[0270] 3. Pharmacogenomics

[0271] The GAP-4 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on GAP-4 activity (e.g., GAP-4 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) GAP-4- or VR5-associated disorders (e.g., GTP hydrolysis-related disorders; disorders related to GTP/GDP levels) associated with aberrant or unwanted GAP-4 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a GAP-4 molecule or GAP-4 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a GAP-4 molecule or GAP-4 modulator.

[0272] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0273] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase 11/111 drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however,.the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[0274] Alternatively, a method termed the “candidate gene approach”, can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., a GAP-4 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[0275] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C 19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

[0276] Alternatively, a method termed the “gene expression profiling”, can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a GAP-4 molecule or GAP-4 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[0277] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a GAP-4 molecule or GAP-4 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0278] VI. Electronic Apparatus Readable Media and Arrays

[0279] Electronic apparatus readable media comprising GAP-4 sequence information is also provided. As used herein, “GAP-4 sequence information” refers to any nucleotide and/or amino acid sequence information particular to the GAP-4 molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like. Moreover, information “related to” said GAP-4 sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantitative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon GAP-4 sequence information of the present invention.

[0280] As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

[0281] As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the GAP-4 sequence information.

[0282] A variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium. For example, the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of data processor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the GAP-4 sequence information.

[0283] By providing GAP-4 sequence information in readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

[0284] The present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4-associated disease or disorder, wherein the method comprises the steps of determining GAP-4 sequence information associated with the subject and based on the GAP-4 sequence information, determining whether the subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[0285] The present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a GAP-4-associated disease or disorder or a pre-disposition to a disease associated with a GAP-4 wherein the method comprises the steps of determining GAP-4 sequence information associated with the subject, and based on the GAP-4 sequence information, determining whether the subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. The method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.

[0286] The present invention also provides in a network, a method for determining whether a subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4 associated disease or disorder associated with-GAP-4, said method comprising the steps of receiving GAP-4 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to GAP-4 and/or a GAP-4-associated disease or disorder, and based on one or more of the phenotypic information, the GAP-4 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4-associated disease or disorder (e.g., cancer, a cardiovascular disorder, or a CNS disorder). The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0287] The present invention also provides a business method for determining whether a subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4-associated disease or disorder, said method comprising the steps of receiving information related to GAP-4 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to GAP-4 and/or related to a GAP-4-associated disease or disorder, and based on one or more of the phenotypic information, the GAP-4 information, and the acquired information, determining whether the subject has a GAP-4-associated disease or disorder or a pre-disposition to a GAP-4-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0288] The invention also includes an array comprising a GAP-4 sequence of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be GAP-4. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

[0289] In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0290] In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a GAP-4-associated disease or disorder, progression of GAP-4-associated disease or disorder, and processes, such a cellular transformation associated with the GAP-4-associated disease or disorder.

[0291] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of GAP-4 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0292] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including GAP-4) that could serve as a molecular target for diagnosis or therapeutic intervention.

[0293] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.

EXAMPLES

Example 1

[0294] Identification and Characterization of Human GAP-4 CDNA

[0295] In this example, the identification and characterization of the gene encoding human GAP-4 (clone 26649) is described.

[0296] Isolation of the Human GAP-4 cDNA

[0297] The invention is based, at least in part, on the discovery of a human gene encoding a novel protein, referred to herein as GAP-4. The entire sequence of the human clone 26649 was determined and found to contain an open reading frame termed human “GAP-4.” The nucleotide sequence encoding the human GAP-4 protein is shown in FIGS. 1A-E and is set forth as SEQ ID NO:1. The protein encoded by this nucleic acid comprises about 881 amino acids and has the amino acid sequence shown in FIGS. 1A-E and set forth as SEQ ID NO:2. The coding region (open reading frame) of SEQ ID NO:1 is set forth as SEQ ID NO:3. Clone 26649, comprising the coding region of human GAP-4, was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209, on May 9, 2000, and assigned Accession No. PTA-1851.

[0298] Analysis of the Human GAP-4 Molecule

[0299] A search for domain consensus sequences was performed using the amino acid sequence of GAP-4 and a database of HMMs (the Pfam database, release 2.1) using the default parameters (described above). The search revealed a RhoGAP domain (Pfam Accession Number PF00620) within SEQ ID NO:2 at residues 266-415 (see FIG. 3).

[0300] A search was performed against the ProDom database resulting in the identification of a portion of the deduced amino acid sequence of human GAP-4 (SEQ ID NO:2) which has a 50% identity to ProDom Accession Number PD109560 (“protein SH3-binding 3BP-1 GTPase activation O75160”) over residues 87 to 268. In addition, human GAP-4 is 69% identical to ProDom Accession Number PD178857 (“O75160_human // KIAA0672”) over residues 1 to 82. Human GAP-4 is also 39% identical to ProDom Accession Number PD000780 (“protein GTPase domain SH2 activation zinc 3-kinase SH3 phosphatidylinositol regulatory”) over residues 265-408. In addition, human GAP-4 is 31% identical to ProDom Accession Number PD216166 (“075160_human // KIAA0672”) over residues 413-788. Human GAP-4 is also 32% identical to ProDom Accession Number PD006324 (“SH3 domain protein domain-containing containing SH3P13 SH3P8 EEN-B2-L4 SH3GL3”) over residues 178-241 and 26% identical over residues 86 to 154. In addition, human GAP-4 is 31% identical to ProDom Accession Number PD057258 (“putative preoptic regulatory factor-2 precursor PORF-2 hypothalamus testis hormone signal”) over residues 377-446. Human GAP-4 is further 20% identical to ProDom Accession Number PD074799 (“O60311_human // KIAA0565 protein”) over residues 24-194. In addition, human GAP-4 is 20% identical to ProDom Accession Number PD008784 (“protein dystrophin structural actin-binding calcium cytoskeleton repeat alternative splicing utrophin”) over residues 3-194. Furthermore, human GAP-4 is 20% identical to ProDom Accession Number PD136871 (“nuclear migration protein JNM1 coiled coil microtubules karyogamy”) over residues 61-242, and 14% identical to ProDom entry “protein coiled coil chain myosin repeat heavy ATP-binding filament heptad” over residues 1-194. The results of this search are shown in FIGS. 4A-D.

[0301] A search was also performed against the Prosite database, and resulted in the identification of several possible N-glycosylation sites at residues 13-16, 449-452, 463-466, 470-473, 593-596, and 874-877. In addition, within the human GAP-4 protein two cAMP and cGMP dependant phosphorylation sites were identified at residues 494-497 and 699-702. In addition, protein kinase C phosphorylation sites were identified within the human GAP-4 protein at residues 38-40, 46-48, 150-152, 175-177, 261-263, 546-548, 628-630, and 667-669. This search also identified casein kinase II phosphorylation sites at residues 60-63, 83-86, 96-99, 109-112, 171,174, 175-178, 214-217, 233-236, 252-255, 261-264, 308-311, 349-352, 415-418, 468-471, 547-550, 570-573, and 820-823 of human GAP-4. A tyrosine phosphorylation site motif was also identified in the human GAP-4 protein at residues 117-124. The search also identified the presence of N-myristoylation site motifs at residues 56-61, 290-295, 322-327, 511-516, 556-561, 616,621, 652-657, 683-688, 782-787, and 817-822. In addition, the search identified an aminoacyl-transfer RNA (class-I) synthetase signature sequence at residues 784-794.

[0302] An analysis of the possible cellular localization of the GAP-4 protein based on its amino acid sequence was performed using the methods and algorithms described in Nakai and Kanehisa (1992) Genomics 14:897-911, and at http://psort.nibb.acjp. The results from this analysis predict that the GAP-4 protein is found in the nucleus, the mitochondria, the cytoplasm and peroxisomes.

EXAMPLE 2

[0303] Expression of Recombinant GAP-4 Protein in Bacterial Cells

[0304] In this example, GAP-4 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, GAP-4 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression ofthe GST-GAP-4 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

EXAMPLE 3

[0305] Expression of Recombinant GAP-4 Protein in COS Cells

[0306] To express the GAP-4 gene in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire GAP-4 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[0307] To construct the plasmid, the GAP-4 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the GAP-4 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the GAP-4 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the GAP-4 gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[0308] COS cells are subsequently transfected with the GAP-4-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of the GAP-4 polypeptide is detected by radiolabeling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S -methionine (or 35S -cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[0309] Alternatively, DNA containing the GAP-4 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the GAP-4 polypeptide is detected by radiolabeling and immunoprecipitation using a GAP-4 specific monoclonal antibody.

[0310] II. 32591, A Novel Human GTPase Activating Molecule and Uses Therefor

Background of the Invention

[0311] The family of G proteins encompasses a diverse array of proteins which regulate a complex range of biological processes, including the regulation of protein synthesis, vesicular and nuclear transport, regulation of the cell cycle, differentiation, and cytoskeletal rearrangements. The common motif among this important family of proteins is the presence of a GTP-binding domain (Alberts et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., New York, N.Y. pp. 206-207, 641). These proteins act as molecular switches that can cycle between active (GTP-bound) and inactive (GDP-bound) states (Bourne et al. (1990) Nature, 348:125-132). In the active state, G proteins are able to interact with a broad range of effector molecules. These effector molecules constitute components of a variety of signaling cascades. The lifetime of the active state of a G protein is determined by the rate at which the bound GTP is converted to GDP by the GTP-hydrolytic activity (GTPase activity) that is intrinsic to most G proteins. Upon hydrolysis of the bound GTP, the G protein reverts to the inactive state. This intrinsic enzymatic activity is accelerated by orders of magnitude in the presence of a family of molecules which interact with G proteins called “GTPase-activating proteins” (GAPs) (Scheffzek et al. (1998) Trends Biochem Sci., 23:257-262; Gamblin and Smerdon (1998) Curr. Opinion in Struct. Biol. 8:195-201). The members of this family of molecules appear to interact with domains of a given G protein, causing conformational changes which activate GTPase activity. The opposing transition from GDP-bound inactive state to GTP-bound active state appears to be facilitated by another class of molecules known as guanine-nucleotide-exchange factors (GEFs).

[0312] It is the regulated cycling between active and inactive states of G proteins that allows for proper transduction of many vital cellular signals. Indeed, the regulation of GTP/GDP levels in the cell by G proteins, and their accessory GAP molecules, has been implicated in a number of diseases, including atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, type I neurofibromatosis, Wiskott-Aldrich syndrome, cystic fibrosis, Microphthalmia with linear skin defects syndrome, and viral infection (Meijt, (1996) Mol. Cell. Biochem. 157:31-38; Olson, (1996) J. Mol. Med. 74:563-571; Wilson et al. (1988) J. Cell Biol. 107:69-77; Gutmann and Collins, (1993) Neuron 10:335-343; Kolluri et al. (1996), PNAS 93:5615-5618; Schaefer et al., (1997) Genomics 46:268-277; Tan et al., (1993) Biol. Chem. 268:27291-27298).

[0313] Several GAP family members have been identified to date, including C. elegans gap-1 and gap-2 (Hajnal et al. (1997) Genes Dev., 11:2715-2728; Hayashizaki et al. (1998) Genes Cells 3:189-202), bovine GAP-1 and GAP-3 (Nice et al. (1992) J. Biol. Chem. 267:1546-1553), and Drosophila Gap1 (Gaul et al. (1992) Cell 68:1007-1019).

SUMMARY OF THE INVENTION

[0314] The present invention is based, at least in part, on the discovery of a novel family of GTPase activating proteins, referred to herein interchangeably as “GTPase Activating Protein-5,” “G Protein Activating Protein-5,” or “GAP-5” nucleic acid and protein molecules. The GAP-5 molecules of the present invention are useful as targets for developing modulating agents to regulate a variety of cellular processes which are influenced by the regulated hydrolysis of GTP to GDP and the resulting GTP/GDP ratios. These processes include transduction of intracellular signaling, structuring of the cytoskeleton, vesicular trafficking, and progression through the cell cycle. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding GAP-5 proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of GAP-5-encoding nucleic acids.

[0315] In one embodiment, a GAP-5 nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:4 or 6 or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or a complement thereof.

[0316] In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown SEQ ID NO:4 or 6, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 1-342 of SEQ ID NO:4. In another embodiment, the nucleic acid molecule includes SEQ ID NO:6 and nucleotides 3649-4431 of SEQ ID NO:4. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:4 or 6. In another preferred embodiment, the nucleic acid molecule includes a fragment of at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, or more nucleotides (e.g., contiguous nucleotides) of the nucleotide sequence of SEQ ID NO:4 or 6, or a complement thereof.

[0317] In another embodiment, a GAP-5 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:5 or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195. In a preferred embodiment, a GAP-5 nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the entire length of the amino acid sequence of SEQ ID NO:5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195.

[0318] In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human GAP-5. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195.

[0319] Another embodiment of the invention features nucleic acid molecules, preferably GAP-5 nucleic acid molecules, which specifically detect GAP-5 nucleic acid molecules relative to nucleic acid molecules encoding non-GAP-5 proteins. For example, in one embodiment, such a nucleic acid molecule is at least 50-100, 100-500, 500-1000, 1000-1500, 1500-2000, 2000-2500, 2500-3000, 3000-3500, or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:4 or 6, the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or a complement thereof.

[0320] In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:5, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:4 or 6 under stringent conditions.

[0321] Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a GAP-5 nucleic acid molecule, e.g., the coding strand of a GAP-5 nucleic acid molecule.

[0322] Another aspect of the invention provides a vector comprising a GAP-5 nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably a GAP-5 protein family member, by culturing a host cell in a suitable medium, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.

[0323] Another aspect of this invention features isolated or recombinant GAP-5 proteins and polypeptides. In preferred embodiments, the isolated GAP-5 protein family member includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0324] In a preferred embodiment, the GAP-5 protein family member has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the amino acid sequence of SEQ ID NO:5, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, and includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0325] In another preferred embodiment, the GAP-5 protein family member modulates GTPase activity, and includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0326] In yet another preferred embodiment, the GAP-5 protein family member is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4 or 6, and includes at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain.

[0327] In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:5, wherein the fragment comprises at least 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:5, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number PTA-2195. In another embodiment, the protein, preferably a GAP-5 protein, has the amino acid sequence of SEQ ID NO:5.

[0328] In another embodiment, the invention features an isolated GAP-5 protein family member which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to a nucleotide sequence of SEQ ID NO:4 or 6, or a complement thereof. This invention further features an isolated protein, preferably a GAP-5 protein, which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4 or 6, or a complement thereof.

[0329] The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-GAP-5 polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably GAP-5 proteins. In addition, the GAP-5 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

[0330] In another aspect, the present invention provides a method for detecting the presence of a GAP-5 nucleic acid molecule, protein or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a GAP-5 nucleic acid molecule, protein or polypeptide such that the presence of a GAP-5 nucleic acid molecule, protein or polypeptide is detected in the biological sample.

[0331] In another aspect, the present invention provides a method for detecting the presence of GAP-5 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of GAP-5 activity such that the presence of GAP-5 activity is detected in the biological sample.

[0332] In another aspect, the invention provides a method for modulating GAP-5 activity comprising contacting a cell capable of expressing GAP-5 with an agent that modulates GAP-5 activity such that GAP-5 activity in the cell is modulated. In one embodiment, the agent inhibits GAP-5 activity. In another embodiment, the agent stimulates GAP-5 activity. In one embodiment, the agent is an antibody that specifically binds to a GAP-5 protein. In another embodiment, the agent modulates expression of GAP-5 by modulating transcription of a GAP-5 gene or translation of a GAP-5 mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a GAP-5 mRNA or a GAP-5 gene.

[0333] In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted GAP-5 protein or nucleic acid expression or activity by administering an agent which is a GAP-5 modulator to the subject. In one embodiment, the GAP-5 modulator is a GAP-5 protein. In another embodiment the GAP-5 modulator is a GAP-5 nucleic acid molecule. In yet another embodiment, the GAP-5 modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant or unwanted GAP-5 protein or nucleic acid expression is a GTP hydrolysis-related disorder, such as atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, cystic fibrosis and viral infection.

[0334] The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a GAP-5 protein; (ii) mis-regulation of the GAP-5 gene; and (iii) aberrant post-translational modification of a GAP-5 protein, wherein a wild-type form of the gene encodes a protein with a GAP-5 activity.

[0335] In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of a GAP-5 protein, by providing an indicator composition comprising a GAP-5 protein having GAP-5 activity, contacting the indicator composition with a test compound, and detennining the effect of the test compound on GAP-5 activity in the indicator composition to identify a compound that modulates the activity of a GAP-5 protein.

[0336] Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0337] The present invention is based, at least in part, on the discovery of a novel family of GTPase activating proteins, referred to herein interchangeably as “GTPase Activating Protein-5,” “G Protein Activating Protein-5,” or “GAP-5.” GAP-5 is a GTPase-associating protein which resembles members of the GAP (GTPase activating protein) family of proteins (described in, for example, Scheffzek et al. (1998) Trends Biochem Sci., 23:257-262) that normally activate the hydrolysis of GTP into GDP by GTPases.

[0338] The GAP-5 molecules of the present invention play a role in GTP hydrolysis and regulation of GTP/GDP levels. As used herein, the term “GTP hydrolysis” includes the dephosphorylation of GTP, resulting in the formation of GDP or other forms of guanine. GTP hydrolysis is mediated by GTPases, e.g., Rho-GTPases, ras-GTPases, rac-GTPases, and rab-GTPases. As used herein, the term “regulation of GTP/GDP levels” includes cellular mechanisms involved in regulating and influencing the levels, e.g., intracellular levels, of GTP and GDP. Such mechanisms include the hydrolysis of GTP to GDP (GTP hydrolysis) in response to biological cues, e.g., by a GTPase. The maintenance of GTP/GDP levels is particularly important for a cell's signaling needs. Thus, the GAP-5 molecules, by participating in GTP hydrolysis and regulation of GTP/GDP levels, may modulate GTP hydrolysis and GTP/GDP levels and provide novel diagnostic targets and therapeutic agents to control GTP hydrolysis-related disorders.

[0339] As used herein, the term “GTP hydrolysis-related disorders” includes disorders, diseases, or conditions which are characterized by aberrant, e.g., upregulated or downregulated, GTP hydrolysis and/or aberrant, e.g., unregulated or downregulated, GTP and/or GDP levels. Examples of such disorders may include cardiovascular disorders, e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, or arrhythmia.

[0340] Other examples of GTP hydrolysis-related disorders include disorders of the central nervous system, e.g., cystic fibrosis, type I neurofibromatosis, cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[0341] Still other examples of GTP hydrolysis-related disorders include cellular proliferation, growth, differentiation, or migration disorders. Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes. As used herein, a “cellular proliferation, growth, differentiation, or migration process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.

[0342] Still other examples of GTP hydrolysis-related disorders include disorders of the immune system, such as Wiskott-Aldrich syndrome, viral infection, autoimmune disorders or immune deficiency disorders, e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency. Other examples of GTP hydrolysis-related disorders include congenital malformalities, including facio-genital dysplasia; and skin disorders, including microphthalmia with linear skin defects syndrome.

[0343] The term “family” when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin. Members of a family may also have common functional characteristics.

[0344] For example, the family of GAP-5 proteins comprise preferably at least one, two, three, or more “transmembrane domains.” As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 10, 15, 20, 25, 30, 35, 40, 45 or more amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have a helical structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acid residues of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al., (1996) Annual Rev. Neurosci. 19: 235-63, the contents of which are incorporated herein by reference. Amino acid residues 909-929 of the human GAP-5 polypeptide (SEQ ID NO:5) comprise transmembrane domains.

[0345] In another embodiment, a GAP-5 molecule of the present invention is identified based on the presence of a “RhoGAP domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “RhoGAP domain” includes a protein domain having an amino acid sequence of about 150 amino acid residues and having a bit score for the alignment of the sequence to the RhoGAP domain (HMM) of at least 169. Preferably, a RhoGAP domain includes at least about 130-200, more preferably about 145-180 amino acid residues, or about 155-175 amino acids and has a bit score for the alignment of the sequence to the RhoGAP domain (HMM) of at least 100, 150, 160, 170, 180, 190, 200, or greater. The RhoGAP domain has been assigned the PFAM Accession PF00620 (http://genome.wustl.edu/Pfam/.html). RhoGAP domains are involved in protein-protein interactions and are described in, for example, Musacchio et al., (1996) PNAS, 93:14373-14378, the contents of which are incorporated herein by reference.

[0346] To identify the presence of an RhoGAP domain in a GAP-5 protein and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 2.1) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. NatlL. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference. A search was performed against the HMM database resulting in the identification of a RhoGAP domain in the amino acid sequence of SEQ ID NO:5 (at about residues 266-415). The results of this search are set forth in FIGS. 6A-B.

[0347] Isolated GAP-5 proteins of the present invention, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:5, or are encoded by a 5 nucleotide sequence sufficiently identical to SEQ ID NO:4 or 6. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.

[0348] As used interchangeably herein, a “GAP-5 activity”, “biological activity of GAP-5,” or “functional activity of GAP-5,” includes an activity exerted by a GAP-5 protein, polypeptide or nucleic acid molecule on a GAP-5-responsive cell or tissue, or on a GAP-5 protein substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a GAP-5 activity is a direct activity, such as an association with a GAP-5-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which a GAP-5 protein binds or interacts in nature, such that GAP-5- mediated function is achieved. A GAP-5 target molecule can be a non-GAP-5 molecule or a GAP-5 protein or polypeptide of the present invention. In an exemplary embodiment, a GAP-5 target molecule is a GAP-5 ligand, e.g., a GTPase. Alternatively, a GAP-5 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the GAP-5 protein with a GAP-5 ligand, e.g., a GTPase. Preferably, a GAP-5 activity is the ability to modulate the hydrolysis of GTP via, e.g., interactions with GTPase molecules.

[0349] Accordingly, another embodiment of the invention features isolated GAP-5 polypeptides having a GAP-5 activity. Preferred proteins are GAP-5 proteins having at least one or more of the following domains: a RhoGAP domain, and/or a transmembrane domain, and, preferably, a GAP-5 activity. Additional preferred GAP-5 proteins have at least one RhoGAP domain, and/or at least one transmembrane domain and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4 or 6.

[0350] The nucleotide sequence of the isolated human GAP-5 cDNA and the predicted amino acid sequence of the human GAP-5 polypeptide are shown in FIGS. 5A-D and in SEQ ID NO:4 and SEQ ID NO:5, respectively. A plasmid containing the nucleotide sequence encoding human GAP-5 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on Jul. 7, 2000 and assigned Accession Number PTA-2195.

[0351] These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112.

[0352] The human GAP-5 gene, which is approximately 4431 nucleotides in length, encodes a protein having a molecular weight of approximately 121 kD and which is approximately 1101 amino acid residues in length.

[0353] Various aspects of the invention are described in further detail in the following subsections:

[0354] I. Isolated Nucleic Acid Molecules

[0355] One aspect of the invention pertains to isolated nucleic acid molecules that encode GAP-5 proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify GAP-5-encoding nucleic acid molecules (e.g., GAP-5 mRNA) and fragments for use as PCR primers for the amplification or mutation of GAP-5 nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0356] The term “isolated nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated GAP-5 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0357] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, as a hybridization probe, GAP-5 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0358] Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195.

[0359] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GAP-5 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0360] In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:4. The sequence of SEQ ID NO:4 corresponds to the human GAP-5 cDNA. This cDNA comprises sequences encoding the human GAP-5 protein (i.e., “the coding region”, from nucleotides 343-3648), as well as 5′ untranslated sequences (nucleotides 1-342) and 3′ untranslated sequences (nucleotides 3649-4431). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:4 (e.g., nucleotides 343-3648, corresponding to SEQ ID NO:6). The isolated nucleic molecule of the invention can consist of the nucleic acid sequence shown in SEQ ID NO:4 or 6.

[0361] In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, thereby forming a stable duplex.

[0362] In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:4 or 6, or the entire length of the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or a portion of any of these nucleotide sequences.

[0363] Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a GAP-5 protein, e.g., a biologically active portion of a GAP-5 protein. The nucleotide sequence determined from the cloning of the GAP-5 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other GAP-5 family members, as well as GAP-5 homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert-of the plasmid deposited with ATCC as Accession Number PTA-2195, of an anti-sense sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or of a naturally occurring allelic variant or mutant of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195. In one embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195.

[0364] Probes based on the GAP-5 nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a GAP-5 protein, such as by measuring a level of a GAP-5-encoding nucleic acid in a sample of cells from a subject, e.g., detecting GAP-5 mRNA levels or determining whether a genomic GAP-5 gene has been mutated or deleted.

[0365] A nucleic acid fragment encoding a “biologically active portion of a GAP-5 protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, which encodes a polypeptide having a GAP-5 biological activity (the biological activities of the GAP-5 proteins are described herein), expressing the encoded portion of the GAP-5 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the GAP-5 protein.

[0366] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, due to degeneracy of the genetic code and, thus, encode the same GAP-5 proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2 195. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:5.

[0367] In addition to the GAP-5 nucleotide sequences shown in SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2 195, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the GAP-5 proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the GAP-5 genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a GAP-5 protein, preferably a mammalian GAP-5 protein, and can further include non-coding regulatory sequences, and introns.

[0368] Allelic variants of human GAP-5 include both functional and non-functional GAP-5 proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the human GAP-5 protein that maintain the ability to bind a GAP-5 ligand or substrate (e.g., a GTPase) and/or modulate GTP hydrolysis and/or GTPase signaling mechanisms, and/or disorders related to regulation of levels of GTP/GDP. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:5, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.

[0369] Non-functional allelic variants are naturally occurring amino acid sequence variants of the human GAP-5 proteins that do not have the ability to either bind a GAP-5 ligand or substrate (e.g., a GTPase) and/or modulate GTP hydrolysis and/or GTPase signaling mechanisms, and/or disorders related to regulation of levels of GTP/GDP. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:5, or a substitution, insertion or deletion in critical residues or critical regions.

[0370] The present invention further provides non-human orthologues of the human GAP-5 protein. Orthologues of the human GAP-5 protein are proteins that are isolated from non-human organisms and possess the same GAP-5 ligand binding and/or modulation of GTPase activity and/or GTPase related signaling mechanisms and/or modulation of GTP/GDP levels. Orthologues of the human GAP-5 protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:5.

[0371] Moreover, nucleic acid molecules encoding other GAP-5 family members and, thus, which have a nucleotide sequence which differs from the GAP-5 sequences of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195 are intended to be within the scope of the invention. For example, another GAP-5 cDNA can be identified based on the nucleotide sequence of human GAP-5. Moreover, nucleic acid molecules encoding GAP-5 proteins from different species, and which, thus, have a nucleotide sequence which differs from the GAP-5 sequences of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195 are intended to be within the scope of the invention. For example, a mouse GAP-5 cDNA can be identified based on the nucleotide sequence of a human GAP-5.

[0372] Nucleic acid molecules corresponding to natural allelic variants and homologues of the GAP-5 cDNAs of the invention can be isolated based on their homology to the GAP-5 nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the GAP-5 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the GAP-5 gene.

[0373] Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195. In other embodiment, the nucleic acid is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500 or more nucleotides in length.

[0374] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4×sodium chloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in 1×SSC,.at about 65-70° C. (or hybridization in 1×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 0.3×SSC, at about 65-70° C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide at about 40-45° C.) followed by one or more washes in 2×SSC, at about 50-60° C. Ranges intermediate to the above-recited values, e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassed by the present invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm (° C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH2PO4, 7% SDS at about 65° C., followed by one or more washes at 0.02M NaH2PO4, 1% SDS at 65° C., see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or alternatively 0.2×SSC, 1% SDS).

[0375] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:4 or 6 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0376] In addition to naturally-occurring allelic variants of the GAP-5 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, thereby leading to changes in the amino acid sequence of the encoded GAP-5 proteins, without altering the functional ability of the GAP-5 proteins. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of GAP-5 (e.g., the sequence of SEQ ID NO:5) without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the GAP-5 proteins of the present invention, e.g., those present in the RhoGAP domain(s) or the transmembrane domain(s), are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the GAP-5 proteins of the present invention and other members of the GAP-5 family are not likely to be amenable to alteration.

[0377] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding GAP-5 proteins that contain changes in amino acid residues that are not essential for activity. Such GAP-5 proteins differ in amino acid sequence from SEQ ID NO:5, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to SEQ ID NO:5.

[0378] An isolated nucleic acid molecule encoding a GAP-5 protein identical to the protein of SEQ ID NO:5, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a GAP-5 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a GAP-5 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GAP-5 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0379] In another preferred embodiment, a mutant GAP-5 protein can be assayed for the ability to (1) interact with a non-GAP-5 protein molecule, e.g., a GTPase or a GAP-5 ligand or substrate; (2) modulate a GAP-5-dependent signal transduction pathway; (3) modulate GTPase-dependant signal transduction; (4) modulate GTP hydrolysis activity; (5) modulate levels of GTP/GDP.

[0380] In addition to the nucleic acid molecules encoding GAP-5 proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire GAP-5 coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding GAP-5. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human GAP-5 corresponds to SEQ ID NO:6). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding GAP-5. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0381] Given the coding strand sequences encoding GAP-5 disclosed herein (e.g., SEQ ID NO:6), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of GAP-5 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of GAP-5 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GAP-5 mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex fommed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0382] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a GAP-5 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of l0 antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0383] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An (α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ,-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et aL (1987) FEBSLett. 215:327-330).

[0384] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave GAP-5 mRNA transcripts to thereby inhibit translation of GAP-5 mRNA. A ribozyme having specificity for a GAP-5-encoding nucleic acid can be designed based upon the nucleotide sequence of a GAP-5 cDNA disclosed herein (i.e., SEQ ID NO:4 or 6, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195). For example, a derivative of a Tetrahymena L-19 IVS RNA can be*constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a GAP-5-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, GAP-5 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0385] Alternatively, GAP-5 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory and/or 5′ untranslated region of the GAP-5 nucleotides (e.g., the GAP-5 promoter and/or enhancers; e.g., nucleotides 1-126 of SEQ ID NO:4) to form triple helical structures that prevent transcription of the GAP-5 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

[0386] In yet another embodiment, the GAP-5 nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0387] PNAs of GAP-5 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of GAP-5 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0388] In another embodiment, PNAs of GAP-5 can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of GAP-5 nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn P.J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser, K.H. et al (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

[0389] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/098 10) or the blood-brain barrier (see, e.g., PCT Publication No.

[0390] W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0391] II. Isolated GAP-5 Proteins and Anti-GAP-5 Antibodies

[0392] One aspect of the invention pertains to isolated GAP-5 proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-GAP-5 antibodies. In one embodiment, native GAP-5 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, GAP-5 proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a GAP-5 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[0393] An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GAP-5 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of GAP-5 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of GAP-5 protein having less than about 30% (by dry weight) of non-GAP-5 protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-GAP-5 protein, still more preferably less than about 10% of non-GAP-5 protein, and most preferably less than about 5% non-GAP-5 protein. When the GAP-5 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0394] The language “substantially free of chemical precursors or other chemicals” includes preparations of GAP-5 protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of GAP-5 protein having less than about 30% (by dry weight) of chemical precursors or non-GAP-5 chemicals, more preferably less than about 20% chemical precursors or non-GAP-5 chemicals, still more preferably less than about 10% chemical precursors or non-GAP-5 chemicals, and most preferably less than about 5% chemical precursors or non-GAP-5 chemicals.

[0395] As used herein, a “biologically active portion” of a GAP-5 protein includes a fragment of a GAP-5 protein which participates in an interaction between a GAP-5 molecule and a non-GAP-5 molecule, e.g., a GTPase. Biologically active portions of a GAP-5 protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the GAP-5 protein, e.g., the amino acid sequence shown in SEQ ID NO:5, which include less amino acids than the full length GAP-5 proteins, and exhibit at least one activity of a GAP-5 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the GAP-5 protein, e.g., interacting with GTPase molecules, modulating GTPase activity, and/or modulating GTP/GDP levels. A biologically active portion of a GAP-5 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200, 500, or more amino acids in length. Biologically active portions of a GAP-5 protein can be used as targets for developing agents which modulate a GAP-5 mediated activity, e.g., modulation of GTP hydrolysis or modulation of GTP/GDP levels.

[0396] In one embodiment, a biologically active portion of a GAP-5 protein comprises at least one RhoGAP domain, and/or at least one transmembrane domain. It is to be understood that a preferred biologically active portion of a GAP-5 protein of the present invention may contain at least one RhoGAP domain. Another preferred biologically active portion of a GAP-5 protein may contain at least one transmembrane domain. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native GAP-5 protein.

[0397] In a preferred embodiment, the GAP-5 protein has an amino acid sequence shown in SEQ ID NO:5. In other embodiments, the GAP-5 protein is substantially identical to SEQ ID NO:5, and retains the functional activity of the protein of SEQ ID NO:5, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the GAP-5 protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more identical to SEQ ID NO:5.

[0398] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the GAP-5 amino acid sequence of SEQ ID NO:5 having 1101 amino acid residues, at least 331, preferably at least 441, more preferably at least 541, even more preferably at least 661, and even more preferably at least 771, 882 or 992 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0399] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Myers and Miller, 1988, Comput. Appl. Biosci. 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0400] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to GAP-5 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=100, wordlength=3 to obtain amino acid sequences homologous to GAP-5 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0401] The invention also provides GAP-5 chimeric or fusion proteins. As used herein, a GAP-5 “chimeric protein” or “fusion protein” comprises a GAP-5 polypeptide operatively linked to a non-GAP-5 polypeptide. A “GAP-5 polypeptide” includes a polypeptide having an amino acid sequence corresponding to GAP-5, whereas a “non-GAP-5 peptide” includes a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to a GAP-5 protein, e.g., a protein which is different from the GAP-5 protein and which is derived from the same or a different organism. Within a GAP-5 fusion protein the GAP-5 polypeptide can correspond to all or a portion of a GAP-5 protein. In a preferred embodiment, a GAP-5 fusion protein comprises at least one biologically active portion of a GAP-5 protein. In another preferred embodiment, a GAP-5 fusion protein comprises at least two biologically active portions of a GAP-5 protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the GAP-5 polypeptide and the non-GAP-5 polypeptide are fused in-frame to each other. The non-GAP-5 polypeptide can be fused to the N-terminus or C-terminus of the GAP-5 polypeptide.

[0402] For example, in one embodiment, the fusion protein is a GST-GAP-5 fusion protein in which the GAP-5 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant GAP-5.

[0403] In another embodiment, the fusion protein is a GAP-5 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of GAP-5 can be increased through use of a heterologous signal sequence.

[0404] The GAP-5 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The GAP-5 fusion proteins can be used to affect the bioavailability of a GAP-5 ligand or substrate. Use of GAP-5 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a GAP-5 protein; (ii) mis-regulation of the GAP-5 gene; and (iii) aberrant post-translational modification of a GAP-5 protein.

[0405] Moreover, the GAP-5-fusion proteins of the invention can be used as immunogens to produce anti-GAP-5 antibodies in a subject, to purify GAP-5 ligands and in screening assays to identify molecules which inhibit the interaction of GAP-5 with a GAP-5 ligand or substrate.

[0406] Preferably, a GAP-5 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A GAP-5-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GAP-5 protein.

[0407] The present invention also pertains to variants of the GAP-5 proteins which function as either GAP-5 agonists (mimetics) or as GAP-5 antagonists. Variants of the GAP-5 proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a GAP-5 protein. An agonist of the GAP-5 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a GAP-5 protein. An antagonist of a GAP-5 protein can inhibit one or more of the activities of the naturally occurring form of the GAP-5 protein by, for example, competitively modulating a GAP-5-mediated activity of a GAP-5 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the GAP-5 protein.

[0408] In one embodiment, variants of a GAP-5 protein which function as either GAP-5 agonists (mimetics) or as GAP-5 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a GAP-5 protein for GAP-5 protein agonist or antagonist activity. In one embodiment, a variegated library of GAP-5 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of GAP-5 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GAP-5 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GAP-5 sequences therein. There are a variety of methods which can be used to produce libraries of potential GAP-5 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential GAP-5 sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[0409] In addition, libraries of fragments of a GAP-5 protein coding sequence can be used to generate a variegated population of GAP-5 fragments for screening and subsequent selection of variants of a GAP-5 protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a GAP-5 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the GAP-5 protein.

[0410] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of GAP-5 proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GAP-5 variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering 6(3):327-331).

[0411] In one embodiment, cell based assays can be exploited to analyze a variegated GAP-5 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a neuronal cell line, which ordinarily responds to GAP-5 in a particular GAP-5 ligand-dependent manner. The transfected cells are then contacted with a GAP-5 ligand and the effect of expression of the mutant on signaling by the GAP-5 ligand can be detected, e.g., by monitoring GTPase activity, GTPase-related signaling mechanisms, or the activity of a GAP-5-regulated transcription factor. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the GAP-5 ligand, and the individual clones further characterized. In related cell-based assays, changes in GTP/GDP levels (i.e., signal transduction) can be measured in live cells which express GAP-5 molecules of the invention. Such an assay can be used for screening compound libraries for useful ligands which interact with GAP-5, or can be used to identify variants of GAP-5 which have useful properties. Other cell based assay include those which can monitor fluxes in intracellular calcium levels which result from GTPase-mediated signaling, e.g., flow cytometry (Valet and Raffael, 1985, Naturwiss., 72:600-602). Also within the scope of the invention are assays and models which utilize GAP-5 nucleic acids to create transgenic organisms for identifying useful pharmaceutical compounds or variants of the GAP-5 molecules.

[0412] An isolated GAP-5 protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind GAP-5 using standard techniques for polyclonal and monoclonal antibody preparation. A full-length GAP-5 protein can be used or, alternatively, the invention provides antigenic peptide fragments of GAP-5 for use as immunogens. The antigenic peptide of GAP-5 comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:5 and encompasses an epitope of GAP-5 such that an antibody raised against the peptide forms a specific immune complex with GAP-5. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0413] Preferred epitopes encompassed by the antigenic peptide are regions of GAP-5 that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity (see FIG. 8).

[0414] A GAP-5 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed GAP-5 protein or a chemically synthesized GAP-5 polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic GAP-5 preparation induces a polyclonal anti-GAP-5 antibody response.

[0415] Accordingly, another aspect of the invention pertains to anti-GAP-5 antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as GAP-5. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind GAP-5. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GAP-5. A monoclonal antibody composition thus typically displays a single binding affinity for a particular GAP-5 protein with which it immunoreacts.

[0416] Polyclonal anti-GAP-5 antibodies can be prepared as described above by immunizing a suitable subject with a GAP-5 immunogen. The anti-GAP-5 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized GAP-5. If desired, the antibody molecules directed against GAP-5 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-GAP-5 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lemer (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a GAP-5 immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds GAP-5.

[0417] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-GAP-5 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lemer, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind GAP-5, e.g., using a standard ELISA assay.

[0418] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-GAP-5 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with GAP-5 to thereby isolate immunoglobulin library members that bind GAP-5. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0419] Additionally, recombinant anti-GAP-5 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0420] An anti-GAP-5 antibody (e.g., monoclonal antibody) can be used to isolate GAP-5 by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-GAP-5 antibody can facilitate the purification of natural GAP-5 from cells and of recombinantly produced GAP-5 expressed in host cells. Moreover, an anti-GAP-5 antibody can be used to detect GAP-5 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the GAP-5 protein. Anti-GAP-5 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0421] III. Recombinant Expression Vectors and Host Cells

[0422] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a GAP-5 protein (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0423] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GAP-5 proteins, mutant forms of GAP-5 proteins, fusion proteins, and the like).

[0424] The recombinant expression vectors of the invention can be designed for expression of GAP-5 proteins in prokaryotic or eukaryotic cells. For example, GAP-5 proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0425] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0426] Purified fusion proteins can be utilized in GAP-5 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for GAP-5 proteins, for example. In a preferred embodiment, a GAP-5 fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

[0427] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polyrnerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0428] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0429] In another embodiment, the GAP-5 expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corporation, San Diego, Calif.).

[0430] Alternatively, GAP-5 proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0431] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0432] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0433] The expression characteristics of an endogenous GAP-5 gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous GAP-5 gene. For example, an endogenous GAP-5 gene which is normally “transcriptionally silent”, i.e., a GAP-5 gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism. Alternatively, a transcriptionally silent, endogenous GAP-5 gene may be activated by insertion of a promiscuous regulatory element that works across cell types.

[0434] A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous GAP-5 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

[0435] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to GAP-5 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0436] Another aspect of the invention pertains to host cells into which a GAP-5 nucleic acid molecule of the invention is introduced, e.g., a GAP-5 nucleic acid molecule within a recombinant expression vector or a GAP-5 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0437] A host cell can be any prokaryotic or eukaryotic cell. For example, a GAP-5 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0438] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0439] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a GAP-5 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0440] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a GAP-5 protein. Accordingly, the invention further provides methods for producing a GAP-5 protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a GAP-5 protein has been introduced) in a suitable medium such that a GAP-5 protein is produced. In another embodiment, the method further comprises isolating a GAP-5 protein from the medium or the host cell.

[0441] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GAP-5-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous GAP-5 sequences have been introduced into their genome or homologous recombinant animals in which endogenous GAP-5 sequences have been altered. Such animals are useful for studying the function and/or activity of a GAP-5 and for identifying and/or evaluating modulators of GAP-5 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GAP-5 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0442] A transgenic animal of the invention can be created by introducing a GAP-5-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The GAP-5 cDNA sequence of SEQ ID NO:4 or 6 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human GAP-5 gene, such as a mouse or rat GAP-5 gene, can be used as a transgene. Alternatively, a GAP-5 gene homologue, such as another GAP-5 family member, can be isolated based on hybridization to the GAP-5 cDNA sequences of SEQ ID NO:4 or 6, or the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195 (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a GAP-5 transgene to direct expression of a GAP-5 protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a GAP-5 transgene in its genome and/or expression of GAP-5 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a GAP-5 protein can further be bred to other transgenic animals carrying other transgenes.

[0443] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a GAP-5 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GAP-5 gene. The GAP-5 gene can be a human gene (e.g., the cDNA of SEQ ID NO:4 or 6), but more preferably, is a non-human homologue of a human GAP-5 gene (e.g., a CDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:4 or 6). For example, a mouse GAP-5 gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous GAP-5 gene in the mouse genome.

[0444] In a preferred embodiment, the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous GAP-5 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous GAP-5 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GAP-5 protein). In the homologous recombination nucleic acid molecule, the altered portion of the GAP-5 gene is flanked at its 5′ and 3′ ends by additional nucleic acid sequence of the GAP-5 gene to allow for homologous recombination to occur between the exogenous GAP-5 gene carried by the homologous recombination nucleic acid molecule and an endogenous GAP-5 gene in a cell, e.g., an embryonic stem cell. The additional flanking GAP-5 nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GAP-5 gene has homologously recombined with the endogenous GAP-5 gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

[0445] In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0446] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0447] IV. Pharmaceutical Compositions

[0448] The GAP-5 nucleic acid molecules, fragments of GAP-5 proteins, and anti-GAP-5 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0449] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0450] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0451] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a GAP-5 protein or an anti-GAP-5 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0452] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0453] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0454] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0455] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0456] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0457] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0458] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0459] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography. As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0460] In a preferred example, a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

[0461] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.

[0462] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0463] Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0464] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0465] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0466] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0467] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0468] V. Uses and Methods of the Invention

[0469] The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, a GAP-5 protein of the invention has one or more of the following activities: (I) it interacts with a non-GAP-5 protein molecule, e.g., a GTPase or a GAP-5 ligand; (2) it modulated a GAP-5-dependent signal transduction pathway; (3) it modulates GTP/GDP levels; and (4) it modulates GTPase signaling mechanisms, and, thus, can be used to, for example, (1) modulate the interaction with a non-GAP-5 protein molecule, e.g., a GTPase; (2) activate a GAP-5-dependent signal transduction pathway; (3) modulate GTP/GDP levels; and (4) modulate GTPase signaling mechanisms.

[0470] The isolated nucleic acid molecules of the invention can be used, for example, to express GAP-5 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GAP-5 mRNA (e.g., in a biological sample) or a genetic alteration in a GAP-5 gene, and to modulate GAP-5 activity, as described further below. The GAP-5 proteins can be used to treat disorders characterized by insufficient or excessive production of a GAP-5 ligand or substrate or production of GAP-5 inhibitors. In addition, the GAP-5 proteins can be used to screen for naturally occurring GAP-5 ligands or substrates to screen for drugs or compounds which modulate GAP-5 activity, as well as to treat disorders characterized by insufficient or excessive production of GAP-5 protein or production of GAP-5 protein forms which have decreased, aberrant or unwanted activity compared to GAP-5 wild type protein (e.g., GTP hydrolysis-related disorders and/or disorders related to GTP/GDP levels). Moreover, the anti-GAP-5 antibodies of the invention can be used to detect and isolate GAP-5 proteins, regulate the bioavailability of GAP-5 proteins, and modulate GAP-5 activity.

[0471] A. Screening Assays:

[0472] The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to GAP-5 proteins, have a stimulatory or inhibitory effect on, for example, GAP-5 expression or GAP-5 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a GAP-5 ligand or substrate.

[0473] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates or ligands of a GAP-5 protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a GAP-5 protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

[0474] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994) J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0475] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

[0476] In one embodiment, an assay is a cell-based assay in which a cell which expresses a GAP-5 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate GAP-5 activity is determined. Determining the ability of the test compound to modulate GAP-5 activity can be accomplished by monitoring, for example, changes in intracellular calcium concentration by, e.g., flow cytometry, or by the activity of a GAP-5-regulated transcription factor. The cell, for example, can be of mammalian origin, e.g., a neuronal cell.

[0477] The ability of the test compound to modulate GAP-5 binding to a ligand or substrate or to bind to GAP-5 can also be determined. Determining the ability of the test compound to modulate GAP-5 binding to a ligand or substrate can be accomplished, for example, by coupling the GAP-5 ligand or substrate with a radioisotope or enzymatic label such that binding of the GAP-5 ligand or substrate to GAP-5 can be determined by detecting the labeled GAP-5 ligand or substrate in a complex. Determining the ability of the test compound to bind GAP-5 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to GAP-5 can be determined by detecting the labeled GAP-5 compound in a complex. For example, compounds (e.g., GAP-5 ligands or substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0478] It is also within the scope of this invention to determine the ability of a compound (e.g., a GAP-5 ligand or substrate) to interact with GAP-5 without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with GAP-5 without the labeling of either the compound or the GAP-5. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and GAP-5.

[0479] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a GAP-5 target molecule (e.g., a GAP-5 ligand or substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GAP-5 target molecule. Determining the ability of the test compound to modulate the activity of a GAP-5 target molecule can be accomplished, for example, by determining the ability of the GAP-5 protein to bind to or interact with the GAP-5 target molecule.

[0480] Determining the ability of the GAP-5 protein or a biologically active fragment thereof, to bind to or interact with a GAP-5 target molecule (e.g., a GTPase) can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the GAP-5 protein to bind to or interact with a GAP-5 target molecule or GTPase can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting the ability of the GTPase to hydrolyze GTP, or by detecting induction of a cellular second messenger of the target (i.e., intracellular Ca2+, diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response such as changes in cytoskeletal structure or nuclear transport. GTPase activity may also be determined by, for example, capillary electrophoresis without radioisotope, as described in Kawata, et al. (2000) Tohoku J. Exp. Med. 192(1):67-79, HPLC as described in Shimada, et al. (1995) Seikagaku 67(6):475-7, or any other methods known in the art.

[0481] In yet another embodiment, an assay of the present invention is a cell-free assay in which a GAP-5 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the GAP-5 protein or biologically active portion thereof is determined. Preferred biologically active portions of the GAP-5 proteins to be used in assays of the present invention include fragments which participate in interactions with non-GAP-5 molecules, e.g., fragments with high surface probability scores. Binding of the test compound to the GAP-5 protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the GAP-5 protein or biologically active portion thereof with a known compound which binds GAP-5 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GAP-5 protein, wherein determining the ability of the test compound to interact with a GAP-5 protein comprises determining the ability of the test compound to preferentially bind to GAP-5 or biologically active portion thereof as compared to the known compound.

[0482] In another embodiment, the assay is a cell-free assay in which a GAP-5 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GAP-5 protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a GAP-5 protein can be accomplished, for example, by determining the ability of the GAP-5 protein to bind to a GAP-5 target molecule by one of the methods described above for determining direct binding. Determining the ability of the GAP-5 protein to bind to a GAP-5 target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[0483] In an alternative embodiment, determining the ability of the test compound to modulate the activity of a GAP-5 protein can be accomplished by determining the ability of the GAP-5 protein to further modulate the activity of a downstream effector of a GAP-5 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.

[0484] In yet another embodiment, the cell-free assay involves contacting a GAP-5 protein or biologically active portion thereof with a known compound which binds the GAP-5 protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the GAP-5 protein, wherein determining the ability of the test compound to interact with the GAP-5 protein comprises determining the ability of the GAP-5 protein to preferentially bind to or modulate the activity of a GAP-5 target molecule.

[0485] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either GAP-5 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a GAP-5 protein, or interaction of a GAP-5 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/GAP-5 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or GAP-5 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.

[0486] Alternatively, the complexes can be dissociated from the matrix, and the level of GAP-5 binding or activity determined using standard techniques.

[0487] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a GAP-5 protein or a GAP-5 target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated GAP-5 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with GAP-5 protein or target molecules but which do not interfere with binding of the GAP-5 protein to its target molecule can be derivatized to the wells of the plate, and unbound target or GAP-5 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GAP-5 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the GAP-5 protein or target molecule.

[0488] In another embodiment, modulators of GAP-5 expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of GAP-5 mRNA or protein in the cell is determined. The level of expression of GAP-5 mRNA or protein in the presence of the candidate compound is compared to the level of expression of GAP-5 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of GAP-5 expression based on this comparison. For example, when expression of GAP-5 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GAP-5 mRNA or protein expression. Alternatively, when expression of GAP-5 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GAP-5 mRNA or protein expression. The level of GAP-5 mRNA or protein expression in the cells can be determined by methods described herein for detecting GAP-5 mRNA or protein.

[0489] In yet another aspect of the invention, the GAP-5 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with GAP-5 (“GAP-5-binding proteins” or “GAP-5-bp”) and are involved in GAP-5 activity. Such GAP-5-binding proteins are also likely to be involved in the propagation of signals by the GAP-5 proteins or GAP-5 targets as, for example, downstream elements of a GAP-5-mediated signaling pathway. Alternatively, such GAP-5-binding proteins are likely to be GAP-5 inhibitors.

[0490] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a GAP-5 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a GAP-5-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the GAP-5 protein.

[0491] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a GAP-5 protein can be confirmed in vivo, e.g., in an animal such as an animal model for cancer or cardiovascular disease.

[0492] Examples of animal models of cancer include transplantable models (e.g., xenografts of colon tumors such as Co-3, AC3603 or WiDr or into immunocompromised mice such as SCID or nude mice); transgenic models (e.g., B66-Min/+ mouse); chemical induction models, e.g., carcinogen (e.g., azoxymethane, 2-dimethylhydrazine, or N-nitrosodimethylamine) treated rats or mice; models of liver metastasis from colon cancer such as that described by Rashidi et al. (2000) Anticancer Res. 20(2A):715; and cancer cell implantation or inoculation models as described in, for example, Fingert, et al. (1987) Cancer Res. 46(14):3824-9 and Teraoka, et al. (1995) Jpn. J. Cancer Res. 86(5):419-23.

[0493] Examples of animal models for cardiovascular disease include mouse models for renal ischemic reperfusion injury (IRI) such as that described in Burne et al. (2000) Transplantation 69(5):1023-5; animal models of congestive heart failure (CHF) such as that described in Smith, et al. (2000) J. Pharmacol. Toxicol. Methods 43(2): 125; animal models of restenosis such as that described in Hehrlein et al. (2000) Eur Heart 21(24):2056-62; and animal models of heart failure such as that described in Arnolda et al. (1999) Aust. N. Z. J. Med. 29(3):403-9.

[0494] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a GAP-5 modulating agent, an antisense GAP-5 nucleic acid molecule, a GAP-5-specific antibody, or a GAP-5-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0495] B. Detection Assays

[0496] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[0497] 1. Chromosome Mapping

[0498] Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the GAP-5 nucleotide sequences, described herein, can be used to map the location of the GAP-5 genes on a chromosome. The mapping of the GAP-5 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

[0499] Briefly, GAP-5 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GAP-5 nucleotide sequences. Computer analysis of the GAP-5 sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GAP-5 sequences will yield an amplified fragment.

[0500] Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

[0501] PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the GAP-5 nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a GAP-5 sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

[0502] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

[0503] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[0504] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[0505] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GAP-5 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[0506] 2. Tissue Typing

[0507] The GAP-5 sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0508] Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the GAP-5 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[0509] Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The GAP-5 nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:4 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 75-100 bases. If predicted coding sequences, such as those in SEQ ID NO:6 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0510] If a panel of reagents from GAP-5 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[0511] 3. Use of Partial GAP-5 Sequences in Forensic Biology

[0512] DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[0513] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:4 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the GAP-5 nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:4, having a length of at least 20 bases, preferably at least 30 bases.

[0514] The GAP-5 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such GAP-5 probes can be used to identify tissue by species and/or by organ type.

[0515] In a similar fashion, these reagents, e.g., GAP-5 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[0516] C. Predictive Medicine:

[0517] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining GAP-5 protein and/or nucleic acid expression as well as GAP-5 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted GAP-5 expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GAP-5 protein, nucleic acid expression or activity. For example, mutations in a GAP-5 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GAP-5 protein, nucleic acid expression or activity.

[0518] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GAP-5 in clinical trials.

[0519] These and other agents are described in further detail in the following sections.

[0520] 1. Diagnostic Assays

[0521] An exemplary method for detecting the presence or absence of GAP-5 protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting GAP-5 protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes GAP-5 protein such that the presence of GAP-5 protein or nucleic acid is detected in the biological sample. A preferred agent for detecting GAP-5 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GAP-5 mRNA or genomic DNA. The nucleic acid probe can be, for example, the GAP-5 nucleic acid set forth in SEQ ID NO:4 or 6, or the DNA insert of the plasmid deposited with ATCC as Accession Number PTA-2195, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GAP-5 mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[0522] A preferred agent for detecting GAP-5 protein is an antibody capable of binding to GAP-5 protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect GAP-5 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of GAP-5 mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of GAP-5 protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of GAP-5 genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of GAP-5 protein include introducing into a subject a labeled anti-GAP-5 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0523] In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.

[0524] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting GAP-5 protein, mRNA, or genomic DNA, such that the presence of GAP-5 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GAP-5 protein, mRNA or genomic DNA in the control sample with the presence of GAP-5 protein, mRNA or genomic DNA in the test sample.

[0525] The invention also encompasses kits for detecting the presence of GAP-5 in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting GAP-5 protein or mRNA in a biological sample; means for determining the amount of GAP-5 in the sample; and means for comparing the amount of GAP-5 in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GAP-5 protein or nucleic acid.

[0526] 2. Prognostic Assays

[0527] The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted GAP-5 expression or activity. As used herein, the term “aberrant” includes a GAP-5 expression or activity which deviates from the wild type GAP-5 expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant GAP-5 expression or activity is intended to include the cases in which a mutation in the GAP-5 gene causes the GAP-5 gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional GAP-5 protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a GAP-5 ligand, e.g., a GTPase, or one which interacts with a non-GAP-5 ligand, e.g. a non-GTPase molecule. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as aberrant hydrolysis of GTP or aberrant levels of GTP/GDP or aberrant GTPase-related signaling. For example, the term unwanted includes a GAP-5 expression or activity which is undesirable in a subject.

[0528] The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in GAP-5 protein activity or nucleic acid expression, such as disorders related to GTP/GDP levels, e.g., atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, type 1 neurofibromatosis, Wiskott-Aldrich syndrome, cystic fibrosis, microphthalmia with linear skin defects syndrome, and viral infection. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in GAP-5 protein activity or nucleic acid expression, such as GTP hydrolysis-related disorders, e.g., cardiovascular disorders, such as atherosclerosis, hypertension, and heart disease; disorders of the central nervous system, such as cystic fibrosis, type 1 neurofibromatosis, Alzheimer's disease; cell growth disorders such as cancers (e.g., carcinoma, sarcoma, or leukemia), tumor angiogenesis and metastasis, skeletal dysplasia, hepatic disorders, hematopoietic and/or myeloproliferative disorders; immune disorders such as Wiskott-Aldrich syndrome, viral infection, autoimmune disorders, immune deficiency disorders (e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency); skin disorders such as microphthalmia with linear skin defects syndrome; and congenital and/or developmental abnormalities such as facio-genital dysplasia. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted GAP-5 expression or activity in which a test sample is obtained from a subject and GAP-5 protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of GAP-5 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted GAP-5 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

[0529] Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted GAP-5 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a GTP hydrolysis-related disorder or a disorder related to GTP/GDP levels. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted GAP-5 expression or activity in which a test sample is obtained and GAP-5 protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of GAP-5 protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted GAP-5 expression or activity).

[0530] The methods of the invention can also be used to detect genetic alterations in a GAP-5 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in GAP-5 protein activity or nucleic acid expression, such as disorders related to GTP/GDP levels, e.g. atherosclerosis, hypertension, faciogenital dysplasia, oncogenesis and metastasis, heart disease, Alzheimer's disease, type 1 neurofibromatosis, Wiskott-Aldrich syndrome, cystic fibrosis, microphthalmia with linear skin defects syndrome, and viral infection. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a GAP-5-protein, or the mis-expression of the GAP-5 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a GAP-5 gene; 2) an addition of one or more nucleotides to a GAP-5 gene; 3) a substitution of one or more nucleotides of a GAP-5 gene, 4) a chromosomal rearrangement of a GAP-5 gene; 5) an alteration in the level of a messenger RNA transcript of a GAP-5 gene, 6) aberrant modification of a GAP-5 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a GAP-5 gene, 8) a non-wild type level of a GAP-5 protein, 9) allelic loss of a GAP-5 gene, and 10) inappropriate post-translational modification of a GAP-5 protein. As described herein,-there are a large number of assays known in the art which can be used for detecting alterations in a GAP-5 gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.

[0531] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in the GAP-5-gene (see Abravaya et al. (1995) Nucleic Acids Res.23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a GAP-5 gene under conditions such that hybridization and amplification of the GAP-5-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[0532] Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0533] In an alternative embodiment, mutations in a GAP-5 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0534] In other embodiments, genetic mutations in GAP-5 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in GAP-5 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0535] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the GAP-5 gene and detect mutations by comparing the sequence of the sample GAP-5 with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162;and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

[0536] Other methods for detecting mutations in the GAP-5 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type GAP-5 sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

[0537] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in GAP-5 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a GAP-5 sequence, e.g., a wild-type GAP-5 sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[0538] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in GAP-5 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control GAP-5 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electropboretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0539] In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[0540] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[0541] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0542] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a GAP-5 gene.

[0543] Furthermore, any cell type or tissue in which GAP-5 is expressed may be utilized in the prognostic assays described herein.

[0544] 3. Monitoring of Effects during Clinical Trials

[0545] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a GAP-5 protein (e.g., the modulation of GTPase activity, GTP hydrolysis, the modulation of GTPase-related signaling mechanisms, the regulation of GTP/GDP levels) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase GAP-5 gene expression, protein levels, or upregulate GAP-5 activity, can be monitored in clinical trials of subjects exhibiting decreased GAP-5 gene expression, protein levels, or downregulated GAP-5 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease GAP-5 gene expression, protein levels, or suppress GAP-5 activity, can be monitored in clinical trials of subjects exhibiting increased GAP-5 gene expression, protein levels, or upregulated GAP-5 activity. In such clinical trials, the expression or activity of a GAP-5 gene, and preferably, other genes that have been implicated in, for example, a GAP-5-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[0546] For example, and not by way of limitation, genes, including GAP-5, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates GAP-5 activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on GAP-5-associated disorders (e.g., GTP hydrolysis-related disorder, disorders related to GTP/GDP levels), for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of GAP-5 and other genes implicated in the GAP-5-associated disorder, respectively. The levels of gene expression (e.g., a gene expression pattern) can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of GAP-5 or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.

[0547] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a GAP-5 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the GAP-5 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the GAP-5 protein, mRNA, or genomic DNA in the pre-administration sample with the GAP-5 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of GAP-5 to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of GAP-5 to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, GAP-5 expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.

[0548] D. Methods of Treatment:

[0549] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted GAP-5 expression or activity, e.g., a GTP hydrolysis-related disorder. “Treatment”, as used herein, is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of disease or disorder or the predisposition toward a disease or disorder. A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the GAP-5 molecules of the present invention or GAP-5 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0550] 1. Prophylactic Methods

[0551] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted GAP-5 expression or activity, by administering to the subject a GAP-5 or an agent which modulates GAP-5 expression or at least one GAP-5 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted GAP-5 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the GAP-5 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of GAP-5 aberrancy, for example, a GAP-5, GAP-5 agonist or GAP-5 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[0552] 2. Therapeutic Methods

[0553] Another aspect of the invention pertains to methods of modulating GAP-5 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a GAP-5 or agent that modulates one or more of the activities of GAP-5 protein activity associated with the cell. An agent that modulates GAP-5 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a GAP-5 protein (e.g., a GAP-5 ligand or substrate), a GAP-5 antibody, a GAP-5 agonist or antagonist, a peptidomimetic of a GAP-5 agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more GAP-5 activities. Examples of such stimulatory agents include active GAP-5 protein and a nucleic acid molecule encoding GAP-5 that has been introduced into the cell. In another embodiment, the agent inhibits one or more GAP-5 activities. Examples of such inhibitory agents include antisense GAP-5 nucleic acid molecules, anti-GAP-5 antibodies, and GAP-5 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a GAP-5 protein or nucleic acid molecule such as a GTP hydrolysis-related disorder. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) GAP-5 expression or activity. In another embodiment, the method involves administering a GAP-5 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted GAP-5 expression or activity.

[0554] Stimulation of GAP-5 activity is desirable in situations in which GAP-5 is abnormally downregulated and/or in which increased GAP-5 activity is likely to have a beneficial effect. Likewise, inhibition of GAP-5 activity is desirable in situations in which GAP-5 is abnormally upregulated and/or in which decreased GAP-5 activity is likely to have a beneficial effect.

[0555] 3. Pharmacogenomics

[0556] The GAP-5 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on GAP-5 activity (e.g., GAP-5 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) GAP-5-associated disorders (e.g., GTP hydrolysis-related disorders; disorders related to GTP/GDP levels) associated with aberrant or unwanted GAP-5 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a GAP-5 molecule or GAP-5 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a GAP-5 molecule or GAP-5 modulator.

[0557] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0558] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[0559] Alternatively, a method termed the “candidate gene approach”, can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., a GAP-5 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[0560] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

[0561] Alternatively, a method termed the “gene expression profiling”, can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a GAP-5 molecule or GAP-5 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[0562] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a GAP-5 molecule or GAP-5 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0563] VI. Electronic Apparatus Readable Media and Arrays

[0564] Electronic apparatus readable media comprising GAP-5 sequence information is also provided. As used herein, “GAP-5 sequence information” refers to any nucleotide and/or amino acid sequence information particular to the GAP-5 molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like. Moreover, information “related to” said GAP-5 sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantitative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon GAP-5 sequence information of the present invention.

[0565] As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

[0566] As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the GAP-5 sequence information.

[0567] A variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium. For example, the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of data processor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the GAP-5 sequence information.

[0568] By providing GAP-5 sequence information in readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

[0569] The present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5-associated disease or disorder, wherein the method comprises the steps of determining GAP-5 sequence information associated with the subject and based on the GAP-5 sequence information, determining whether the subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[0570] The present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a GAP-5-associated disease or disorder or a pre-disposition to a disease associated with a GAP-5 wherein the method comprises the steps of determining GAP-5 sequence information associated with the subject, and based on the GAP-5 sequence information, determining whether the subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. The method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.

[0571] The present invention also provides in a network, a method for determining whether a subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5 associated disease or disorder associated with GAP-5, said method comprising the steps of receiving GAP-5 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to GAP-5 and/or a GAP-5-associated disease or disorder, and based on one or more of the phenotypic information, the GAP-5 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5-associated disease or disorder (e.g., a cardiovascular disorder, a CNS disorder, or a) cellular proliferation, growth, differentiation, or migration disorder.) The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0572] The present invention also provides a business method for determining whether a subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5-associated disease or disorder, said method comprising the steps of receiving information related to GAP-5 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to GAP-5 and/or related to a GAP-5-associated disease or disorder, and based on one or more of the phenotypic information, the GAP-5 information, and the acquired information, determining whether the subject has a GAP-5-associated disease or disorder or a pre-disposition to a GAP-5-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0573] The invention also includes an array comprising a GAP-5 sequence of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be GAP-5. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

[0574] In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0575] In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a GAP-5-associated disease or disorder, progression of GAP-5-associated disease or disorder, and processes, such a cellular transformation associated with the GAP-5-associated disease or disorder.

[0576] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of GAP-5 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0577] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including GAP-5) that could serve as a molecular target for diagnosis or therapeutic intervention.

[0578] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.

EXAMPLES

Example 1

[0579] Identification and Characterization of Human GAP-5 cDNA

[0580] In this example, the identification and characterization of the gene encoding human GAP-5 (clone Fbh32591FL) is described.

[0581] Isolation of the Human GAP-5 cDNA

[0582] The invention is based, at least in part, on the discovery of a human gene encoding a novel protein, referred to herein as GAP-5. The entire sequence of the human clone 32591 was determined and found to contain an open reading frame termed human “GAP-5.” The nucleotide sequence encoding the human GAP-5 protein is shown in FIGS. 5A-D and is set forth as SEQ ID NO:4. The protein encoded by this nucleic acid comprises about 1101 amino acids and has the amino acid sequence shown in FIGS. 5A-D and set forth as SEQ ID NO:5. The coding region (open reading frame) of SEQ ID NO:4 is set forth as SEQ ID NO:6. Clone Fbh32591FL, comprising the coding region of human GAP-5, was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209, on Jul. 7, 2000 and assigned Accession No. PTA-2195.

[0583] Analysis of the Human GAP-5 Molecule

[0584] A search for domain consensus sequences was performed using the amino acid sequence of GAP-5 and a database of HMMs (the Pfam database, release 2.1) using the default parameters (described above). The search revealed a RhoGAP domain (Pfam Accession Number PF00620) within SEQ ID NO:5 at residues 34-186 (see FIGS. 6A-B).

[0585] A search was performed using the amino acid sequence of GAP-5 and the ProDom database, which resulted in the identification a 41% identity between GAP-5 and ProDom entry “P85A(4) P85B(4) CHIN2//protein GTPase domain SH2 activation Zinc 3-kinase SH3 Phosphatidylinositol regulatory” over residues 33 to 185. The results of this search are shown in FIG. 7.

[0586] A search was also performed against the Prosite database, and resulted in the identification of several possible N-glycosylation sites at residues 189-192, 362-365, and 437-440. In addition, within the human GAP-5 protein two cAMP and cGMP dependant protein kinase phosphorylation sites were identified at residues 9-12 and 280-283. In addition, protein kinase C phosphorylation sites were identified within the human GAP-5 protein at residues 12-14, 72-74, 107-109, 283-285, 302-304, 317-319, 321-323, 349-351, 388-390, 401-403, 988-990, 1043-1045, and 1082-1084. This search also identified casein kinase II phosphorylation sites at residues 12-15, 29-32, 39-42, 209-212, 221-224, 240-243, 271-274, 298-301, 382-385, 402-405, 489-492, 511-514, 517-520, 542-545, 576-579, 611-614, 681-684, 709-712, 860-863, 883-886, 974-977, 1020-1023, and 1048-1051. A tyrosine phosphorylation site motif was also identified in the human GAP-5 protein at residues 800-807. The search also identified the presence of N-myristoylation site motifs at residues 48-53, 58-63, 217-222, 234-239, 380-385, 387-392, 400-405, 409-414, 525-530, 631-636, 677-682, 697-702, 722-727, 864-869, 878-883, 921-926, 981-986, 992-997, 1014-1019, 1024-1029, and 1056-1061. This search also revealed a single amidation site at residues 6-9.

[0587] An analysis of the possible cellular localization of the GAP-5 protein based on its amino acid sequence was performed using methods and algorithms similar to those described in Nakai and Kanehisa (1992) Genomics 14:897-911, and at http://psort.nibb.ac.jp. The results from this analysis predict that the GAP-5 protein is found in the nucleus, the cytoplasm, the mitochondria, and in cytoskeletal components.

[0588] Tissue Distribution of Human GAP-5 mRNA Using Taqman™ Analysis

[0589] This example describes the tissue distribution of human GAP-5 mRNA in a variety of cells and tissues, as determined using the TaqMan™ procedure. The Taqman™ procedure is a quantitative, reverse transcription PCR-based approach for detecting mRNA. The RT-PCR reaction exploits the 5′ nuclease activity of AmpliTaq Gold™ DNA Polymerase to cleave a TaqMan™ probe during PCR. Briefly, cDNA was generated from the samples of interest, e.g., various human normal and cancer samples, and used as the starting material for PCR amplification. In addition to the 5′ and 3′ gene-specific primers, a gene-specific oligonucleotide probe (complementary to the region being amplified) was included in the reaction (i.e., the Taqman™ probe). The TaqMan™ probe includes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5′ end of the probe (such as FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2′,7′-tetrachlorofluorescein), JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′ end of the probe.

[0590] During the PCR reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5′-3′ nucleolytic activity of the AmpliTaq™ Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3′ end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. RNA was prepared using the trizol method and treated with DNase to remove contaminating genomic DNA. cDNA was synthesized using standard techniques. Mock cDNA synthesis in the absence of reverse transcriptase resulted in samples with no detectable PCR amplification of the control gene confirms efficient removal of genomic DNA contamination.

[0591] Highest expression of GAP-5 mRNA was detected in the mammary gland, natural killer cells, bone marrow, fetal kidney, CHF heart, fetal thymus, fetal spleen, esophagus, erythroleukemia cells, CD3 treated T cells, CD3 IL-4/IL-10 treated T cells, CD3, TNFg/TNFa treated T cells, Burkitt's lymphoma B cells, placenta, small intestine, fetal liver, spleen, thymus, normal megakaryocytes, Th-2 induced T-cell, colon carcinoma tissue, d8 dendritic cells, IBD colon, lung squamous cell carcinoma tissue, and Thl cells. Lesser expression was also detected in the pituitary, congenital heart failure tissue, lung carcinoma tissue, embryonic keratinocytes, lung, HMC-1, CHT127, CHT1221, tissue obtained from a colon to liver metastasis, normal breast tissue, stomach, Th-1 induced T-cell, and cervical cancer tissue.

Example 2

[0592] Expression of Recombinant GAP-5 Protein in Bacterial Cells

[0593] In this example, GAP-5 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, GAP-5 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-GAP-5 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 3

[0594] Expression of Recombinant GAP-5 Protein in COS Cells

[0595] To express the GAP-5 gene in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used: This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire GAP-5 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[0596] To construct the plasmid, the GAP-5 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the GAP-5 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the GAP-5 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the GAP-5 gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB 101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[0597] COS cells are subsequently transfected with the GAP-5-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of the GAP-5 polypeptide is detected by radiolabeling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S -methionine (or 35S -cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both. the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[0598] Alternatively, DNA containing the GAP-5 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the GAP-5 polypeptide is detected by radiolabeling and immunoprecipitation using a GAP-5 specific monoclonal antibody.

[0599] III. 57809 and 57798, Novel Human Cadherin Molecules and Uses Therefor

BACKGROUND OF THE INVENTION

[0600] Cadherins form a superfamily of membrane glycoproteins which are involved in intercellular adhesion. The cadherin superfamily includes classical cadherins type 1 (e.g., E-cadherin) and type 2 (e.g., cadherin 11), desmosomal cadherins (e.g., desmogleins and desmocollins), and protocadherins (e.g., fat-like cadherins). Cadherins are important in forming cell junction adhesions, e.g., adherens junctions and desmosomes, and in the maintenance of cell-cell interactions. In addition to a role in cell adhesion, cadherins mediate signaling events that affect cell differentiation, proliferation, migration and survival.

[0601] Typically, cadherin molecules have three major regions, an extracellular domain that mediates specific adhesion, a transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain serves to link cadherins to the cytoskeleton via a cadherin-associated complex (CAC), and to aggregate the cadherin proteins at sites of cell-cell attachment (Nagafuchi et al. (1989) Cell Reg. 1:37-44). Cadherin mediated cell adhesions are supported by the formation of lateral, cooperative cadherin cis dimers which are stabilized by attachment to the cytoskeleton, as well as the trans interactions in which they engage, e.g., homophilic interactions with cadherin molecules on apposed cells (Steinberg, M. S. et al. (1999) Curr. Opin. Cell Biol. 11:554-560). Cadherin mediated cell adhesion can be transiently modulated by the Rho family of small GTPases (e.g., rho, rac, cdc42) which 35 regulate the actin cytoskeleton, as well as by tyrosine kinases and phosphatases (Tepass, U. (1999) Curr. Opin. Cell Biol. 11:540-548).

[0602] The cadherin cytoplasmic domain interacts with catenins (e.g., a, β and γ, p120ctn), proteins that connect cadherins to the cytoskeleton, as well as other integral membrane proteins and peripheral cytoplasmic proteins (Steinberg, M. S. supra; Provost, E. et al. (1999) Curr. Opin. Cell Biol. 11:567-572). The catenin proteins are regulated by phosphorylation and may be involved in the modulation of cell proliferation and differentiation, as well as cell division. For example, β-catenin has an established role in the wnt signal transduction pathway in which it participates in the regulation of gene expression as a cotranscriptional regulator of the LEF/TCF family of transcription factors. Thus, cadherins are involved in signal transduction between the cell surface and the nucleus, and influence gene expression. Genetic analysis has revealed that 13-catenin is involved in Xenopus and Drosophila embryonic development (e.g., in the establishment of dorsal-ventral and anterior-posterior axes), and acts as a protooncogene in may tumor types (Miller, J. R. et al. (1999) Oncogene 18:7860-7872; Tepass, U. supra).

[0603] Cell adhesion molecules are critical to the development of multi-cellular organisms. The spatio-temporal pattern of cadherin expression in developing tissues suggests an essential role in the establishment and maintenance of cell and tissue boundaries during differentiation, and in morphogenetic events such as adhesive contact formation, cell sorting, axonal patterning, neural plate induction, epithelial planar polarization, germ layer formation, organogenesis, and gastrulation (Tepass, U. supra). Alterations in cadherin expression or function correlates with morphoregulatory processes such as cell migration, cell differentiation and tissue rearrangement, as well as pathological states such as tumor formation and metastasis (Steinberg et al. supra; Behrens, J. (1999) Cancer Metastasis Rev. 18:15-30). Aberrant cadherin expression or function disrupts embryonic morphogenesis and may alter the characteristics of differentiated cells (Heasman et al. (1994) Development 120:49-57; Steinberg et al. supra; Behrens, J. supra).

SUMMARY OF THE INVENTION

[0604] The present invention is based, at least in part, on the discovery of novel members of the family of cadherin molecules, referred to herein as “CDHN” nucleic acid and protein molecules (e.g., CDHN-1 and CDHN-2). The CDHN nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cellular proliferation, growth, adhesion, differentiation, or migration. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding CDHN proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of CDHN-encoding nucleic acids.

[0605] In one embodiment, a CDHN nucleic acid molecule of the invention is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more indentical to the nucleotide sequence (e.g., to the entire length of the nucleotide sequence) shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a complement thereof.

[0606] In a preferred embodiment, the isolated nucleic acid molecule includes the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or a complement thereof. In another embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 1-111 of SEQ ID NO:7. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO:9 and nucleotides 2887-3181 of SEQ ID NO:7. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:7 or 9. In another embodiment, the nucleic acid molecule includes SEQ ID NO:12 and nucleotides 1-161 of SEQ ID NO:I0. In yet a further embodiment, the nucleic acid molecule includes SEQ ID NO:12 and nucleotides 2655-2938 of SEQ ID NO:10. In another preferred embodiment, the nucleic acid molecule consists of the nucleotide sequence shown in SEQ ID NO:10 or 12.

[0607] In another embodiment, a CDHN nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:8 or 11, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In a preferred embodiment, a CDHN nucleic acid molecule includes a nucleotide sequence encoding a protein having an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the amino acid sequence of SEQ ID NO:8 or 11, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0608] In another preferred embodiment, an isolated nucleic acid molecule encodes the amino acid sequence of human CDHN-1 or human CDHN-2. In yet another preferred embodiment, the nucleic acid molecule includes a nucleotide sequence encoding a protein having the amino acid sequence of SEQ ID NO:8 or 11, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In yet another preferred embodiment, the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000,2100,2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 2938, 3000, 3100, 3181 or more nucleotides in length. In a further preferred embodiment, the nucleic acid molecule is at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 2938, 3000, 3100, 3181 or more nucleotides in length and encodes a protein having a CDHN activity (as described herein).

[0609] Another embodiment of the invention features nucleic acid molecules, preferably CDHN nucleic acid molecules, which specifically detect CDHN nucleic acid molecules relative to nucleic acid molecules encoding non-CDHN proteins. For example, in one embodiment, such a nucleic acid molecule is at least 20, 30, 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000 or more nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a complement thereof.

[0610] In preferred embodiments, the nucleic acid molecules are at least 15 nucleotides (e.g., 15 contiguous nucleotides) in length and hybridize under stringent conditions to the nucleotide molecules set forth in SEQ ID NO:7, 9, 10, or 12.

[0611] In other preferred embodiments, the nucleic acid molecule encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:8 or 11, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______, wherein the nucleic acid molecule hybridizes to a nucleic acid molecule comprising SEQ ID NO:7 or 9, or SEQ ID NO:10 or 12, respectively, under stringent conditions.

[0612] Another embodiment of the invention provides an isolated nucleic acid molecule which is antisense to a CDHN nucleic acid molecule, e.g., the coding strand of a CDHN nucleic acid molecule.

[0613] Another aspect of the invention provides a vector comprising a CDHN nucleic acid molecule. In certain embodiments, the vector is a recombinant expression vector. In another embodiment, the invention provides a host cell containing a vector of the invention. In yet another embodiment, the invention provides a host cell containing a nucleic acid molecule of the invention. The invention also provides a method for producing a protein, preferably a CDHN protein, by culturing in a suitable medium, a host cell, e.g., a mammalian host cell such as a non-human mammalian cell, of the invention containing a recombinant expression vector, such that the protein is produced.

[0614] Another aspect of this invention features isolated or recombinant CDHN proteins and polypeptides. In one embodiment, an isolated CDHN protein includes at least one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide. In a preferred embodiment, an isolated CDHN protein includes at least one, preferably two, three, four, five or more, cadherin domains. In another preferred embodiment, an isolated CDHN protein includes at least one, preferably two, three, four, five or more, cadherin domains, and at least one or more of the following domains: a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide. In a further preferred embodiment, an isolated CDHN protein includes at least one, preferably two, three, four, five, or six CA domains. In another preferred embodiment, an isolated CDHN protein includes at least one, preferably two, three, four, five, or six CA domains, and at least one or more of the following domains: a cadherin domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide.

[0615] In a preferred embodiment, a CDHN protein includes at least one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO:8 or 11, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In another preferred embodiment, a CDHN protein includes at least one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide, and has a CDHN activity (as described herein).

[0616] In yet another preferred embodiment, a CDHN protein includes at least one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12.

[0617] In another embodiment, the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:8 or 11, wherein the fragment comprises at least 15 amino acids (e.g., contiguous amino acids) of the amino acid sequence of SEQ ID NO:8 or 11, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number ______. In another embodiment, a CDHN protein has the amino acid sequence of SEQ ID NO:8 or 11.

[0618] In another embodiment, the invention features a CDHN protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:7, 9, 10, or 12, or a complement thereof. This invention further features a CDHN protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12, or a complement thereof.

[0619] The proteins of the present invention or portions thereof, e.g., biologically active portions thereof, can be operatively linked to a non-CDHN polypeptide (e.g., heterologous amino acid sequences) to form fusion proteins. The invention further features antibodies, such as monoclonal or polyclonal antibodies, that specifically bind proteins of the invention, preferably CDHN proteins. In addition, the CDHN proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.

[0620] In another aspect, the present invention provides a method for detecting the presence of a CDHN nucleic acid molecule, protein, or polypeptide in a biological sample by contacting the biological sample with an agent capable of detecting a CDHN nucleic acid molecule, protein, or polypeptide such that the presence of a CDHN nucleic acid molecule, protein or polypeptide is detected in the biological sample.

[0621] In another aspect, the present invention provides a method for detecting the presence of CDHN activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of CDHN activity such that the presence of CDHN activity is detected in the biological sample.

[0622] In another aspect, the invention provides a method for modulating CDHN activity comprising contacting a cell capable of expressing CDHN with an agent that modulates CDHN activity such that CDHN activity in the cell is modulated. In one embodiment, the agent inhibits CDHN activity. In another embodiment, the agent stimulates CDHN activity. In one embodiment, the agent is an antibody that specifically binds to a CDHN protein. In another embodiment, the agent modulates expression of a CDHN by modulating transcription of a CDHN gene or translation of a CDHN mRNA. In yet another embodiment, the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a CDHN mRNA or a CDHN gene.

[0623] In one embodiment, the methods of the present invention are used to treat a subject having a disorder characterized by aberrant or unwanted CDHN protein or nucleic acid expression or activity by administering an agent which is a CDHN modulator to the subject. In one embodiment, the CDHN modulator is a CDHN protein. In another embodiment the CDHN modulator is a CDHN nucleic acid molecule. In yet another embodiment, the CDHN modulator is a peptide, peptidomimetic, or other small molecule. In a preferred embodiment, the disorder characterized by aberrant or unwanted CDHN protein or nucleic acid expression is a cadherin-associated disorder, e.g., a central nervous system (CNS) disorder, a cardiovascular disorder, a musculoskeletal disorder, a gastrointestinal disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, adhesion, or migration disorder.

[0624] The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a CDHN protein; (ii) mis-regulation of the gene; and (iii) aberrant post-translational modification of a CDHN protein, wherein a wild-type form of the gene encodes a protein with a CDHN activity.

[0625] In another aspect the invention provides methods for identifying a compound that binds to or modulates the activity of a CDHN protein, by providing an indicator composition comprising a CDHN protein having CDHN activity, contacting the indicator composition with a test compound, and determining the effect of the test compound on CDHN activity in the indicator composition to identify a compound that modulates the activity of a CDHN protein.

[0626] Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0627] The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as “cadherin” or “CDHN” nucleic acid and protein molecules, which are novel members of a family of cell adhesion molecules. These novel molecules are capable of mediating cell-cell and/or cell-substrate interactions. Thus, these novel CDHN molecules may play a role in or function in a variety of cellular processes, e.g., growth, proliferation, differentiation, adhesion, migration, signal transduction, cytoskeletal organization, transcriptional regulation, and inter- or intra-cellular communication.

[0628] As used herein, the term “cadherin” includes a molecule which is involved in cell-cell and/or cell-matrix adhesion. A variety of tissue-specific forms of cadherins have been identified including epithelial (E-cadherin), neural (N-cadherin), placental (P-cadherin), retinal (R-cadherin), vascular endothelial (VE-cadherin), kidney (K-cadherin), osteoblast (OB-cadherin), brain (BR-cadherin), muscle (M-cadherin) and liver-intestine (LI-cadherin), and cadherin subtype expression is correlated with the terminal differentiation of multiple cell types. Cadherin molecules have been shown to be involved in a variety of cellular adhesive events including cell sorting and patterning, multicellular organization, morphogenetic events during embryonic development, organogenesis, tissue remodeling, angiogenesis, tumorigenesis or metastasis. As cadherins, the CDHN molecules of the present invention provide novel diagnostic targets and therapeutic agents to control cadherin-associated disorders.

[0629] As used herein, a “cadherin-associated disorder” or a “CDHN associated disorder” includes a disorder, disease or condition which is caused or characterized by a misregulation (e.g., downregulation or upregulation) of a CDHN-mediated activity. Cadherin-associated disorders can detrimentally affect cellular functions such as cellular proliferation, growth, differentiation, adhesion, migration, or inter- or intra-cellular communication; tissue development, integrity and function, such as cardiac function, neuronal function, or musculoskeletal function. Examples of cadherin-associated disorders include central nervous system (CNS) disorders such as cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, myasthenia gravis, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldt disease; neurological developmental disorders such as neural tube defects, arrhinencephaly, spina bifida, adrenoleukodystrophy, Walker-Warburg syndrome, Miller-Dieker syndrome, Meckel-Gruber syndrome, meningomyelocele, Arnold-Chirai malformation, anencephaly, heterotopias, agyria, polymicrogyria, hydrocephalus, Zellweger syndrome, lissencephaly, cerebral palsy; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, autism, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Further CNS-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[0630] Further examples of cadherin-associated disorders include cardiac-related disorders. Cardiovascular system disorders in which the CDHN molecules of the invention may be directly or indirectly involved include arteriosclerosis, atherosclerosis, angiogenesis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, coronary microembolism, coronary artery ligation, vascular heart disease, atrial fibrillation, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, cardiomyopathy, myocardial infarction, coronary artery disease, and arrhythmia; and cardiovascular developmental disorders (e.g., arteriovenous malformations, arteriovenous fistulae, Raynaud's syndrome, neurogenic thoracic outlet syndrome, causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrial septal defects, atrioventricular canal, coarctation of the aorta, ebsteins anomaly, hypoplastic left heart syndrome, interruption of the aortic arch, mitral valve prolapse, ductus arteriosus, patent foramen ovale, partial anomalous pulmonary venous return, pulmonary atresia with ventricular septal defect, pulmonary atresia without ventricular septal defect, persistence of the fetal circulation, pulmonary valve stenosis, single ventricle, total anomalous pulmonary venous return, transposition of the great vessels, tricuspid atresia, truncus arteriosus, ventricular septal defects). CDHN mediated or related disorders also include disorders of the musculoskeletal system such as paralysis and muscle weakness, e.g., ataxia, myotonia, spinal muscle atrophy, myopathy, and myokymia; and musculoskeletal developmental disorders (e.g., cleft palate, midline skull defects, muscular dystrophies, Klippel-Feil syndrome).

[0631] CDHN disorders also include cellular proliferation, growth, differentiation, adhesion, or migration disorders. Cellular proliferation, growth, differentiation, adhesion, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, adhesion, or migration processes. As used herein, a “cellular proliferation, growth, differentiation, adhesion, or migration process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. The CDHN molecules of the present invention are involved in adhesive and signaling mechanisms which are known to be involved in cellular proliferation, growth, differentiation, adhesion, and migration processes. Thus, the CDHN molecules may modulate cellular proliferation, growth, differentiation, adhesion, or migration, and may play a role in disorders characterized by aberrantly regulated growth, differentiation, adhesion, or migration. Such disorders include cancer, e.g., carcinoma, sarcoma, lymphoma or leukemia, examples of which include, but are not limited to, breast, endometrial, ovarian, uterine, hepatic, gastrointestinal, prostate, colorectal, and lung cancer, melanoma, neurofibromatosis, adenomatous polyposis of the colon, Wilms' tumor, nephroblastoma, teratoma, rhabdomyosarcoma; tumor invasion, angiogenesis and metastasis; skeletal dysplasia; hematopoietic and/or myeloproliferative disorders.

[0632] CDHN-associated or related disorders also include inflammatory or immune system disorders, examples of which include, but are not limited to inflammatory bowel disease, ulcerative colitis, Crohn's disease, leukocyte adhesion deficiency II syndrome, peritonitis, chronic obstructive pulmonary disease, lung inflammation, asthma, nephritis, amyloidosis, rheumatoid arthritis, chronic bronchitis, sarcoidosis, scleroderma, lupus, polymyositis, Reiter's syndrome, psoriasis, pelvic inflammatory disease, inflammatory breast disease, orbital inflammatory disease, immune deficiency disorders (e.g., common variable immunodeficiency, congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, selective IgA deficiency, chronic mucocutaneous candidiasis, severe combined immunodeficiency), wound healing, and autoimmune disorders (e.g., pemphigus vulgaris, paraneoplastic pemphigus).

[0633] A CDHN associated disorder also includes a hematopoietic or thrombotic disorder, for example, disseminated intravascular coagulation, thromboembolic vascular disease, anemia, lymphoma, leukemia, neutrophilia, neutropenia, myeloproliferative disorders, thrombocytosis, thrombocytopenia, von Willebrand disease, thalassemia, and hemophilia.

[0634] In addition, CDHN associated disorders include gastrointestinal and digestive disorders including, but not limited to, esophageal disorders such as atresia and fistulas, stenosis, achalasia, esophageal rings and webs, hiatal hernia, lacerations, esophagitis, diverticulae, systemic sclerosis (scleroderma), varices, Barrett's esophagus, Mallory Weiss syndrome, esophageal tumors such as squamous cell carcinomas and adenocarcinomas, stomach disorders such as diaphragmatic hernias, pyloric stenosis, dyspepsia, gastritis, acute gastric erosion and ulceration, peptic ulcers, stomach tumors such as carcinomas and sarcomas, small intestine disorders such as congenital atresia and stenosis, diverticula, Meckel's diverticulum, Hirschsprung disease, pancreatic rests, insulin dependent diabetes mellitus, ischemic bowel disease, infective enterocolitis, Crohn's disease, tumors of the small intestine such as carcinomas and sarcomas, disorders of the colon such as malabsorption, obstructive lesions such as hernias, megacolon, diverticular disease, melanosis coli, ischemic injury, celiac disease, hemorrhoids, angiodysplasia of right colon, inflammations of the colon such as ulcerative colitis, tumors of the colon such as polyps and sarcomas, and abdominal wall defects; as well as hepatic disorders (e.g., cholestasis, cirrhosis, and hyperbilirubinemia) and renal disorders (e.g., renal failure, renal neoplasms, renal osteodystrophy, renal dysplasia, polycystic disease, and glomerulonephritis).

[0635] CDHN-associated or related disorders also include disorders affecting tissues in which CDHN (e.g., CDHN-1 or CDHN-2) protein is expressed. In one embodiment, a CDHN associated disorder is a disorder associated with aberrant cell patterning, differentiation and/or development in a tissue (e.g., an embryonic tissue) in which CDHN is expressed.

[0636] As used herein, a “cadherin-mediated activity” or a “CDHN-mediated activity” includes an activity which involves cadherin mediated adhesion or signal transduction. Cadherin-mediated activities include cell-cell and cell-matrix interactions, cell adhesion and migration, inter- and intra-cellular signaling.

[0637] The term “family” when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin, e.g., monkey proteins. Members of a family may also have common functional characteristics.

[0638] A CDHN protein of the present invention includes a protein which comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain . In one embodiment, an extracellular domain of a CDHN protein may comprise at least one or more of the following domains: a cadherin domain, a CA domain, and/or a cadherins extracellular repeated domain signature pattern.

[0639] For example, the family of CDHN proteins comprises at least one “cadherin domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “cadherin domain” includes a protein domain having an amino acid sequence of about 50-200 amino acid residues, preferably about 60-170 amino acid residues, more preferably about 70-140 amino acid residues, and more preferably about 80-110 amino acid residues, having a bit score for the alignment of the sequence to the cadherin domain (HMM) of at least about 14, more preferably 25, 27, 33, 40, 42, 49, 64, 75, 79 or greater. Cadherin domains are described in, for example, in Takeichi, M. (1990) Ann. Rev. Biochem., 59:237-252; Takeichi, M. (1987) Trends Genet., 3:213-217; and Mahoney et al. (1991) Cell, 67:853-868, the contents of which are incorporated herein by reference.

[0640] To identify the presence of a cadherin domain in a CDHN protein, and to make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of known protein domains (e.g., the HMM database). The cadherin domain (HMM) has been assigned the PFAM Accession PF00028 (http://genome.wustl.edu/Pfam/html). A search was performed against the HMM database resulting in the identification of cadherin domains in the amino acid sequence of human CDHN-1 at about residues 187-284, 298-390, 513-603, 617-706 and 724-817 of SEQ ID NO:11. The results of the search are set forth in FIGS. 12A-B. Cadherin domains were also identified in the amino acid sequence of human CDHN-2 at about residues 27-119, 133-234,244-329,343-442, 457-558 and 571-659 of SEQ ID NO:11. The results of the search are set forth in FIGS. 18A-B.

[0641] In one embodiment, a cadherin domain includes at least about 50-200 amino acid residues and has at least about 50-60% homology with a cadherin domain of human CDHN (e.g., residues 187-284, 298-390, 513-603, 617-706 and 724-817 of SEQ ID NO:8, or residues 27-119, 133-234, 244-329, 343-442, 457-558 and 571-659 of SEQ ID NO:11). Preferably, a cadherin domain includes at least about 70-140 amino acid residues, or about 80-110 amino acid residues, and has at least 60-70% homology, preferably about 70-80%, or about 80-90% homology with a cadherin domain of human CDHN (e.g., residues 187-284, 298-390, 513-603, 617-706 and 724-817 of SEQ ID NO:8, or residues 27-119, 133-234, 244-329, 343-442, 457-558 and 571-659 of SEQ ID NO:11).

[0642] Accordingly, CDHN proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a cadherin domain of human CDHN are within the scope of the invention.

[0643] In another embodiment, a CDHN protein of the present invention is identified based on the presence of at least one “CA domain” or “cadherin repeat domain” in the protein or corresponding nucleic acid molecule. As used herein, the term “CA domain” or “cadherin repeat domain” includes a protein domain having an amino acid sequence of about 40-130 amino acid residues, preferably about 50-120 amino acid residues, more preferably about 60-110 amino acid residues, and more preferably about 70-100 amino acid residues, having a bit score for the alignment of the sequence to the CA domain (HMM) of at least about 2, more preferably 6, 10, 23, 35, 45, 57, 58, 66, 67, 75, 85, 99, 103 or greater. Cadherin repeat domains are described in, for example, in Yap, A S. et al. (1997) Ann. Rev. Cell. Dev. Biol., 1:119-146; Overduin, M. et al. (1995) Science 267: 386-389; Shapiro, L. et al. (1995) Nature 374: 327-337; Shapiro, L. et al. (1995) Proc. Natl. Acad. Sci. USA 92: 6793-6797; and Takeichi, M. (1988) Development 102: 639-655, the contents of which are incorporated herein by reference.

[0644] To identify the presence of a CA domain in a CDHN protein, and to make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of known protein domains (e.g., the HMM database). The CA domain (HMM) has been assigned the Prosite Profile PS50268 (http://smart.embl-heidelberg.de). A search was performed against the HMM database resulting in the identification of CA domains in the amino acid sequence of human CDHN-1 at about residues 205-291, 215-397, 427-506, 530-610, 634-713 and 740-824 of SEQ ID NO:8. The results of the search are set forth in FIGS. 13A-B. CA domains were also identified in the amino acid sequence of human CDHN-2 at about residues 47-126, 150-243, 260-336, 360-449, 474-563 and 585-663 of SEQ ID NO:11. The results of the search are set forth in FIGS. 19A-B.

[0645] In one embodiment, a CA domain includes at least about 40-130 amino acid residues and has at least about 50-60% homology with a CA domain of human CDHN (e.g., residues 205-291, 215-397, 427-506, 530-610, 634-713 and 740-824 of SEQ ID NO:8, or residues 47-126, 150-243, 260-336, 360-449, 474-563 and 585-663 of SEQ ID NO:11). Preferably, a CA domain includes at least about 60-110 amino acid residues, or about 70-100 amino acid residues, and has at least 60-70% homology, preferably about 70-80%, or about 80-90% homology with a CA domain of human CDHN (e.g., residues 205-291, 215-397, 427-506, 530-610, 634-713 and 740-824 of SEQ ID NO:8, or residues 47-126, 150-243, 260-336, 360-449, 474-563 and 585-663 of SEQ ID NO:11).

[0646] Accordingly, CDHN proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a CA domain of human CDHN are within the scope of the invention.

[0647] In one embodiment, a CDHN protein comprises the following cadherins extracellular repeated domain signature pattern:

[LIV]-X-[LIV]-X-D-X-N-D-[NH]-X-P (SEQ ID NO:13)

[0648] The signature patterns or consensus patterns described herein are described according to the following designation: all amino acids are indicated according to their universal single letter designation; “X” designates any amino acid; X(n) designates n number of amino acids, e.g., X (2) designates any two amino acids, e.g., X (1-3) designates any of one to three amino acids; and, amino acids in brackets indicates any one of the amino acids within the brackets, e.g., [LIV] indicates any of one of either L (leucine), I (isoleucine), or V (valine). Cadherins extracellular repeated domain signatures comprise asparagine residues, as well as conserved aspartic acid residues. In one embodiment the residues within the cadherins extracellular repeated domain signature pattern of SEQ ID NO:13 may be important for the binding of calcium.

[0649] To identify the presence of a cadherins extracellular repeated domain signature pattern in a CDHN protein, and to make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein is searched against a database of known protein domains. The cadherins extracellular repeated domain signature pattern has been assigned the Prosite Accession Number PS00232 (www.expasy.ch/prosite). CDHN-1 has such a signature pattern at about amino acid residues 170-180, 281-291, 496-506, 600-610 and 703-713 of SEQ ID NO:8. CDHN-2 has such a signature pattern at about amino acid residues 326-336 of SEQ ID NO:11.

[0650] In another embodiment, a CDHN protein of the present invention is identified based on the presence of at least one “transmembrane domain”. As used herein, the term “transmembrane domain” includes an amino acid sequence of about 15 amino acid residues in length which spans the plasma membrane. More preferably, a transmembrane domain includes about at least 20, 25, 30, 35, 40, or 45 amino acid residues and spans the plasma membrane. Transmembrane domains are rich in hydrophobic residues, and typically have an alpha-helical structure. In a preferred embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembrane domain are hydrophobic, e.g., leucines, isoleucines, tyrosines, or tryptophans. Transmembrane domains are described in, for example, Zagotta W. N. et al., (1996) Annual Rev. Neurosci. 19: 235-263, the contents of which are incorporated herein by reference. Amino acid residues 19-35, 42-59, 298-315, 369-393 and 863-886 of the native CDHN-1 protein, and amino acid residues 8-26, 265-282, 336-360, 830-853 of the putative mature CDHN-1 protein are predicted to comprise a transmembrane domain (see FIG. 11). In addition, amino acid residues 540-557, 571-588 and 789-813 of the native CDHN-2 protein, and amino acid residues 519-536, 550-567 and 768-792 of the putative mature CDHN-2 protein are predicted to comprise a transmembrane domain (see FIG. 17). Accordingly, CDHN proteins having at least 50-60% homology, preferably about 60-70%, more preferably about 70-80%, or about 80-90% homology with a transmembrane domain of human CDHN are within the scope of the invention.

[0651] In another embodiment of the invention, a CDHN protein of the present invention is identified based on the presence of a signal peptide. The prediction of such a signal peptide can be made, for example, utilizing the computer algorithm SignalP (Henrik, et al. (1997) Protein Engineering 10:1-6). As used herein, a “signal sequence” or “signal peptide” includes a peptide containing about 15 or more amino acids which occurs at the N-terminus of secretory and membrane bound proteins and which contains a large number of hydrophobic amino acid residues. For example, a signal sequence contains at least about 10-30 amino acid residues, preferably about 15-25 amino acid residues, more preferably about 18-20 amino acid residues, and more preferably about 19 amino acid residues, and has at least about 35-65%, preferably about 38-50%, and more preferably about 40-45% hydrophobic amino acid residues (e.g., Valine, Leucine, Isoleucine or Phenylalanine). Such a “signal sequence”, also referred to in the art as a “signal peptide”, serves to direct a protein containing such a sequence to a lipid bilayer, and is cleaved in secreted and membrane bound proteins. A signal sequence was identified in the amino acid sequence of human CDHN-1 at about amino acids 1-33 of SEQ ID NO:8. A signal sequence was also identified in the amino acid sequence of human CDHN-2 at about amino acids 1-21 of SEQ ID NO:11. Accordingly, the present invention provides a mature CDHN protein lacking the signal peptide, e.g. amino acid residues 34-924 of SEQ ID NO:8 (CDHN-1) or amino acid residues 22-830 of SEQ ID NO:11 (CDHN-2).

[0652] In a preferred embodiment, the CDHN molecules of the invention include at least one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide.

[0653] Isolated proteins of the present invention, preferably CDHN proteins, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:11 or 11, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:7, 9, 10, or 12. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains have at least 30%, 40%, or 50% homology, preferably 60% homology, more preferably 70%-80%, and even more preferably 90-95% homology across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 30%, 40%, or 50%, preferably 60%, more preferably 70-80%, or 90-95% homology and share a common functional activity are defined herein as sufficiently identical.

[0654] As used interchangeably herein, a “CDHN activity”, “biological activity of CDHN” or “CDHN-mediated activity”, includes an activity exerted by a CDHN protein, polypeptide or nucleic acid molecule on a CDHN responsive cell or tissue, or on a CDHN protein substrate, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a CDHN activity is a direct activity, such as an association with a CDHN target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which a CDHN protein binds or interacts in nature, such that CDHN mediated function is achieved. A CDHN target molecule can be a non-CDHN molecule or a CDHN protein or polypeptide of the present invention. In one exemplary embodiment, a CDHN target molecule is a CDHN protein. In another exemplary embodiment, a CDHN target molecule is a CDHN substrate (e.g., a cytoplasmic protein, e.g., a protein containing at least one armadillo repeat). Alternatively, a CDHN activity is an indirect activity, such as a cellular signaling or adhesion activity mediated by interaction of the CDHN protein with a CDHN ligand or substrate. The biological activities of CDHN are described herein. For example, the CDHN proteins of the present invention can have one or more of the following activities: 1) modulation of cell adhesion, e.g., cell-cell and cell-substrate adhesion; 2) modulation of cell growth, proliferation, and/or differentiation; 3) modulation of cell motility, e.g., cell migration and cell invasion; 4) modulation of cytoskeletal organization; 5) modulation and maintenance of multicellular organization, e.g., cell sorting, cell polarization, tissue morphogenesis, tissue integrity; 6) modulation of intra- and/or inter-cellular signaling; and 7) modulation of transcriptional regulation of gene expression.

[0655] Accordingly, another embodiment of the invention features isolated CDHN proteins and polypeptides having a CDHN activity. Other preferred proteins are CDHN proteins having one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide and, preferably, a CDHN activity.

[0656] Additional preferred proteins have at least one or more of the following domains: a cadherin domain, a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12.

[0657] The nucleotide sequence of the isolated human CDHN-1 cDNA and the predicted amino acid sequence of the human CDHN-1 polypeptide are shown in FIGS. 9A-C and in SEQ ID NOs:7 and 8, respectively. The nucleotide sequence of the isolated human CDHN-2 cDNA and the predicted amino acid sequence of the human CDHN-2 polypeptide are shown in FIGS. 15A-C and in SEQ ID NOs:10 and 11, respectively. Plasmids containing the nucleotide sequence encoding human CDHN-1 and CDHN-2 were deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Numbers ______. These deposits will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. These deposits were made merely as a convenience for those of skill in the art and are not an admission that deposits are required under 35 U.S.C. §112.

[0658] The human CDHN-1 gene, which is approximately 3181 nucleotides in length, encodes a protein having a molecular weight of approximately 102 kD and which is approximately 924 amino acid residues in length.

[0659] The human CDHN-2 gene, which is approximately 2938 nucleotides in length, encodes a protein having a molecular weight of approximately 91 kD and which is approximately 830 amino acid residues in length.

[0660] Various aspects of the invention are described in further detail in the following subsections:

[0661] I. Isolated Nucleic Acid Molecules

[0662] One aspect of the invention pertains to isolated nucleic acid molecules that encode CDHN proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify CDHN-encoding nucleic acid molecules (e.g., CDHN mRNA) and fragments for use as PCR primers for the amplification or mutation of CDHN nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0663] The term “isolated nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated CDHN nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0664] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ as a hybridization probe, CDHN nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0665] Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0666] A nucleic acid of the invention can be amplified using cDNA, mRNA or, alternatively, genomic DNA as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to CDHN nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0667] In a preferred embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12. This cDNA may comprise sequences encoding the human CDHN-1 protein (i.e., “the coding region”, from nucleotides 112-2886), as well as 5′ untranslated sequences (nucleotides 1-111) and 3′ untranslated sequences (nucleotides 2887-3181) of SEQ ID NO:7. Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:7 (e.g., nucleotides 112-2886, corresponding to SEQ ID NO:9). This cDNA may comprise sequences encoding the human CDHN-2 protein (i.e., “the coding region”, from nucleotides 162-2654), as well as 5′ untranslated sequences (nucleotides 1-161) and 3′ untranslated sequences (nucleotides 2655-2938) of SEQ ID NO:10. Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:10 (e.g., nucleotides 162-2654, corresponding to SEQ ID NO:12). In another embodiment, an isolated nucleic acid molecule of the invention consists of the nucleic acid sequence of SEQ ID NO:7, 9, 10, or 12.

[0668] In another preferred embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, respectively, thereby forming a stable duplex.

[0669] In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the entire length of the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the entire length of the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion of any of these nucleotide sequences.

[0670] Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a CDHN protein, e.g., a biologically active portion of a CDHN protein. The nucleotide sequences determined from the cloning of the CDHN-1 and CDHN-2 genes allow for the generation of probes and primers designed for use in identifying and/or cloning other CDHN family members, as well as CDHN homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ of an anti-sense sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or of a naturally occurring allelic variant or mutant of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In one embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is greater than 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600,1600-1800, 1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, 2800-3000, 3000 or more nucleotides in length and hybridizes under stringent hybridization conditions to a nucleic acid molecule of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0671] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4×sodium chloride/sodium citrate (SSC), at about 65-70° C. (or alternatively hybridization in 4×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in I×SSC, at about 65-70° C. (or alternatively hybridization in 1×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 0.3×SSC, at about 65-70° C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide at about 40-45° C.) followed by one or more washes in 2×SSC, at about 50-60° C. Ranges intermediate to the above-recited values, e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassed by the present invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH2PO4, 7% SDS at about 65° C., followed by one or more washes at 0.02M NaH2PO4, 1% SDS at 65° C. (see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995), or alternatively 0.2×SSC, 1% SDS.

[0672] Probes based on the CDHN nucleotide sequences can be used to detect transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a CDHN protein, such as by measuring a level of a CDHN-encoding nucleic acid in a sample of cells from a subject e.g., detecting CDHN mRNA levels or determining whether a genomic CDHN gene has been mutated or deleted.

[0673] A nucleic acid fragment encoding a “biologically active portion of a CDHN protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ which encodes a polypeptide having a CDHN biological activity (the biological activities of the CDHN proteins are described herein), expressing the encoded portion of the CDHN protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the CDHN protein.

[0674] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ due to degeneracy of the genetic code and thus encode the same CDHN proteins-as those encoded by the nucleotide sequence shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:8 or 11.

[0675] In addition to the CDHN nucleotide sequences shown in SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of the CDHN proteins may exist within a population (e.g., the human population). Such genetic polymorphism in the CDHN genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a CDHN protein, preferably a mammalian CDHN protein, and can further include non-coding regulatory sequences, and introns.

[0676] Allelic variants of human CDHN proteins include both functional and non-functional CDHN proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the human CDHN protein that maintain the ability to bind a CDHN ligand or substrate and/or modulate cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:8 or 11, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.

[0677] Non-functional allelic variants are naturally occurring amino acid sequence variants of the human CDHN protein that do not have the ability to either bind a CDHN ligand or substrate and/or modulate any of the CDHN activities described herein. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:8 or 11, or a substitution, insertion or deletion in critical residues or critical regions of the protein.

[0678] The present invention further provides non-human orthologues of the human CDHN-1 and CDHN-2 proteins. Orthologues of the human CDHN protein are proteins that are isolated from non-human organisms and possess the same CDHN ligand or substrate binding and/or modulation of cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms. Orthologues of the human CDHN protein can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:8 or 11.

[0679] Moreover, nucleic acid molecules encoding other CDHN family members and, thus, which have a nucleotide sequence which differs from the CDHN sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ are intended to be within the scope of the invention. For example, another CDHN cDNA can be identified based on the nucleotide sequence of human CDHN. Moreover, nucleic acid molecules encoding CDHN proteins from different species, and which, thus, have a nucleotide sequence which differs from the CDHN sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ are intended to be within the scope of the invention. For example, a mouse CDHN cDNA can be identified based on the nucleotide sequence of a human CDHN.

[0680] Nucleic acid molecules corresponding to natural allelic variants and homologues of the CDHN cDNAs of the invention can be isolated based on their homology to the CDHN nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the CDHN cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the CDHN gene. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In other embodiment, the nucleic acid is at least 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950,950-1000, 1000-1100, 1100-1200, 1200-1300, 1300-1400, 1400-1500, 1500-1600, 1600-1800, 1800-2000, 2000-2200, 2200-2400, 2400-2600, 2600-2800, 2800-3000, 3000 or more nucleotides in length. As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% identical to each other typically remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50° C., preferably at 55° C., more preferably at 60° C., and even more preferably at 65° C. Ranges intermediate to the above-recited values, e.g., at 60-65° C. or at 55-60° C. are also intended to be encompassed by the present invention. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:7, 9, 10, or 12 and corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0681] In addition to naturally-occurring allelic variants of the CDHN sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, thereby leading to changes in the amino acid sequence of the encoded CDHN protein, without altering the functional ability of the CDHN protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of CDHN (e.g., the sequence of SEQ ID NO:8 or 11) without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the CDHN proteins of the present invention, e.g., those present in a cadherin domain, a CA domain, or a cadherins extracellular repeated domain signature pattern, are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the CDHN proteins of the present invention and other members of the CDHN family are not likely to be amenable to alteration.

[0682] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding CDHN proteins that contain changes in amino acid residues that are not essential for activity. Such CDHN proteins differ in amino acid sequence from SEQ ID NO:8 or 11, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:8 or 11.

[0683] An isolated nucleic acid molecule encoding a CDHN protein identical to the protein of SEQ ID NO:8 or 11 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a CDHN protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a CDHN coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for CDHN biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0684] In a preferred embodiment, a mutant CDHN protein can be assayed for the ability to: 1) modulate of cell adhesion, e.g., cell-cell and cell-substrate adhesion; 2) modulate cell growth, proliferation, and/or differentiation; 3) modulate of cell motility, e.g., cell migration and cell invasion; 4) modulate cytoskeletal organization; 5) modulate and maintain multicellular organization, e.g., cell sorting, cell polarization, tissue morphogenesis, tissue integrity; 6) modulate intra- and/or inter-cellular signaling; and 7) modulate transcriptional regulation of gene expression.

[0685] In addition to the nucleic acid molecules encoding CDHN proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire CDHN coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding a CDHN. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human CDHN-1 corresponds to SEQ ID NO:9, the coding region of human CDHN-2 corresponds to SEQ ID NO:12). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding a CDHN. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0686] Given the coding strand sequences encoding CDHN-1 and CDHN-2 disclosed herein (e.g., SEQ ID NO:9 and 12), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of CDHN mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of CDHN mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of CDHN mRNA. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0687] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a CDHN protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0688] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual P-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0689] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave CDHN mRNA transcripts to thereby inhibit translation of CDHN mRNA. A ribozyme having specificity for a CDHN-encoding nucleic acid can be designed based upon the nucleotide sequence of a CDHN cDNA disclosed herein (i.e., SEQ ID NO:7, 9, 10, or 12, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a CDHN-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, CDHN mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0690] Alternatively, CDHN gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the CDHN (e.g., the CDHN promoter and/or enhancers; e.g., nucleotides 1-111 of SEQ ID NO:7 or nucleotides 1-161 of SEQ ID NO:10) to form triple helical structures that prevent transcription of the CDHN gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6): 569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

[0691] In yet another embodiment, the CDHN nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0692] PNAs of CDHN nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of CDHN nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0693] In another embodiment, PNAs of CDHN can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of CDHN nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser, K. H. et al (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

[0694] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0695] Alternatively, the expression characteristics of an endogenous CDHN gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous CDHN gene. For example, an endogenous CDHN gene which is normally “transcriptionally silent”, i.e., a CDHN gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism. Alternatively, a transcriptionally silent, endogenous CDHN gene may be activated by insertion of a promiscuous regulatory element that works across cell types.

[0696] A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous CDHN gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

[0697] II. Isolated CDHN Proteins and Anti-CDHN Antibodies

[0698] One aspect of the invention pertains to isolated CDHN proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-CDHN antibodies. In one embodiment, native CDHN proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, CDHN proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a CDHN protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[0699] An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the CDHN protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of CDHN protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of CDHN protein having less than about 30% (by dry weight) of non-CDHN protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-CDHN protein, still more preferably less than about 10% of non-CDHN protein, and most preferably less than about 5% non-CDHN protein. When the CDHN protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0700] The language “substantially free of chemical precursors or other chemicals” includes preparations of CDHN protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of CDHN protein having less than about 30% (by dry weight) of chemical precursors or non-CDHN chemicals, more preferably less than about 20% chemical precursors or non-CDHN chemicals, still more preferably less than about 10% chemical precursors or non-CDHN chemicals, and most preferably less than about 5% chemical precursors or non-CDHN chemicals.

[0701] As used herein, a “biologically active portion” of a CDHN protein includes a fragment of a CDHN protein which participates in an interaction between CDHN molecules, or in an interaction between a CDHN molecule and a non-CDHN molecule. Biologically active portions of a CDHN protein include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the CDHN protein, e.g., the amino acid sequence shown in SEQ ID NO:8 or 11, which include less amino acids than the full length CDHN protein, and exhibit at least one activity of a CDHN protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the CDHN protein, e.g., modulation of cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms. A biologically active portion of a CDHN protein can be a polypeptide which is, for example, 25, 50, 75, 100, 125, 150, 175, 200, 250, 300, 400, 500 or more amino acids in length. Biologically active portions of a CDHN protein can be used as targets for developing agents which modulate a CDHN mediated activity, e.g., cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms.

[0702] In one embodiment, a biologically active portion of a CDHN protein comprises at least one, preferably two, three, four, five or more cadherin domains. In another embodiment, a biologically active portion of a CDHN protein comprises at least one, preferably two, three, four, five or six CA domains. In another embodiment, a biologically active portion of a CDHN protein of the present invention may contain at least one, preferably two, three, four, five or more, cadherin domains, and at least one or more of the following domains: a CA domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide. In a further embodiment, a biologically active portion of a CDHN protein of the present invention may contain at least one, preferably two, three, four, five, or six CA domains, and at least one or more of the following domains: a cadherin domain, a cadherins extracellular repeated domain signature pattern, a transmembrane domain, or a signal peptide. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native CDHN protein.

[0703] In a preferred embodiment, the CDHN protein has an amino acid sequence shown in SEQ ID NO:8 or 11. In other embodiments, the CDHN protein is substantially identical to SEQ ID NO:8 or 11 and retains the functional activity of the protein of SEQ ID NO:8 or 11, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. Accordingly, in another embodiment, the CDHN protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:8 or 11.

[0704] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the CDHN amino acid sequence of SEQ ID NO:8 having 924 amino acid residues, at least 277, preferably at least 370, more preferably at least 462, even more preferably at least 555, and even more preferably at least 647 or more amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0705] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0706] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to CDHN nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=100, wordlength=3 to obtain amino acid sequences homologous to CDHN protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0707] The invention also provides CDHN chimeric or fusion proteins. As used herein, a CDHN “chimeric protein” or “fusion protein” comprises a CDHN polypeptide operatively linked to a non-CDHN polypeptide. A “CDHN polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a CDHN (e.g., CDHN-1, CDHN-2) molecule, whereas a “non-CDHN polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the CDHN protein, e.g., a protein which is different from the CDHN protein and which is derived from the same or a different organism. Within a CDHN fusion protein the CDHN polypeptide can correspond to all or a portion of a CDHN protein. In a preferred embodiment, a CDHN fusion protein comprises at least one biologically active portion of a CDHN protein. In another preferred embodiment, a CDHN fusion protein comprises at least two biologically active portions of a CDHN protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the CDHN polypeptide and the non-CDHN polypeptide are fused in-frame to each other. The non-CDHN polypeptide can be fused to the N-terminus or C-terminus of the CDHN polypeptide.

[0708] For example, in one embodiment, the fusion protein is a GST-CDHN fusion protein in which the CDHN sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant CDHN.

[0709] In another embodiment, the fusion protein is a CDHN protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of CDHN can be increased through use of a heterologous signal sequence.

[0710] The CDHN fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The CDHN fusion proteins can be used to affect the bioavailability of a CDHN ligand or substrate. Use of CDHN fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a CDHN protein; (ii) mis-regulation of a CDHN gene; and (iii) aberrant post-translational modification of a CDHN protein.

[0711] Moreover, the CDHN fusion proteins of the invention can be used as immunogens to produce anti-CDHN antibodies in a subject, to purify CDHN ligands and in screening assays to identify molecules which inhibit the interaction of CDHN with a CDHN substrate.

[0712] Preferably, a CDHN chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A CDHN-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the CDHN protein.

[0713] The present invention also pertains to variants of the CDHN proteins which function as either CDHN agonists (mimetics) or as CDHN antagonists. Variants of the CDHN proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a CDHN protein. An agonist of the CDHN proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a CDHN protein. An antagonist of a CDHN protein can inhibit one or more of the activities of the naturally occurring form of the CDHN protein by, for example, competitively modulating a CDHN-mediated activity of a CDHN protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the CDHN protein.

[0714] In one embodiment, variants of a CDHN protein which function as either CDHN agonists (mimetics) or as CDHN antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a CDHN protein for CDHN protein agonist or antagonist activity. In one embodiment, a variegated library of CDHN variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of CDHN variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential CDHN sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of CDHN sequences therein. There are a variety of methods which can be used to produce libraries of potential CDHN variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential CDHN sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[0715] In addition, libraries of fragments of a CDHN protein coding sequence can be used to generate a variegated population of CDHN fragments for screening and subsequent selection of variants of a CDHN protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a CDHN coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the CDHN protein.

[0716] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of CDHN proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify CDHN variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering 6(3): 327-331).

[0717] In one embodiment, cell based assays can be exploited to analyze a variegated CDHN library. For example, a library of expression vectors can be transfected into a cell line, e.g., a mammalian cell line, which ordinarily responds to a CDHN ligand in a particular CDHN ligand-dependent manner. The transfected cells are then contacted with a CDHN ligand and the effect of expression of the mutant on, e.g., modulation of cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms can be detected. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the CDHN ligand, and the individual clones further characterized.

[0718] An isolated CDHN protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind CDHN using standard techniques for polyclonal and monoclonal antibody preparation. A full-length CDHN protein can be used or, alternatively, the invention provides antigenic peptide fragments of CDHN for use as immunogens. The antigenic peptide of CDHN comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:8 or 11 and encompasses an epitope of CDHN such that an antibody raised against the peptide forms a specific immune complex with the CDHN protein. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0719] Preferred epitopes encompassed by the antigenic peptide are regions of CDHN that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity (see, for example, FIGS. 10 and 16).

[0720] A CDHN immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed CDHN protein or a chemically synthesized CDHN polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic CDHN preparation induces a polyclonal anti-CDHN antibody response.

[0721] Accordingly, another aspect of the invention pertains to anti-CDHN antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as a CDHN. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind CDHN molecules. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of CDHN. A monoclonal antibody composition thus typically displays a single binding affinity for a particular CDHN protein with which it immunoreacts.

[0722] Polyclonal anti-CDHN antibodies can be prepared as described above by immunizing a suitable subject with a CDHN immunogen. The anti-CDHN antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized CDHN. If desired, the antibody molecules directed against CDHN can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-CDHN antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem.255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lemer (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a CDHN immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds CDHN.

[0723] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-CDHN monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind CDHN, e.g., using a standard ELISA assay.

[0724] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-CDHN antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with CDHN to thereby isolate immunoglobulin library members that bind CDHN. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[0725] Additionally, recombinant anti-CDHN antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[0726] An anti-CDHN antibody (e.g., monoclonal antibody) can be used to isolate CDHN by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-CDHN antibody can facilitate the purification of natural CDHN from cells and of recombinantly produced CDHN expressed in host cells. Moreover, an anti-CDHN antibody can be used to detect CDHN protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the CDHN protein. Anti-CDHN antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[0727] III. Recombinant Expression Vectors and Host Cells

[0728] Another aspect of the invention pertains to vectors, preferably expression vectors, containing a nucleic acid encoding a CDHN protein (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host-cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0729] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., CDHN proteins, mutant forms of CDHN proteins, fusion proteins, and the like).

[0730] The recombinant expression vectors of the invention can be designed for expression of CDHN proteins in prokaryotic or eukaryotic cells. For example, CDHN proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0731] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[0732] Purified fusion proteins can be utilized in CDHN activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for CDHN proteins, for example. In a preferred embodiment, a CDHN fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

[0733] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[0734] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[0735] In another embodiment, the CDHN expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corporation, San Diego, Calif.).

[0736] Alternatively, CDHN proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[0737] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufinan et al (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0738] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[0739] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to CDHN mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the anti sense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

[0740] Another aspect of the invention pertains to host cells into which a CDHN nucleic acid molecule of the invention is introduced, e.g., a CDHN nucleic acid molecule within a recombinant expression vector or a CDHN nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0741] A host cell can be any prokaryotic or eukaryotic cell. For example, a CDHN protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[0742] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[0743] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a CDHN protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[0744] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a CDHN protein. Accordingly, the invention further provides methods for producing a CDHN protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a CDHN protein has been introduced) in a suitable medium such that a CDHN protein is produced. In another embodiment, the method further comprises isolating a CDHN protein from the medium or the host cell.

[0745] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which CDHN coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous CDHN sequences have been introduced into their genome or homologous recombinant animals in which endogenous CDHN sequences have been altered. Such animals are useful for studying the function and/or activity of a CDHN and for identifying and/or evaluating modulators of CDHN activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous CDHN gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[0746] A transgenic animal of the invention can be created by introducing a CDHN-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The CDHN cDNA sequence of SEQ ID NO:7 or 10 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human CDHN gene, such as a mouse or rat CDHN gene, can be used as a transgene. Alternatively, a CDHN gene homologue, such as another CDHN family member, can be isolated based on hybridization to the CDHN cDNA sequences of SEQ ID NO:7, 9, 10, or 12, or the DNA insert of the plasmid deposited with ATCC as Accession Number ______ (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a CDHN transgene to direct expression of a CDHN protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a CDHN transgene in its genome and/or expression of CDHN mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a CDHN protein can further be bred to other transgenic animals carrying other transgenes.

[0747] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a CDHN gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the CDHN gene. The CDHN gene can be a human gene (e.g., the cDNA of SEQ ID NO:9 or 12), but more preferably, is a non-human homologue of a human CDHN gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:7 or 10). For example, a mouse CDHN gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous CDHN gene in the mouse genome. In a preferred embodiment, the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous CDHN gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous CDHN gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous CDHN protein). In the homologous recombination nucleic acid molecule, the altered portion of the CDHN gene is flanked at its 5′ and 3′ ends by additional nucleic acid sequence of the CDHN gene to allow for homologous recombination to occur between the exogenous CDHN gene carried by the homologous recombination nucleic acid molecule and an endogenous CDHN gene in a cell, e.g., an embryonic stem cell. The additional flanking CDHN nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced CDHN gene has homologously recombined with the endogenous CDHN gene are selected (see e.g., Li, E. et al (1992) Cell 69:915). The selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

[0748] In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[0749] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[0750] IV. Pharmaceutical Compositions

[0751] The CDHN nucleic acid molecules, fragments of CDHN proteins, and anti-CDHN antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0752] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0753] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0754] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a CDHN protein or an anti-CDHN antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0755] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0756] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0757] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0758] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0759] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0760] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0761] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0762] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0763] As defined herein, a therapeutically effective amount of protein or polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a protein, polypeptide, or antibody can include a single treatment or, preferably, can include a series of treatments.

[0764] In a preferred example, a subject is treated with antibody, protein, or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody, protein, or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

[0765] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e., including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.

[0766] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[0767] Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[0768] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[0769] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Arnon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[0770] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[0771] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0772] V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, a CDHN protein of the invention has one or more of the following activities: 1) modulation of cell adhesion, e.g., cell-cell and cell-substrate adhesion; 2) modulation of cell growth, proliferation, and/or differentiation; 3) modulation of cell motility, e.g., cell migration and cell invasion; 4) modulation of cytoskeletal organization; 5) modulation and maintenance of multicellular organization, e.g., cell sorting, cell polarization, tissue morphogenesis, tissue integrity; 6) modulation of intra- and/or inter-cellular signaling; and 7) modulation of transcriptional regulation of gene expression. The isolated nucleic acid molecules of the invention can be used, for example, to express CDHN protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect CDHN mRNA (e.g., in a biological sample) or a genetic alteration in a CDHN gene, and to modulate CDHN activity, as described further below. The CDHN proteins can be used to treat disorders characterized by insufficient or excessive production of a CDHN substrate or production of CDHN inhibitors. In addition, the CDHN proteins can be used to screen for naturally occurring CDHN substrates, to screen for drugs or compounds which modulate CDHN activity, as well as to treat disorders characterized by insufficient or excessive production of CDHN protein or production of CDHN protein forms which have decreased, aberrant or unwanted activity compared to CDHN wild type protein (e.g., cadherin-associated disorders, such as central nervous system (CNS) disorders, cardiovascular disorders, musculoskeletal disorders, gastrointestinal disorders, inflammatory or immune system disorders, or cell proliferation, growth, differentiation, adhesion, or migration disorders).

[0773] Moreover, the anti-CDHN antibodies of the invention can be used to detect and isolate CDHN proteins, regulate the bioavailability of CDHN proteins, and modulate CDHN activity.

[0774] A. Screening Assays:

[0775] The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to CDHN proteins, have a stimulatory or inhibitory effect on, for example, CDHN expression or CDHN activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of a CDHN substrate.

[0776] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a CDHN protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a CDHN protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

[0777] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[0778] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

[0779] In one embodiment, an assay is a cell-based assay in which a cell which expresses a CDHN protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate CDHN activity is determined. Determining the ability of the test compound to modulate CDHN activity can be accomplished by monitoring, for example, cell aggregation, adhesion and/or motility in a cell which expresses CDHN. The cell, for example, can be of mammalian origin, e.g., an epithelial or neuronal cell. The ability of the test compound to modulate CDHN binding to a substrate or to bind to CDHN can also be determined. Determining the ability of the test compound to modulate CDHN binding to a substrate can be accomplished, for example, by coupling the CDHN substrate with a radioisotope or enzymatic label such that binding of the CDHN substrate to CDHN can be determined by detecting the labeled CDHN substrate in a complex. Alternatively, CDHN could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate CDHN binding to a CDHN substrate in a complex. Determining the ability of the test compound to bind CDHN can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to CDHN can be determined by detecting the labeled compound in a complex. For example, compounds (e.g., CDHN substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[0780] It is also within the scope of this invention to determine the ability of a compound (e.g., a CDHN substrate) to interact with CDHN without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with CDHN without the labeling of either the compound or the CDHN. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and CDHN.

[0781] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a CDHN target molecule (e.g., a CDHN substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the CDHN target molecule. Determining the ability of the test compound to modulate the activity of a CDHN target molecule can be accomplished, for example, by determining the ability of the CDHN protein to bind to or interact with the CDHN target molecule.

[0782] Determining the ability of the CDHN protein, or a biologically active fragment thereof, to bind to or interact with a CDHN target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the CDHN protein to bind to or interact with a CDHN target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular response (i.e., cell proliferation, differentiation, adhesion, migration and/or signal transduction), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response.

[0783] In yet another embodiment, an assay of the present invention is a cell-free assay in which a CDHN protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the CDHN protein or biologically active portion thereof is determined. Preferred biologically active portions of the CDHN proteins to be used in assays of the present invention include fragments which participate in interactions with non-CDHN molecules, e.g., fragments with high surface probability scores (see, for example, FIGS. 10 and 16). Binding of the test compound to the CDHN protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the CDHN protein or biologically active portion thereof with a known compound which binds the CDHN to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the CDHN protein, wherein determining the ability of the test compound to interact with the CDHN protein comprises determining the ability of the test compound to preferentially bind to the CDHN or biologically active portion thereof as compared to the known compound.

[0784] In another embodiment, the assay is a cell-free assay in which a CDHN protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the CDHN protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a CDHN protein can be accomplished, for example, by determining the ability of the CDHN protein to bind to a CDHN target molecule by one of the methods described above for determining direct binding. Determining the ability of the CDHN protein to bind to a CDHN target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[0785] In an alternative embodiment, determining the ability of the test compound to modulate the activity of a CDHN protein can be accomplished by determining the ability of the CDHN protein to further modulate the activity of a downstream effector of a CDHN target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.

[0786] In yet another embodiment, the cell-free assay involves contacting a CDHN protein or biologically active portion thereof with a known compound (e.g., a CDHN substrate) which binds the CDHN protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the CDHN protein, wherein determining the ability of the test compound to interact with the CDHN protein comprises determining the ability of the CDHN protein to preferentially bind to or modulate the activity of a CDHN target protein, e.g., associate with the cytoskeleton via a CDHN substrate.

[0787] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either CDHN or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a CDHN protein, or interaction of a CDHN protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/CDHN fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or CDHN protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of CDHN binding or activity determined using standard techniques.

[0788] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a CDHN protein or a CDHN target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated CDHN protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with CDHN protein or target molecules but which do not interfere with binding of the CDHN protein to its target molecule can be derivatized to the wells of the plate, and unbound target or CDHN protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the CDHN protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the CDHN protein or target molecule.

[0789] In another embodiment, modulators of CDHN expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of CDHN mRNA or protein in the cell is determined. The level of expression of CDHN mRNA or protein in the presence of the candidate compound is compared to the level of expression of CDHN mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of CDHN expression based on this comparison. For example, when expression of CDHN mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of CDHN mRNA or protein expression. Alternatively, when expression of CDHN mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of CDHN mRNA or protein expression. The level of CDHN mRNA or protein expression in the cells can be determined by methods described herein for detecting CDHN mRNA or protein.

[0790] In yet another aspect of the invention, the CDHN proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with CDHN (“CDHN binding proteins” or “CDHN-bp”) and are involved in CDHN activity. Such CDHN binding proteins are also likely to be involved in the propagation of signals by the CDHN proteins or CDHN targets as, for example, downstream elements of a CDHN-mediated signaling pathway. Alternatively, such CDHN binding proteins are likely to be CDHN inhibitors.

[0791] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a CDHN protein, or a biologically active portion thereof, is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a CDHN-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the CDHN protein.

[0792] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a CDHN protein can be confirmed in vivo, e.g., in an animal such as an animal model for cellular transformation, tumorigenesis and/or metastasis.

[0793] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a CDHN modulating agent, an antisense CDHN nucleic acid molecule, a CDHN-specific antibody, or a CDHN binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[0794] B. Detection Assays

[0795] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[0796] 1. Chromosome Mapping

[0797] Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the CDHN nucleotide sequences, described herein, can be used to map the location of the CDHN genes on a chromosome. The mapping of the CDHN sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

[0798] Briefly, CDHN genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the CDHN nucleotide sequences. Computer analysis of the CDHN sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the CDHN sequences will yield an amplified fragment.

[0799] Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

[0800] PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the CDHN nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a CDHN sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

[0801] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

[0802] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[0803] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[0804] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with a CDHN gene can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[0805] 2. Tissue Typing

[0806] The CDHN sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[0807] Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the CDHN nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[0808] Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The CDHN nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:7 or 10 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:9 or 12 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[0809] If a panel of reagents from CDHN nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[0810] 3. Use of CDHN Sequences in Forensic Biology

[0811] DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[0812] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:7 or SEQ ID NO:10 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the CDHN nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:7 or SEQ ID NO:10 having a length of at least 20 bases, preferably at least 30 bases.

[0813] The CDHN nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such CDHN probes can be used to identify tissue by species and/or by organ type.

[0814] In a similar fashion, these reagents, e.g., CDHN primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[0815] C. Predictive Medicine:

[0816] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining CDHN protein and/or nucleic acid expression as well as CDHN activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted CDHN expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with CDHN protein, nucleic acid expression or activity. For example, mutations in a CDHN gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with CDHN protein, nucleic acid expression or activity.

[0817] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of CDHN in clinical trials.

[0818] These and other agents are described in further detail in the following sections.

[0819] 1. Diagnostic Assays

[0820] An exemplary method for detecting the presence or absence of CDHN protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting CDHN protein or nucleic acid (e.g., mRNA, or genomic DNA) that encodes CDHN protein such that the presence of CDHN protein or nucleic acid is detected in the biological sample. A preferred agent for detecting CDHN mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to CDHN mRNA or genomic DNA. The nucleic acid probe can be, for example, the CDHN nucleic acid set forth in SEQ ID NO:7, 9, 10, or 12, or the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to CDHN mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[0821] A preferred agent for detecting CDHN protein is an antibody capable of binding to CDHN protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect CDHN mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of CDHN mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of CDHN protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of CDHN genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of CDHN protein include introducing into a subject a labeled anti-CDHN antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[0822] In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.

[0823] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting CDHN protein, mRNA, or genomic DNA, such that the presence of CDHN protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of CDHN protein, mRNA or genomic DNA in the control sample with the presence of CDHN protein, mRNA or genomic DNA in the test sample.

[0824] The invention also encompasses kits for detecting the presence of a CDHN in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting a CDHN protein or mRNA in a biological sample; means for determining the amount of CDHN in the sample; and means for comparing the amount of CDHN in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect CDHN protein or nucleic acid.

[0825] 2. Prognostic Assays

[0826] The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted CDHN expression or activity. As used herein, the term “aberrant” includes a CDHN expression or activity which deviates from the wild type CDHN expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant CDHN expression or activity is intended to include the cases in which a mutation in the CDHN gene causes the CDHN gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional CDHN protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a CDHN substrate, or one which interacts with a non-CDHN substrate. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as cellular proliferation. For example, the term unwanted includes a CDHN expression or activity which is undesirable in a subject.

[0827] The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in CDHN protein activity or nucleic acid expression, such as a central nervous system (CNS) disorder, a cardiovascular disorder, a musculoskeletal disorder, a gastrointestinal disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, adhesion, or migration disorder. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in CDHN protein activity or nucleic acid expression, such as a central nervous system (CNS) disorder, a cardiovascular disorder, a musculoskeletal disorder, a gastrointestinal disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, adhesion, or migration disorder. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted CDHN expression or activity in which a test sample is obtained from a subject and CDHN protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of CDHN protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted CDHN expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., cerebrospinal fluid or serum), cell sample, or tissue.

[0828] Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted CDHN expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a central nervous system (CNS) disorder, a cardiovascular disorder, a musculoskeletal disorder, a gastrointestinal disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, adhesion, or migration disorder. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted CDHN expression or activity in which a test sample is obtained and CDHN protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of CDHN protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted CDHN expression or activity).

[0829] The methods of the invention can also be used to detect genetic alterations in a CDHN gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in CDHN protein activity or nucleic acid expression, such as a central nervous system (CNS) disorder, a cardiovascular disorder, a musculoskeletal disorder, a gastrointestinal disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, adhesion, or migration disorder. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a CDHN protein, or the mis-expression of the CDHN gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a CDHN gene; 2) an addition of one or more nucleotides to a CDHN gene; 3) a substitution of one or more nucleotides of a CDHN gene, 4) a chromosomal rearrangement of a CDHN gene; 5) an alteration in the level of a messenger RNA transcript of a CDHN gene, 6) aberrant modification of a CDHN gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a CDHN gene, 8) a non-wild type level of a CDHN protein, 9) allelic loss of a CDHN gene, and 10) inappropriate post-translational modification of a CDHN protein. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in a CDHN gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.

[0830] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in a CDHN gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a CDHN gene under conditions such that hybridization and amplification of the CDHN gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[0831] Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[0832] In an alternative embodiment, mutations in a CDHN gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[0833] In other embodiments, genetic mutations in CDHN can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in CDHN can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[0834] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the CDHN gene and detect mutations by comparing the sequence of the sample CDHN with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

[0835] Other methods for detecting mutations in a CDHN gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type CDHN sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

[0836] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in CDHN cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a CDHN sequence, e.g., a wild-type CDHN sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[0837] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in CDHN genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control CDHN nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[0838] In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[0839] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1 989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[0840] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[0841] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a CDHN gene.

[0842] Furthermore, any cell type or tissue in which CDHN is expressed may be utilized in the prognostic assays described herein.

[0843] 3. Monitoring of Effects during Clinical Trials

[0844] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a CDHN protein (e.g., the modulation of cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase CDHN gene expression, protein levels, or upregulate CDHN activity, can be monitored in clinical trials of subjects exhibiting decreased CDHN gene expression, protein levels, or downregulated CDHN activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease CDHN gene expression, protein levels, or downregulate CDHN activity, can be monitored in clinical trials of subjects exhibiting increased CDHN gene expression, protein levels, or upregulated CDHN activity. In such clinical trials, the expression or activity of a CDHN gene, and preferably, other genes that have been implicated in, for example, a CDHN-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[0845] For example, and not by way of limitation, genes, including CDHN, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates CDHN activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on CDHN-associated disorders (e.g., disorders characterized by deregulated cell proliferation, differentiation, adhesion, migration and/or signaling mechanisms), for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of CDHN and other genes implicated in the CDHN-associated disorder, respectively. The levels of gene expression (e.g., a gene expression pattern) can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of CDHN or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.

[0846] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a CDHN protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the CDHN protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the CDHN protein, mRNA, or genomic DNA in the pre-administration sample with the CDHN protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of CDHN to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of CDHN to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, CDHN expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.

[0847] D. Methods of Treatment:

[0848] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted CDHN expression or activity, e.g., a cadherin-associated disorder such as a central nervous system (CNS) disorder, a cardiovascular disorder, a musculoskeletal disorder, a gastrointestinal disorder, an inflammatory or immune system disorder, or a cell proliferation, growth, differentiation, adhesion, or migration disorder. With regard to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”). Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the CDHN molecules of the present invention or CDHN modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[0849] 1. Prophylactic Methods

[0850] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted CDHN expression or activity, by administering to the subject a CDHN or an agent which modulates CDHN expression or at least one CDHN activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted CDHN expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the CDHN aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of CDHN aberrancy, for example, a CDHN, CDHN agonist or CDHN antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[0851] 2. Therapeutic Methods

[0852] Another aspect of the invention pertains to methods of modulating CDHN expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell with a CDHN or agent that modulates one or more of the activities of CDHN protein activity associated with the cell. An agent that modulates CDHN protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a CDHN protein (e.g., a CDHN substrate), a CDHN antibody, a CDHN agonist or antagonist, a peptidomimetic of a CDHN agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more CDHN activities. Examples of such stimulatory agents include active CDHN protein and a nucleic acid molecule encoding a CDHN that has been introduced into the cell. In another embodiment, the agent inhibits one or more CDHN activities. Examples of such inhibitory agents include antisense CDHN nucleic acid molecules, anti-CDHN antibodies, and CDHN inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a CDHN protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) CDHN expression or activity. In another embodiment, the method involves administering a CDHN protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted CDHN expression or activity.

[0853] Stimulation of CDHN activity is desirable in situations in which CDHN is abnormally downregulated and/or in which increased CDHN activity is likely to have a beneficial effect. Likewise, inhibition of CDHN activity is desirable in situations in which CDHN is abnormally upregulated and/or in which decreased CDHN activity is likely to have a beneficial effect.

[0854] 3. Pharmacogenomics

[0855] The CDHN molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on CDHN activity (e.g., CDHN gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) CDHN-associated disorders (e.g., central nervous system (CNS) disorders, cardiovascular disorders; musculoskeletal disorders, gastrointestinal disorders, inflammatory or immune system disorders, or cell proliferation, growth, differentiation, adhesion, or migration disorders) associated with aberrant or unwanted CDHN activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a CDHN molecule or CDHN modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a CDHN molecule or CDHN modulator.

[0856] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[0857] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a-Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[0858] Alternatively, a method termed the “candidate gene approach”, can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., a CDHN protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[0859] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

[0860] Alternatively, a method termed the “gene expression profiling”, can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a CDHN molecule or CDHN modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[0861] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a CDHN molecule or CDHN modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[0862] VI. Electronic Apparatus Readable Media and Arrays

[0863] Electronic apparatus readable media comprising CDHN sequence information is also provided. As used herein, “CDHN sequence information” refers to any nucleotide and/or amino acid sequence information particular to the CDHN molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like. Moreover, information “related to” said CDHN sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantitative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding, or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact discs; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; and general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon CDHN sequence information of the present invention.

[0864] As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatuses; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

[0865] As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the CDHN sequence information.

[0866] A variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium. For example, the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, represented in the form of an ASCII file, or stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of dataprocessor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the CDHN sequence information.

[0867] By providing CDHN sequence information in readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

[0868] The present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder, wherein the method comprises the steps of determining CDHN sequence information associated with the subject and based on the CDHN sequence information, determining whether the subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder, and/or recommending a particular treatment for the disease, disorder, or pre-disease condition.

[0869] The present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a CDHN associated disease or disorder or a pre-disposition to a disease associated with CDHN wherein the method comprises the steps of determining CDHN sequence information associated with the subject, and based on the CDHN sequence information, determining whether the subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. The method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.

[0870] The present invention also provides in a network, a method for determining whether a subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder associated with CDHN, said method comprising the steps of receiving CDHN sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to CDHN and/or a CDHN associated disease or disorder, and based on one or more of the phenotypic information, the CDHN information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0871] The present invention also provides a business method for determining whether a subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder, said method comprising the steps of receiving information related to CDHN (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to CDHN and/or related to a CDHN associated disease or disorder, and based on one or more of the phenotypic information, the CDHN information, and the acquired information, determining whether the subject has a CDHN associated disease or disorder or a pre-disposition to a CDHN associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[0872] The invention also includes an array comprising a.CDHN sequence of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be CDHN. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

[0873] In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[0874] In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a CDHN associated disease or disorder, progression of CDHN associated disease or disorder, and processes, such a cellular transformation associated with the CDHN associated disease or disorder.

[0875] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of CDHN expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[0876] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including CDHN) that could serve as a molecular target for diagnosis or therapeutic intervention.

[0877] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.

EXAMPLES

Example 1

[0878] Identification and Characterization of Human CDHN cDNAs

[0879] In this example, the identification and characterization of the genes encoding human CDHN-1 (clone Fbh57798) and CDHN-2 (clone Fbh57809) is described.

[0880] Isolation of the CDHN cDNAs

[0881] The invention is based, at least in part, on the discovery of human genes encoding novel proteins, referred to herein as CDHN-1 and CDHN-2. The entire sequences of human clones Fbh57798 and Fbh57809 were determined and found to contain open reading frames termed human “CDHN-1” and “CDHN-2”, respectively.

[0882] The nucleotide sequence encoding the human CDHN-1 protein is shown in FIGS. 9A-C and is set forth as SEQ ID NO:7. The protein encoded by this nucleic acid comprises about 924 amino acids and has the amino acid sequence shown in FIGS. 9A-C and set forth as SEQ ID NO:8. The coding region (open reading frame) of SEQ ID NO:7 is set forth as SEQ ID NO:9.

[0883] The nucleotide sequence encoding the human CDHN-2 protein is shown in FIGS. 15A-C and is set forth as SEQ ID NO:10. The protein encoded by this nucleic acid comprises about 830 amino acids and has the amino acid sequence shown in FIGS. 15A-C and set forth as SEQ ID NO:11. The coding region (open reading frame) of SEQ ID NO:10 is set forth as SEQ ID NO:12.

[0884] Clones Fbh57798 and Fbh57809, comprising the coding region of human CDHN-1 and CDHN-2, respectively, were deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______, and assigned Accession No. ______.

[0885] Analysis of the Human CDHN Molecules

[0886] The amino acid sequences of human CDHN-1 and CDHN-2 were analyzed using the program PSORT (http://www. psort.nibb.ac.jp) to predict the localization of the proteins within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence. The results of the analyses show that human CDHN-1 (SEQ ID NO:8) may be localized to the mitochondria, to the endoplasmic reticulum, to the nucleus, or to the cytoplasm. The results of the analyses further show that human CDHN-2 (SEQ ID NO:11) may be localized to the cytoplasm, to the nucleus, to the mitochondria, to the Golgi, to the endoplasmic reticulum, to secretory vesicles, or to peroxisomes.

[0887] The amino acid sequences of human CDHN-1 and CDHN-2 were also analyzed by the SignalP program (Henrik, et al. (1997) Protein Engineering 10:1-6) for the presence of a signal peptide. These analyses revealed the presence of a signal peptide in the amino acid sequence of CDHN-1 (SEQ ID NO:8) from residues 1-33, and a signal peptide in the amino acid sequence of CDHN-2 (SEQ ID NO: 11) from amino acid residues 1-21.

[0888] Searches of the amino acid sequences of CDHN-1 and CDHN-2 were performed against the Memsat database (FIGS. 11 and 17). These searches resulted in the identification of five transmembrane domains in the amino acid sequence of human CDHN-1 (SEQ ID NO:8) at about residues 19-35, 42-59, 298-315, 369-393 and 863-886 in the native molecule, and the identification of four transmembrane domains in the amino acid sequence of the predicted mature CDHN-1 protein at about residues 8-26, 265-282, 336-360 and 830-853 (FIG. 11). These searches further identified three transmembrane domains in the amino acid sequence of human CDHN-2 (SEQ ID NO:11) at about residues 540-557, 571-588 and 789-813 in the native molecule, and about residues 519-536, 550-567 and 768-792 of the predicted mature protein (FIG. 17).

[0889] Searches were performed against the Prosite database, and resulted in the identification of several possible glycosylation sites within the human CDHN proteins. For example, N-linked glycosylation sites were identified at about residues 108-111, 299-302, 305-308, 653-656, 721-724, 776-779, 817-820 and 822-825 of human CDHN-1 (SEQ ID: NO:8), as well as at about residues 519-522, 604-607 and 724-727 of human CDHN-2 (SEQ ID NO:11). These searches further identified putative phosphorylation sites within the human CDHN proteins. Protein kinase C phosphorylation sites were identified at about amino acid residues 12-14, 219-221, 333-335, 366-368, 428-430, 464-466, 581-583, 609-611, 662-664, 698-700, 767-769 and 850-852, casein kinase II phosphorylation sites were identified at about residues 44-47, 57-60, 82-85, 116-119, 144-147, 362-365, 428-431, 516-519, 533-536, 568-571, 601-604, 635-638, 778-781 and 824-827, and tyrosine phosphorylation sites were identified at about residues 37-43, 430-436, 572-580 and 796-802 of human CDHN-1 (SEQ ID NO:8). Furthermore, protein kinase C phosphorylation sites were identified at about residues 3-5, 597-599, 643-645, and 679-681, and casein kinase II phosphorylation sites were identified at about amino acid residues 153-156, 199-202, 234-237, 266-269, 313-316, 339-342, 361-364, 433-436, 460-463, 477-480 and 535-538, of human CDHN-2 (SEQ ID NO:11). The searches also identified the presence of N-myristoylation site motifs at about amino acid residues 48-53, 101-106, 129-134, 309-314, 377-382, 665-670, 690-695, 734-739 and 881-886 of human CDHN-1 (SEQ ID NO:8), and at about amino acid residues 140-145, 159-164, 354-359, 369-374, 426-431, 468-473, 627-632, 647-652, 685-690 and 790-795 of human CDHN-2 (SEQ ID NO: 11). In addition, these searches identified the presence of cadherins extracellular repeated domain signature motifs at about amino acid residues 170-180, 281-291, 496-506, 600-610 and 703-713 of human CDHN-1 (SEQ ID NO:8), and at about amino acid residues 326-336 of human CDHN-2 (SEQ ID NO:11). Furthermore, the search identified a leucine zipper pattern at about amino acid residues 796-817 of human CDHN-2 (SEQ ID NO:8).

[0890] Searches of the amino acid sequences of CDHN-1 and CDHN-2 were also performed against the HMM (PFAM) database (FIGS. 12A-B and 18A-B). This search resulted in the identification of “cadherin” domains in the amino acid sequence of CDHN-1 (SEQ ID NO:8) at about residues 187-284, 298-390, 513-603, 617-706, and 724-817. This search also resulted in the identification of “cadherin” domains at about residues 27-119, 133-234, 244-329, 343-442, 457-558 and 571-659 of human CDHN-2 (SEQ ID NO:11).

[0891] Searches of the amino acid sequences of CDHN-1 and CDHN-2 were also performed against the HMM (SMART) database (FIGS. 13A-B and 19A-B). This search resulted in the identification of “CA” domains in the amino acid sequence of CDHN-1 (SEQ ID NO:8) at about residues 205-291, 315-397, 427-506, 530-610, 634-713 and 740-824. This search also resulted in the identification of “CA” domains at about residues 47-126, 150-243, 260-336, 360-449, 474-563 and 585-663 of human CDHN-2 (SEQ ID NO:11).

[0892] Searches of the amino acid sequences of CDHN-1 and CDHN-2 were also performed against the ProDom database (FIGS. 14A-H and 20A-I). These searches resulted in the local alignment of the human CDHN-1 protein (SEQ ID NO:8) with p99.2 (671) FAT(32) Q14517(28) O88277(27) over amino acid residues 191-293 [score=147], over amino acid residues 555-612 [score=139], over amino acid residues 632-713 [score 126], over amino acid residues 305-389 [score=74], over amino acid residues 728-822 [score=67], over amino acid residues 466-512 [score=56], over amino acid residues 168-182 [score=54], and over amino acid residues 93-123 [score=49]. These searches also resulted in the local alignment of CDHN-1 with p99.2 (1) Q19319_CAEEL over amino acid residues 527-825 [score=150], over amino acid residues 312-619 [score=94], over amino acid residues 629-814 [score=82], and over amino acid residues 207-509 [score=78]. In addition, these searches resulted in the local alignment of CDHN-1 with p99.2 (1) P81137_MANSE over amino acid residues 168-610 [score=154], over amino acid residues 411-775 [score=133], over amino acid residues 383-721 [score=116], and over amino acid residues 251-274 [score=37]. Furthermore, these searches resulted in the local alignment of CDHN-1 with p99.2 (1) O01909_CAEEL over amino acid residues 600-713 [score=139], over amino acid residues 189-398 [score=136], over amino acid residues 170-310 [score=123], over amino acid residues 500-626 [score=111], and over amino acid residues 673-830 [score=86]; and with p99.2 (1) O93508_BRARE over amino acid residues 739-831 [score=109], over amino acid residues 506-604 [score=89], over amino acid residues 610-707 [score=80], over amino acid residues 291-362 [score=79], and over amino acid residues 191-285 [score=76].

[0893] These searches resulted in the local alignment of the human CDHN-2 protein (SEQ ID NO:11) with p99.2 (3) O75309(1) 088338(1) Q28634(1) over amino acid residues 559-693 [score=583], and over amino acid residues 125-177 [score=72]. These searches also resulted in the local alignment of CDHN-2 with p99.2 (3) 075309(1) Q28634(1) O88338(1) over amino acid residues 1-62 [score=291]. In addition, these searches resulted in the local alignment of CDHN-2 with p99.2 (3) 075309 088338(1) Q28634(1) over amino acid residues 782-830 [score=210]; and with p99.2(38) CAD1(4) DSC1(3) CAD2(3) over amino acid residues 677-781 [score=204]. These searches resulted in the local alignment of CDHN-2 with p99.2 (671) FAT(32) Q14517(28) O88277(27) over amino acid residues 346-451 [score=145], over amino acid residues 60-128 [score=102], over amino acid residues 282-340 [score=79], and over amino acid residues 152-242 [score=77]. Furthermore, these searches resulted in the local alignment of CDHN-2 with p99.2 (1) P81137_MANSE over amino acid residues 270-454 [score=128], over amino acid residues 323-452 [score=104], over amino acid residues 354-606 [score=87], over amino acid residues 62-205 [score=87], and over amino acid residues 324-483 [score=69], over amino acid residues 114-182 [score=66], over amino acid residues 612-657 [score=59], over amino acid residues 562-670 [score=56], over amino acid residues 114-127 [score=50], and over amino acid residues 572-608 [score=41]. These searches also resulted in the local alignment of CDHN-2 with p99.2 (1) Q19319_CAEEL over amino acid residues 58-249 [score=115], over amino acid residues 356-650 [score=90], over amino acid residues 267-452 [score=87], and over amino acid residues 206-239 [score=43]. In addition, these searches resulted in the local alignment of CDHN-2 with p99.2(1) O76356_CAEEL over amino acid residues 15-102 [score=79]; with p99.2 (3) CADL(1) Q12864(1) Q15336(1) over amino acid residues 781-828 [score=71]; and with p99.2 (1) ENDR_BOVIN over amino acid residues 612-713 [score=76].

Example 2

[0894] Expression of Recombinant CDHN Protein in Bacterial Cells

[0895] In this example, CDHN is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, CDHN is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression ofthe GST-CDHN fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB 199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 3

[0896] Expression of Recombinant CDHN Protein in COS Cells

[0897] To express the CDHN gene in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire CDHN protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[0898] To construct the plasmid, the CDHN DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the CDHN coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the CDHN coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the CDHN gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[0899] COS cells are subsequently transfected with the CDHN-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of the CDHN polypeptide is detected by radiolabeling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA-specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[0900] Alternatively, DNA containing the CDHN coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the CDHN polypeptide is detected by radiolabeling and immunoprecipitation using a CDHN specific monoclonal antibody.

[0901] IV. 33358, A Novel Human Ankyrin Family Member and Uses Thereof

BACKGROUND OF THE INVENTION

[0902] Protein-protein interactions are critical for virtually all cellular processes. Cell growth, differentiation, and death are mechanisms regulated by the interaction of proteins with one another. Proteins altered in binding specificity may lead to aberrant or absent interactions and are responsible for a variety of diseases; e.g. birth defects, cancer, and heart disease. Various motifs mediating such interactions have been identified in recent years and include death domains, PDZ domains, WW domains, leucine zippers and leucine rich repeats, and ankyrin repeats.

[0903] Ankyrin repeat containing proteins are a diverse family of proteins which include cell cycle proteins, transcription factors, and proteins that mediate development (Blank, V. et al. (1992) Trends Biochem. Sci. 17:135-140, Bork, P. (1993) Proteins 17:363-374). Ankyrin repeats are named for their homology to repeats in the erythrocyte protein ankyrin. Such repeats are 33 amino acids long and are typically found in clusters of four or more. The structure of ankyrin-repeat regions of many proteins have been solved and it is well documented that each ankyrin-repeat forms an L shaped structure whereby two alpha-helices are connected by a beta-hairpin (a helix-loop-helix) (Batchelor, A. H. et al. (1998) Science 279:1037-1041, Zhang Z. et al. (1998) J. Biol. Chem. 273:18681-18684, Jacobs M. D. et al (1998) Cell 95:749-758). The alpha helices are often stacked upon one another forming a scaffold by which the beta-hairpin is exposed and available to bind heterologous proteins.

[0904] Ankyrin-repeat containing proteins are present in nearly all cells. These proteins have been identified as important for diverse activities including regulation of cardiac cellular processes; e.g. cardiogenesis and heart diseases (Zou, Y. et al. (1997) Development 124:793-804, Yang, Y. et al. (1998) Structure 15:619-626, Kuo, H. et al. (1999) Development 126:4223-4234).

SUMMARY OF THE INVENTION

[0905] The present invention is based, at least in part, on the discovery of ankyrin repeat-containing protein family members, referred to herein as “Cardiac/Skeletal Muscle-Restricted Ankyrin-Repeat Containing Protein” or “C/SKARP” nucleic acid and protein molecules. The C/SKARP nucleic acid and protein molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., myogenic cellular processes including, but not limited to cardiac cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding C/SKARP proteins or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of C/SKARP-encoding nucleic acids.

[0906] In one embodiment, the invention features an isolated nucleic acid molecule that includes the nucleotide sequence set forth in SEQ ID NO:14 or 16. In another embodiment, the invention features an isolated nucleic acid molecule that encodes a polypeptide including the amino acid sequence set forth in SEQ ID NO: 15. In another embodiment, the invention features an isolated nucleic acid molecule that includes the nucleotide sequence contained in the plasmid deposited with ATCC® as Accession Number ______.

[0907] In still other embodiments, the invention features isolated nucleic acid molecules including nucleotide sequences that are substantially identical (e.g., 60% identical) to the nucleotide sequence set forth as SEQ ID NO:14 or 16. The invention further features isolated nucleic acid molecules including at least 30 contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NO:14 or 16. In another embodiment, the invention features isolated nucleic acid molecules which encode a polypeptide including an amino acid sequence that is substantially identical (e.g., 60% identical) to the amino acid sequence set forth as SEQ ID NO:15. Also featured are nucleic acid molecules which encode allelic variants of the polypeptide having the amino acid sequence set forth as SEQ ID NO:15. In addition to isolated nucleic acid molecules encoding full-length polypeptides, the present invention also features nucleic acid molecules which encode fragments, for example biologically active or antigenic fragments, of the full-length polypeptides of the present invention (e.g., fragments including at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:15). In still other embodiments, the invention features isolated nucleic acid molecules that are complementary to, are antisense to, or hybridize under stringent conditions to the isolated nucleic acid molecules described herein.

[0908] In a related aspect, the invention provides vectors including the isolated nucleic acid molecules described herein (e.g., C/SKARP-1-encoding nucleic acid molecules). Such vectors can optionally include nucleotide sequences encoding heterologous polypeptides. Also featured are host cells including such vectors (e.g., host cells including vectors suitable for producing C/SKARP-1 nucleic acid molecules and polypeptides).

[0909] In another aspect, the invention features isolated C/SKARP-1 polypeptides and/or biologically active or antigenic fragments thereof. Exemplary embodiments feature a polypeptide including the amino acid sequence set forth as SEQ ID NO:15, a polypeptide including an amino acid sequence at least 60% identical to the amino acid sequence set forth as SEQ ID NO:15, a polypeptide encoded by a nucleic acid molecule including a nucleotide sequence at least 60% identical to the nucleotide sequence set forth as SEQ ID NO:14 or 16. Also featured are fragments of the full-length polypeptides described herein (e.g., fragments including at least 10 contiguous amino acid residues of the sequence set forth as SEQ ID NO:15) as well as fragments of allelic variants of the polypeptide having the amino acid sequence set forth as SEQ ID NO:15.

[0910] The C/SKARP-1 polypeptides and/or biologically active or antigenic fragments thereof, are useful, for example, as reagents or targets in assays applicable to treatment and/or diagnosis of C/SKARP-1 mediated or related disorders. In one embodiment, a C/SKARP-1 polypeptide or fragment thereof has a C/SKARP-1 activity. In another embodiment, a C/SKARP-1 polypeptide or fragment thereof has an ankyrin repeat domain and optionally, has a C/SKARP-1 activity. In a related aspect, the invention features antibodies (e.g., antibodies which specifically bind to any one of the polypeptides, as described herein) as well as fusion polypeptides including all or a fragment of a polypeptide described herein.

[0911] The present invention further features methods for detecting C/SKARP-1 polypeptides and/or C/SKARP-1 nucleic acid molecules, such methods featuring, for example, a probe, primer or antibody described herein. Also featured are kits for the detection of C/SKARP-1 polypeptides and/or C/SKARP-1 nucleic acid molecules. In a related aspect, the invention features methods for identifying compounds which bind to and/or modulate the activity of a C/SKARP-1 polypeptide or C/SKARP-1 nucleic acid molecule described herein. Further featured are methods for modulating a C/SKARP-1 activity.

[0912] Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[0913] The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as “Cardiac/Skeletal Muscle Restricted Ankyrin-Repeat Containing Protein” or “C/SKARP” protein and nucleic acid molecules, which comprise a family of molecules having certain conserved structural and functional features.

[0914] The term “family” when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having at least one common structural domain or motif and having sufficient amino acid or nucleotide sequence homology or identity as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin. Members of a family may also have common functional characteristics.

[0915] For example, a C/SKARP protein of the present invention can include at least one “ankyrin repeat domain” in the polypeptide (or encoded by the corresponding nucleic acid sequence). As used herein, the term “ankyrin repeat domain” includes a protein domain involved in protein-protein interactions having an amino acid sequence of about 190 to 200 (e.g., about 196) amino acid residues in length and including six ankyrin repeats (e.g., including six consecutive copies of an ankyrin repeat). In another embodiment, an ankyrin repeat domain includes at least about 160 to about 170 (e.g., about 163 to 164) amino acid residues, about 125 to 135 (e.g., about 130 to 131 amino acid residues, about 90 to 100 (e.g., about 95 to 99) amino acid residues or about 60 to 70 (e.g., about 65 to 66) amino acid residues and includes five, four, three or two ankyrin repeats, respectively.

[0916] In a preferred embodiment, a C/SKARP polypeptide or protein has an “ankyrin repeat domain” which includes at least about 190 to 200, about 160 to 170, or about 125 to 135 amino acid residues and has at least about 60%, 70% 80% 90% 95%, 99%, or 100% identity with the “ankyrin repeat domain,” of human C/SKARP-1 (e.g., amino acids 64 to 259 of SEQ ID NO:15).

[0917] As used herein, the term “ankyrin repeat” includes a protein motif typically containing about 33 amino acid residues, initially identified in ankyrin and now identified in over 650 distinct proteins and known to have a role in protein-protein interactions (see e.g., Bork (1993) Proteins: Structure, Function, and Genetics 17:363-374). Preferably, an ankyrin repeat has an amino acid sequence of about 25-40 amino acid residues and has a bit score for the alignment of the sequence to an ankyrin repeat (HMM) (e.g., the Pfam ankyrin repeat HMM having Accession Number PF00023) of at least 10. More preferably, an ankyrin repeat includes at least about 30-36, about 31-35 amino acid residues, about 32-34, or typically about 33 amino acid residues, and has a bit score for the alignment of the sequence to an ankyrin repeat (HMM) of at least 12, 14, 16, 18, 20, 22, 24, 26, or greater. In a preferred embodiment, a C/SKARP protein of the present invention has at least one, and preferably two, three, four, five, or most preferably, six or more ankyrin repeats, as defined herein.

[0918] To identify the presence of an ankyrin repeat in a C/SKARP-1 protein, and make the determination that a query protein has a particular profile, the amino acid sequence of the protein is searched against a database of HMMs (e.g., the Pfam database, release 5.3) using the default parameters (http://www.sanger.ac.uk/Software/Pfam/HMM_search). For example, the search can be performed using the hmmsf program (family specific) using the default parameters (e.g., a threshold score of 15) for determining a hit. hmmsf is available as part of the HMMER package of search programs (HMMER 2.1.1, December 1998) which is freely distributed by the Washington University School of Medicine. Alternatively, the threshold score for determining a hit can be lowered (e.g., to 8 bits). A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3)405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al.(1990) Meth. Enzymol. 183:146-159; Gribskov et al.(1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(l994) J. Mol. Biol. 235:1501-1531; and Stultz et al(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

[0919] A search was performed against the HMM database resulting in the identification of six ankyrin repeats in the amino acid sequence of human C/SKARP-1 (SEQ ID NO:15) at about residues 64-96, 97-129, 130-162, 165-194, 195-227, and 229-259 of SEQ ID NO:15. Identification of ankyrin repeats in a C/SKARP protein of the present invention according to the above-described methodologies further facilitates identification of an ankyrin repeat domain, e.g., comprising six ankyrin repeats as defined herein.

[0920] In yet another embodiment, C/SKARP-1 family members include at least one or more transmembrane domains. As used herein, a “transmembrane domain” includes a protein domain having at least about 10 amino acid residues of which about 60% of the amino acid residues contain non-polar side chains, for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. In a preferred embodiment, a “transmembrane domain” includes a protein domain having at least about 13, preferably about 16, more preferably about 19, and even more preferably about 21, 23, 25, 30, 35 or 40 amino acid residues, of which at least about 70%, preferably about 80%, and more preferably about 90% of the amino acid residues contain non-polar side chains, for example, alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine. A transmembrane domain is lipophilic in nature. Predicted transmembrane domains are found, for example, from about amino acid residues 13-35 and 135-151 of SEQ ID NO:15

[0921] In yet another embodiment, C/SKARP-1 family members includes a signal peptide. As used herein, a “signal sequence” includes a peptide of at least about 20 amino acid residues in length which occurs at the N-terminus of secretory and integral membrane proteins and which contains at least 55% hydrophobic amino acid residues. In a preferred embodiment, a signal sequence contains at least about 15-45 amino acid residues, preferably about 20-42 amino acid residues. Signal sequences of 25-35 amino acid residues and 28-32 amino acid residues are also within the scope of the invention. As used herein, a signal sequence has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine, Tryptophan, or Proline). Such a “signal sequence”, also referred to in the art as a “signal peptide”, serves to direct a protein containing such a sequence to a lipid bilayer. A predicted signal peptide is found, for example, from about amino acid residues 1-43 of SEQ ID NO:15 (although this possible signal peptide is not believed to be utilized by the C/SKARP-1 polypeptide of SEQ ID NO:15).

[0922] Isolated proteins of the present invention, for example C/SKARP proteins, preferably have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:15, or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:14 or 16. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity and share a common functional activity are defined herein as sufficiently identical.

[0923] In a preferred embodiment, a C/SKARP protein includes at least one or more ankyrin repeat domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the amino acid sequence of SEQ ID NO:15, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In yet another preferred embodiment, a C/SKARP protein includes at least one or more ankyrin repeat domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:14 or 16. In another preferred embodiment, a C/SKARP protein includes at least one or more ankyrin repeat domain, and has a C/SKARP activity.

[0924] As used herein, a “C/SKARP activity”, “biological activity of C/SKARP” or “functional activity of C/SKARP”, refers to an activity exerted by a C/SKARP protein, polypeptide or nucleic acid molecule on e.g., a C/SKARP-responsive cell or on a C/SKARP target, e.g., a protein activity, as determined in vivo or in vitro. In one embodiment, a C/SKARP activity is a direct activity, such as an association with a C/SKARP target molecule. A “target molecule” or “binding partner” is a molecule with which a C/SKARP protein binds or interacts in nature. In an exemplary embodiment, a C/SKARP target molecule is a protein molecule (e.g., a second C/SKARP protein or a non-C/SKARP protein molecule). A C/SKARP activity can also be an indirect activity, e.g., a cellular signaling activity mediated by interaction of the C/SKARP protein with a C/SKARP target. In a preferred embodiment, the C/SKARP proteins of the present invention have one or more of the following activities: (i) mediation of specific macromolecular interactions; (ii) mediation of interactions between proteins and/or between regions of a single protein; (iii) formation of binding sites for distinct proteins (e.g., non-C/SKARP proteins); (iv) bridging of cellular components; (v) regulation of gene expression (e.g., cardiac gene expression); (vi) modulation of cellular localization (e.g., anchoring C/SKARP binding proteins in a specific cellular localization); (vii) modulation of development and/or differentiation (e.g., myogenic development and/or differentiation, heart development and/or differentiation); (viii) modulation of cardiac maturation and/or morphogenesis; (ix) as a marker (e.g., an early marker) of cardiac and/or myogenic cell lineage; and (x) modulation and/or treatment of cardiac hypertrophy.

[0925] Inhibition or over stimulation of the activity of proteins involved in signaling pathways associated with cellular growth can lead to perturbed cellular growth, which can in turn lead to cellular growth related disorders. As used herein, a “cellular growth related disorder” includes a disorder, disease, or condition characterized by a deregulation, e.g., an upregulation or a downregulation, of cellular growth. Cellular growth deregulation may be due to a deregulation of cellular proliferation, cell cycle progression, cellular differentiation and/or cellular hypertrophy. Examples of cellular growth related disorders include cardiovascular disorders such as heart failure, hypertension, atrial fibrillation, dilated cardiomyopathy, idiopathic cardiomyopathy, or angina; proliferative disorders or differentiative disorders such as cancer, e.g., melanoma, prostate cancer, cervical cancer, breast cancer, colon cancer, or sarcoma.

[0926] As used herein, the term “cardiovascular disorder” includes a disease, disorder, or state involving the cardiovascular system, e.g., the heart, the blood vessels, and/or the blood. A cardiovascular disorder can be caused by an imbalance in arterial pressure, a malfunction of the heart, or an occlusion of a blood vessel, e.g., by a thrombus. Examples of such disorders include hypertension, atherosclerosis, coronary artery spasm, coronary artery disease, valvular disease, arrhythmias, and cardiomyopathies.

[0927] As used herein, the term “congestive heart failure” includes a condition characterized by a diminished capacity of the heart to supply the oxygen demands of the body. Symptoms and signs of congestive heart failure include diminished blood flow to the various tissues of the body, accumulation of excess blood in the various organs, e.g., when the heart is unable to pump out the blood returned to it by the great veins, exertional dyspnea, fatigue, and/or peripheral edema, e.g., peripheral edema resulting from left ventricular dysfunction. Congestive heart failure may be acute or chronic. The manifestation of congestive heart failure usually occurs secondary to a variety of cardiac or systemic disorders that share a temporal or permanent loss of cardiac function. Examples of such disorders include hypertension, coronary artery disease, valvular disease, and cardiomyopathies, e.g., hypertrophic, dilative, or restrictive cardiomyopathies. Congestive heart failure is described in, for example, Cohn J. N. et al. (1998) American Family Physician 57:1901-04, the contents of which are incorporated herein by reference.

[0928] A partial human C/SKARP-1 cDNA has been identified, which is approximately 1538 nucleotides in length, encodes a protein which is approximately 323 amino acid residues in length.

[0929] A plasmid containing the nucleotide sequence encoding human C/SKARP-1 was deposited with American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[0930] Various aspects of the invention are described in further detail in the following subsections:

[0931] I. Isolated Nucleic Acid Molecules

[0932] One aspect of the invention pertains to isolated nucleic acid molecules that encode C/SKARP-1 proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify C/SKARP-1-encoding nucleic acid molecules (e.g., C/SKARP-1 mRNA) and fragments for use as PCR primers for the amplification or mutation of C/SKARP-1 nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[0933] The term “isolated nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regards to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated C/SKARP-1 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[0934] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or portion of the nucleic acid sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, as a hybridization probe, C/SKARP-1 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0935] Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0936] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to C/SKARP-1 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0937] In a one embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:14. The sequence of SEQ ID NO:14 corresponds to the human C/SKARP-1 cDNA. This cDNA comprises sequences encoding the human C/SKARP-1 protein (i.e., “the coding region”, from nucleotides 75-1046), as well as 5′ untranslated sequences (nucleotides 1-74) and 3′ untranslated sequences (nucleotides 1047-1538). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:14 (e.g., nucleotides 75-1046, corresponding to SEQ ID NO:16). Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention comprises SEQ ID NO:16 and nucleotides 1-74 of SEQ ID NO:14. In yet another embodiment, the isolated nucleic acid molecule comprises SEQ ID NO:16 and nucleotides 1047-1538 of SEQ ID NO:14. In yet another embodiment, the nucleic acid molecule consists of the nucleotide sequence set forth as SEQ ID NO:14 or 16.

[0938] In still another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, thereby forming a stable duplex.

[0939] In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more identical to the nucleotide sequence shown in SEQ ID NO:14 or 16 (e.g., to the entire length of the nucleotide sequence), or to the nucleotide sequence (e.g., the entire length of the nucleotide sequence) of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or to a portion or complement of any of these nucleotide sequences. In one embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least (or no greater than) 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1700 or more nucleotides in length and hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0940] Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a C/SKARP-1 protein, e.g., a biologically active portion of a C/SKARP-1 protein. The nucleotide sequence determined from the cloning of the C/SKARP-1 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other C/SKARP-1 family members, as well as C/SKARP-1 homologues from other species. The probe/primer (e.g., oligonucleotide) typically comprises substantially purified oligonucleotide. The oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, of an anti-sense sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or of a naturally occurring allelic variant or mutant of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[0941] Exemplary probes or primers are at least (or no greater than)12 or 15, 20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides in length and/or comprise consecutive nucleotides of an isolated nucleic acid molecule described herein. Also included within the scope of the present invention are probes or primers comprising contiguous or consecutive nucleotides of an isolated nucleic acid molecule described herein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases within the probe or primer sequence. Probes based on the C/SKARP-1 nucleotide sequences can be used to detect (e.g., specifically detect) transcripts or genomic sequences encoding the same or homologous proteins. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a C/SKARP-1 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases when compared to a sequence disclosed herein or to the sequence of a naturally occurring variant. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a C/SKARP-1 protein, such as by measuring a level of a C/SKARP-1-encoding nucleic acid in a sample of cells from a subject e.g., detecting C/SKARP-1 mRNA levels or determining whether a genomic C/SKARP-1 gene has been mutated or deleted.

[0942] A nucleic acid fragment encoding a “biologically active portion of a C/SKARP-1 protein” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, which encodes a polypeptide having a C/SKARP-1 biological activity (the biological activities of the C/SKARP-1 proteins are described herein), expressing the encoded portion of the C/SKARP-1 protein (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the C/SKARP-1 protein. In an exemplary embodiment, the nucleic acid molecule is at least 50-100, 100-250, 250-500, 500-700, 750-1000, 1000-1250, 1250-1500, 1500-1700 or more nucleotides in length and encodes a protein having a GPCR52871 activity (as described herein).

[0943] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, due to degeneracy of the genetic code and thus encode the same C/SKARP-1 proteins as those encoded by the nucleotide sequence shown in SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence which differs by at least 1, but no greater than 5, 10, 20, 50 or 100 amino acid residues from the amino acid sequence shown in SEQ ID NO:15, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number ______. In yet another embodiment, the nucleic acid molecule encodes the amino acid sequence of human GPCR52871. If an alignment is needed for this comparison, the sequences should be aligned for maximum homology.

[0944] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologues (different locus), and orthologues (different organism) or can be non naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[0945] Allelic variants result, for example, from DNA sequence polymorphisms within a population (e.g., the human population) that lead to changes in the amino acid sequences of the C/SKARP-1 proteins. Such genetic polymorphism in the C/SKARP-1 genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a C/SKARP-1 protein, preferably a mammalian C/SKARP-1 protein, and can further include non-coding regulatory sequences, and introns.

[0946] Accordingly, in one embodiment, the invention features isolated nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:15, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:14 or 16, for example, under stringent hybridization conditions.

[0947] Allelic variants of human C/SKARP-1 include both functional and non-functional C/SKARP-1 proteins. Functional allelic variants are naturally occurring amino acid sequence variants of the human C/SKARP-1 protein that maintain the ability to bind a C/SKARP-1 ligand and/or modulate cellular mechanisms associated with cell growth or differentiation. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:15 or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein.

[0948] Non-functional allelic variants are naturally occurring amino acid sequence variants of the human C/SKARP-1 protein that do not have the ability to either bind a C/SKARP-1 ligand and/or modulate cellular mechanisms associated with cell growth or differentiation. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:15 or a substitution, insertion or deletion in critical residues or critical regions.

[0949] The present invention further provides non-human orthologues (e.g., non-human orthologues of the human C/SKARP-1 protein). Orthologues of the human C/SKARP-1 protein are proteins that are isolated from non-human organisms and possess the same C/SKARP-1 ligand binding and/or modulation of cellular mechanisms associated with cell growth or differentiation of the human C/SKARP-1 protein. Orthologues of the human C/SKARP-1 protein can readily be identified as comprising an amino acid sequence that is substantially homologous to SEQ ID NO:15.

[0950] Moreover, nucleic acid molecules encoding other C/SKARP-1 family members and, thus, which have a nucleotide sequence which differs from the C/SKARP-1 sequences of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ are intended to be within the scope of the invention. For example, another C/SKARP-1 cDNA can be identified based on the nucleotide sequence of human C/SKARP-1. Moreover, nucleic acid molecules encoding C/SKARP-1 proteins from different species, and which, thus, have a nucleotide sequence which differs from the C/SKARP-1 sequences of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ are intended to be within the scope of the invention. For example, a mouse C/SKARP-1 cDNA can be identified based on the nucleotide sequence of a human C/SKARP-1.

[0951] Nucleic acid molecules corresponding to natural allelic variants and homologues of the C/SKARP-1 cDNAs of the invention can be isolated based on their homology to the C/SKARP-1 nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the C/SKARP-1 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the C/SKARP-1 gene.

[0952] Orthologues, homologues and allelic variants can be identified using methods known in the art (e.g., by hybridization to an isolated nucleic acid molecule of the present invention, for example, under stringent hybridization conditions). In one embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In another embodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 450, 467, 500, 550, 600, 650, 700, 750, 800, 850, 900, or 950 nucleotides in length.

[0953] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4×sodium chloride/sodium citrate (SSC), at about 65-70° C. (or alternatively hybridization in 4×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in 1×SSC, at about 65-70° C. (or alternatively hybridization in 1×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 0.3×SSC, at about 65-70° C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide at about 40-45° C.) followed by one or more washes in 2×SSC, at about 50-60° C. Ranges intermediate to the above-recited values, e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassed by the present invention. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.1 5M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log10[Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5M NaH2PO4, 7% SDS at about 65° C., followed by one or more washes at 0.02M NaH2PO4, 1% SDS at 65° C., see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or alternatively 0.2×SSC, 1% SDS).

[0954] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:14 or 16 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0955] In addition to naturally-occurring allelic variants of the C/SKARP-1 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, thereby leading to changes in the amino acid sequence of the encoded C/SKARP-1 proteins, without altering the functional ability of the C/SKARP-1 proteins. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of C/SKARP-1 (e.g., the sequence of SEQ ID NO:15) without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the C/SKARP-1 proteins of the preserit invention, e.g., those present in the ankyrin repeat domain, are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the C/SKARP-1 proteins of the present invention and other ankyrin repeat containing kinases are not likely to be amenable to alteration.

[0956] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding C/SKARP-1 proteins that contain changes in amino acid residues that are not essential for activity. Such C/SKARP-1 proteins differ in amino acid sequence from SEQ ID NO:15, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:15 (e.g., to the entire length of SEQ ID NO:15).

[0957] An isolated nucleic acid molecule encoding a C/SKARP-1 protein homologous to the protein of SEQ ID NO:15 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a C/SKARP-1 protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a C/SKARP-1 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for C/SKARP-1 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, the encoded protein can be expressed recombinantly and the activity of the protein can be determined.

[0958] In a preferred embodiment, a mutant C/SKARP-1 protein can be assayed for the ability to 1) regulate transmission of signals from cellular receptors, e.g., cardiac cell growth factor receptors; 2) modulate the entry of cells, e.g., cardiac precursor cells, into mitosis; 3) modulate cellular differentiation; 4) modulate cell death; and 5) regulate cytoskeleton function, e.g., actin bundling.

[0959] In addition to the nucleic acid molecules encoding C/SKARP-1 proteins described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. In an exemplary embodiment, the invention provides an isolated nucleic acid molecule which is antisense to a C/SKARP-1 nucleic acid molecule (e.g., is antisense to the coding strand of a C/SKARP-1 nucleic acid molecule). An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire C/SKARP-1 coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding C/SKARP-1. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human C/SKARP-1 corresponds to SEQ ID NO:16). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding C/SKARP-1. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[0960] Given the coding strand sequences encoding C/SKARP-1 disclosed herein (e.g., SEQ ID NO:16), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of C/SKARP-1 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of C/SKARP-1 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of C/SKARP-1 mRNA (e.g., between the −10 and +10 regions of the start site of a gene nucleotide sequence). An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[0961] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a C/SKARP-1 protein to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[0962] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[0963] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave C/SKARP-1 mRNA transcripts to thereby inhibit translation of C/SKARP-1 mRNA. A ribozyme having specificity for a C/SKARP-1-encoding nucleic acid can be designed based upon the nucleotide sequence of a C/SKARP-1 cDNA disclosed herein (i.e., SEQ ID NO:14 or 16, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a C/SKARP-1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, C/SKARP-1 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[0964] Alternatively, C/SKARP-1 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the C/SKARP-1 (e.g., the C/SKARP-1 promoter and/or enhancers) to form triple helical structures that prevent transcription of the C/SKARP-1 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

[0965] In yet another embodiment, the C/SKARP-1 nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[0966] PNAs of C/SKARP-1 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of C/SKARP-1 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[0967] In another embodiment, PNAs of C/SKARP-1 can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of C/SKARP-1 nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNase H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P. J. et al. (1 996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

[0968] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/0981 0) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[0969] II. Isolated C/SKARP-1 Proteins and Anti-C/SKARP-1 Antibodies

[0970] One aspect of the invention pertains to isolated or recombinant C/SKARP-1 proteins and polypeptides, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-C/SKARP-1 antibodies. In one embodiment, native C/SKARP-1 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, C/SKARP-1 proteins are produced by recombinant DNA techniques. Alternative to recombinant expression, a C/SKARP-1 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[0971] An “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the C/SKARP-1 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of C/SKARP-1 protein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of C/SKARP-1 protein having less than about 30% (by dry weight) of non-C/SKARP-1 protein (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-C/SKARP-1 protein, still more preferably less than about 10% of non-C/SKARP-1 protein, and most preferably less than about 5% non-C/SKARP-1 protein. When the C/SKARP-1 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0972] The language “substantially free of chemical precursors or other chemicals” includes preparations of C/SKARP-1 protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis of the protein. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of C/SKARP-1 protein having less than about 30% (by dry weight) of chemical precursors or non-C/SKARP-1 chemicals, more preferably less than about 20% chemical precursors or non-C/SKARP-1 chemicals, still more preferably less than about 10% chemical precursors or non-C/SKARP-1 chemicals, and most preferably less than about 5% chemical precursors or non-C/SKARP-1 chemicals.

[0973] As used herein, a “biologically active portion” of a C/SKARP-1 protein includes a fragment of a C/SKARP-1 protein which participates in an interaction between a C/SKARP-1 molecule and a non-C/SKARP-1 molecule. Biologically active portions of a C/SKARP-1 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence of the C/SKARP-1 protein, e.g., the amino acid sequence shown in SEQ ID NO:15, which include less amino acids than the full length C/SKARP-1 proteins, and exhibit at least one activity of a C/SKARP-1 protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the C/SKARP-1 protein, e.g., modulating signaling pathways associated with cellular growth and differentiation. A biologically active portion of a C/SKARP-1 protein can be a polypeptide which is, for example, 10, 25, 50, 100, 200 or more amino acids in length. Biologically active portions of a C/SKARP-1 protein can be used as targets for developing agents which modulate a C/SKARP-1 mediated activity, e.g., the modulation of signaling pathways associated with cellular growth and differentiation.

[0974] In one embodiment, a biologically active portion of a C/SKARP-1 protein comprises at least one ankyrin repeat domain. It is to be understood that a preferred biologically active portion of a C/SKARP-1 protein of the present invention may contain at least one ankyrin repeat domain. Another preferred biologically active portion of a C/SKARP-1 protein may contain at least one, two, three, four, five or six ankyrin repeats. Moreover, other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native C/SKARP-1 protein.

[0975] Another aspect of the invention features fragments of the protein having the amino acid sequence of SEQ ID NO:15, for example, for use as immunogens. In one embodiment, a fragment comprises at least 5 amino acids (e.g., contiguous or consecutive amino acids) of the amino acid sequence of SEQ ID NO:15, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number ______. In another embodiment, a fragment comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids (e.g., contiguous or consecutive amino acids) of the amino acid sequence of SEQ ID NO:15, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number ______.

[0976] In a preferred embodiment, a C/SKARP-1 protein has an amino acid sequence shown in SEQ ID NO:15. In other embodiments, the C/SKARP-1 protein is substantially homologous to SEQ ID NO:15, and retains the functional activity of the protein of SEQ ID NO:15, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. In another embodiment, the C/SKARP-1 protein is a protein which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to SEQ ID NO:15.

[0977] In another embodiment, the invention features a C/SKARP-1 protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:14 or 16, or a complement thereof. This invention further features a C/SKARP-1 protein which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:14 or 16, or a complement thereof.

[0978] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the C/SKARP-1 amino acid sequence of SEQ ID NO:15 having 323 amino acid residues, at least 97, preferably at least 129, more preferably at least 161, even more preferably at least 194, and even more preferably at least 226, 258 or 291 amino acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0979] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting example of parameters to be used in conjunction with the GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frame shift gap penalty of 5.

[0980] In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0 or version 2.U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0981] The nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide arches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to C/SKARP-1 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to C/SKARP-1 protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0982] The invention also provides C/SKARP-1 chimeric or fusion proteins. As used herein, a C/SKARP-1 “chimeric protein” or “fusion protein” comprises a C/SKARP-1 polypeptide operatively linked to a non-C/SKARP-1 polypeptide. An “C/SKARP-1 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to C/SKARP-1, whereas a “non-C/SKARP-1 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a protein which is not substantially homologous to the C/SKARP-1 protein, e.g., a protein which is different from the C/SKARP-1 protein and which is derived from the same or a different organism. Within a C/SKARP-1 fusion protein the C/SKARP-1 polypeptide can correspond to all or a portion of a C/SKARP-1 protein. In a preferred embodiment, a C/SKARP-1 fusion protein comprises at least one biologically active portion of a C/SKARP-1 protein. In another preferred embodiment, a C/SKARP-1 fusion protein comprises at least two biologically active portions of a C/SKARP-1 protein. Within the fusion protein, the term “operatively linked” is intended to indicate that the C/SKARP-1 polypeptide and the non-C/SKARP-1 polypeptide are fused in-frame to each other. The non-C/SKARP-1 polypeptide can be fused to the N-terminus or C-terminus of the C/SKARP-1 polypeptide.

[0983] For example, in one embodiment, the fusion protein is a GST-C/SKARP-1 fusion protein in which the C/SKARP-1 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant C/SKARP-1.

[0984] In another embodiment, the fusion protein is a C/SKARP-1 protein containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of C/SKARP-1 can be increased through use of a heterologous signal sequence.

[0985] The C/SKARP-1 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The C/SKARP-1 fusion proteins can be used to affect the bioavailability of a C/SKARP-1 substrate. Use of C/SKARP-1 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a C/SKARP-1 protein; (ii) mis-regulation of the C/SKARP-1 gene; and (iii) aberrant post-translational modification of a C/SKARP-1 protein.

[0986] Moreover, the C/SKARP-1-fusion proteins of the invention can be used as immunogens to produce anti-C/SKARP-1 antibodies in a subject, to purify C/SKARP-1 ligands and in screening assays to identify molecules which inhibit the interaction of C/SKARP-1 with a C/SKARP-1 substrate.

[0987] Preferably, a C/SKARP-1 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragrnents coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A C/SKARP-1-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the C/SKARP-1 protein.

[0988] The present invention also pertains to variants of the C/SKARP-1 proteins which function as either C/SKARP-1 agonists (mimetics) or as C/SKARP-1 antagonists. Variants of the C/SKARP-1 proteins can be generated by mutagenesis, e.g., discrete point mutation or truncation of a C/SKARP-1 protein. An agonist of the C/SKARP-1 proteins can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a C/SKARP-1 protein. An antagonist of a C/SKARP-1 protein can inhibit one or more of the activities of the naturally occurring form of the C/SKARP-1 protein by, for example, competitively modulating a C/SKARP-1-mediated activity of a C/SKARP-1 protein. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the protein has fewer side effects in a subject relative to treatment with the naturally occurring form of the C/SKARP-1 protein.

[0989] In one embodiment, variants of a C/SKARP-1 protein which function as either C/SKARP-1 agonists (mimetics) or as C/SKARP-1 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a C/SKARP-1 protein for C/SKARP-1 protein agonist or antagonist activity. In one embodiment, a variegated library of C/SKARP-1 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of C/SKARP-1 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential C/SKARP-1 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of C/SKARP-1 sequences therein. There are a variety of methods which can be used to produce libraries of potential C/SKARP-1 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential C/SKARP-1 sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[0990] In addition, libraries of fragments of a C/SKARP-1 protein coding sequence can be used to generate a variegated population of C/SKARP-1 fragments for screening and subsequent selection of variants of a C/SKARP-1 protein. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a C/SKARP-1 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the C/SKARP-1 protein.

[0991] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of C/SKARP-1 proteins. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify C/SKARP-1 variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering 6(3):327-331).

[0992] In one embodiment, cell based assays can be exploited to analyze a variegated C/SKARP-1 library. For example, a library of expression vectors can be transfected into a cell line, e.g., a cardiac cell line, which ordinarily responds to a particular ligand in a C/SKARP-1-dependent manner. The transfected cells are then contacted with the ligand and the effect of expression of the mutant on signaling by the ligand can be detected, e.g., by monitoring intracellular calcium, IP3, or diacylglycerol concentration, phosphorylation profile of intracellular proteins, cell proliferation and/or migration, or the activity of a C/SKARP-1-regulated transcription factor. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the ligand, and the individual clones further characterized.

[0993] An isolated C/SKARP-1 protein, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind C/SKARP-1 using standard techniques for polyclonal and monoclonal antibody preparation. A full-length C/SKARP-1 protein can be used or, alternatively, the invention provides antigenic peptide fragments of C/SKARP-1 for use as immunogens. The antigenic peptide of C/SKARP-1 comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:15 and encompasses an epitope of C/SKARP-1 such that an antibody raised against the peptide forms a specific immune complex with C/SKARP-1. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[0994] Preferred epitopes encompassed by the antigenic peptide are regions of C/SKARP-1 that are located on the surface of the protein, e.g., hydrophilic regions, as well as regions with high antigenicity (see, for example, FIG. 23).

[0995] A C/SKARP-1 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed C/SKARP-1 protein or a chemically synthesized C/SKARP-1 polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic C/SKARP-1 preparation induces a polyclonal anti-C/SKARP-1 antibody response.

[0996] Accordingly, another aspect of the invention pertains to anti-C/SKARP-1 antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as C/SKARP-1. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind C/SKARP-1. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of C/SKARP-1. A monoclonal antibody composition thus typically displays a single binding affinity for a particular C/SKARP-1 protein with which it immunoreacts.

[0997] Polyclonal anti-C/SKARP-1 antibodies can be prepared as described above by immunizing a suitable subject with a C/SKARP-1 immunogen. The anti-C/SKARP-1 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized C/SKARP-1. If desired, the antibody molecules directed against C/SKARP-1 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-C/SKARP-1 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1 975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a C/SKARP-1 immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds C/SKARP-1.

[0998] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-C/SKARP-1 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind C/SKARP-1, e.g., using a standard ELISA assay.

[0999] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-C/SKARP-1 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with C/SKARP-1 to thereby isolate immunoglobulin library members that bind C/SKARP-1. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[1000] Additionally, recombinant anti-C/SKARP-1 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[1001] An anti-C/SKARP-1 antibody (e.g., monoclonal antibody) can be used to isolate C/SKARP-1 by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-C/SKARP-1 antibody can facilitate the purification of natural C/SKARP-1 from cells and of recombinantly produced C/SKARP-1 expressed in host cells. Moreover, an anti-C/SKARP-1 antibody can be used to detect C/SKARP-1 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the C/SKARP-1 protein. Anti-C/SKARP-1 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[1002] III. Recombinant Expression Vectors and Host Cells

[1003] Another aspect of the invention pertains to vectors, for example recombinant expression vectors, containing a C/SKARP-1 nucleic acid molecule or vectors containing a nucleic acid molecule which encodes a C/SKARP-1 protein (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[1004] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., C/SKARP-1 proteins, mutant forms of C/SKARP-1 proteins, fusion proteins, and the like).

[1005] Accordingly, an exemplary embodiment provides a method for producing a protein, preferably a C/SKARP-1 protein, by culturing in a suitable medium a host cell of the invention (e.g., a mammalian host cell such as a non-human mammalian cell) containing a recombinant expression vector, such that the protein is produced.

[1006] The recombinant expression vectors of the invention can be designed for expression of C/SKARP-1 proteins in prokaryotic or eukaryotic cells. For example, C/SKARP-1 proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[1007] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[1008] Purified fusion proteins can be utilized in C/SKARP-1 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for C/SKARP-1 proteins, for example. In a preferred embodiment, a C/SKARP-1 fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

[1009] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[1010] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[1011] In another embodiment, the C/SKARP-1 expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corporation, San Diego, Calif.).

[1012] Alternatively, C/SKARP-1 proteins can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[1013] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[1014] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the □-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[1015] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to C/SKARP-1 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986.

[1016] Another aspect of the invention pertains to host cells into which a C/SKARP-1 nucleic acid molecule of the invention is introduced, e.g., a C/SKARP-1 nucleic acid molecule within a vector (e.g., a recombinant expression vector) or a C/SKARP-1 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[1017] A host cell can be any prokaryotic or eukaryotic cell. For example, a C/SKARP-1 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[1018] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[1019] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a C/SKARP-1 protein or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[1020] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a C/SKARP-1 protein. Accordingly, the invention further provides methods for producing a C/SKARP-1 protein using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a C/SKARP-1 protein has been introduced) in a suitable medium such that a C/SKARP-1 protein is produced. In another embodiment, the method further comprises isolating a C/SKARP-1 protein from the medium or the host cell.

[1021] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which C/SKARP-1-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous C/SKARP-1 sequences have been introduced into their genome or homologous recombinant animals in which endogenous C/SKARP-1 sequences have been altered. Such animals are useful for studying the function and/or activity of a C/SKARP-1 and for identifying and/or evaluating modulators of C/SKARP-1 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous C/SKARP-1 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[1022] A transgenic animal of the invention can be created by introducing a C/SKARP-1-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The C/SKARP-1 cDNA sequence of SEQ ID NO:14 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human C/SKARP-1 gene, such as a mouse or rat C/SKARP-1 gene, can be used as a transgene. Alternatively, a C/SKARP-1 gene homologue, such as another C/SKARP-1 family member, can be isolated based on hybridization to the C/SKARP-1 cDNA sequences of SEQ ID NO:14 or 16, or the DNA insert of the plasmid deposited with ATCC as Accession Number ______ (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a C/SKARP-1 transgene to direct expression of a C/SKARP-1 protein to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a C/SKARP-1 transgene in its genome and/or expression of C/SKARP-1 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a C/SKARP-1 protein can further be bred to other transgenic animals carrying other transgenes.

[1023] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a C/SKARP-1 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the C/SKARP-1 gene. The C/SKARP-1 gene can be a human gene (e.g., the cDNA of SEQ ID NO:16), but more preferably, is a non-human homologue of a human C/SKARP-1 gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:14). For example, a mouse C/SKARP-1 gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous C/SKARP-1 gene in the mouse genome. In a preferred embodiment, the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous C/SKARP-1 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous C/SKARP-1 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous C/SKARP-1 protein). In the homologous recombination nucleic acid molecule, the altered portion of the C/SKARP-1 gene is flanked at its 5′ and 3′ ends by additional nucleic acid sequence of the C/SKARP-1 gene to allow for homologous recombination to occur between the exogenous C/SKARP-1 gene carried by the homologous recombination nucleic acid molecule and an endogenous C/SKARP-1 gene in a cell, e.g., an embryonic stem cell. The additional flanking C/SKARP-1 nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced C/SKARP-1 gene has homologously recombined with the endogenous C/SKARP-1 gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Berns et al.

[1024] In another embodiment, transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[1025] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al. (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter GO phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[1026] IV. Pharmaceutical Compositions

[1027] The C/SKARP-1 nucleic acid molecules, C/SKARP-1 proteins, fragments thereof, anti-C/SKARP-1 antibodies, and C/SKARP-1 modulators (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[1028] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[1029] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[1030] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a C/SKARP-1 protein, nucleic acid molecule, anti-C/SKARP-1 antibody, or C/SKARP-1 modulators) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[1031] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[1032] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[1033] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[1034] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[1035] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[1036] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[1037] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[1038] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[1039] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[1040] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[1041] V. Uses and Methods of the Invention The nucleic acid molecules, proteins, protein homologues, antibodies and modulators described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, a C/SKARP-1 protein of the invention has one or more of the following activities: (i) mediation of specific macromolecular interactions; (ii) mediation of interactions between proteins and/or between regions of a single protein; (iii) formation of binding sites for distinct proteins (e.g., non-C/SKARP proteins); (iv) bridging of cellular components; (v) regulation of gene expression (e.g., cardiac gene expression) and, thus, can be used to, for example, (1) modulate cellular localization (e.g., anchoring C/SKARP binding proteins in a specific cellular localization); (2) modulate development and/or differentiation (e.g., myogenic development and/or differentiation, heart development and/or differentiation); (3) modulate cardiac maturation and/or morphogenesis; (4) as a marker (e.g., an early marker) of cardiac and/or myogenic cell lineage; and (5) modulate and/or treat C/SKARP-1-associated or related disorders.

[1042] As used herein, a “C/SKARP-1-associated or related disorder” includes a disorder, disease, or condition which is caused or characterized by a misregulation (e.g., downregulation or upregulation) of C/SKARP-1 activity. The C/SKARP-1 molecules of the present invention may also act as novel diagnostic targets and therapeutic agents for cardiovascular diseases or disorders. Exemplary C/SKARP-related disorders include, but are not limited to, cardiac hypertrophy, cardiac disorders and/or cardiovascular disease (e.g., congestive heart failure, cardiomyopathy and the like. Additional exemplary C/SKARP-1-associated disorders include, but are not limited to disorders such as arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, ischemic disease, arrhythmia, and cardiovascular developmental disorders (e.g., arteriovenous malformations, arteriovenous fistulae, Raynaud's syndrome, neurogenic thoracic outlet syndrome, causalgia/reflex sympathetic dystrophy, hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrial septal defects, atrioventricular canal, coarctation of the aorta, ebsteins anomaly, hypoplastic left heart syndrome, interruption of the aortic arch, mitral valve prolapse, ductus arteriosus, patent foramen ovale, partial anomalous pulmonary venous return, pulmonary atresia with ventricular septal defect, pulmonary atresia without ventricular septal defect, persistence of the fetal circulation, pulmonary valve stenosis, single ventricle, total anomalous pulmonary venous return, transposition of the great vessels, tricuspid atresia, truncus arteriosus, ventricular septal defects). A cardiovascular disease or disorder also includes an endothelial cell disorder. As used herein, an “endothelial cell disorder” includes a disorder characterized by aberrant, unregulated, or unwanted endothelial cell activity, e.g., proliferation, migration, angiogenesis, or vascularization; or aberrant expression of cell surface adhesion molecules or genes associated with angiogenesis, e.g., TIE-2, FLT and FLK. Endothelial cell disorders include tumorigenesis, tumor metastasis, psoriasis, diabetic retinopathy, endometriosis, Grave's disease, ischemic disease (e.g., atherosclerosis), and chronic inflammatory diseases (e.g., rheumatoid arthritis).

[1043] The isolated nucleic acid molecules of the invention can be used, for example, to express C/SKARP-1 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect C/SKARP-1 mRNA (e.g., in a biological sample) or a genetic alteration in a C/SKARP-1 gene, and to modulate C/SKARP-1 activity, as described further below. The C/SKARP-1 proteins can be used to treat disorders characterized by insufficient or excessive production of a C/SKARP-1 substrate or production of C/SKARP-1 inhibitors. In addition, the C/SKARP-1 proteins can be used to screen for naturally occurring C/SKARP-1 substrates, to screen for drugs or compounds which modulate C/SKARP-1 activity, as well as to treat disorders characterized by insufficient or excessive production of C/SKARP-1 protein or production of C/SKARP-1 protein forms which have decreased, aberrant or unwanted activity compared to C/SKARP-1 wild type protein (e.g., C/SKARP-1-associated disorders). Moreover, the anti-C/SKARP-1 antibodies of the invention can be used to detect and isolate C/SKARP-1 proteins, regulate the bioavailability of C/SKARP-1 proteins, and modulate C/SKARP-1 activity.

[1044] A. Screening Assays:

[1045] The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to C/SKARP-1 proteins, have a stimulatory or inhibitory effect on, for example, C/SKARP-1 expression or C/SKARP-1 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of C/SKARP-1 substrate.

[1046] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a C/SKARP-1 protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a C/SKARP-1 protein or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1 997) Anticancer Drug Des. 12:145).

[1047] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[1048] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

[1049] In one embodiment, an assay is a cell-based assay in which a cell which expresses a C/SKARP-1 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate C/SKARP-1 activity is determined. Determining the ability of the test compound to modulate C/SKARP-1 activity can be accomplished by monitoring, for example, intracellular calcium, IP3, or diacylglycerol concentration, phosphorylation profile of intracellular proteins, cell proliferation and/or migration, or the activity of a C/SKARP-1-regulated transcription factor. The cell, for example, can be of mammalian origin, e.g., a cardiac cell.

[1050] The ability of the test compound to modulate C/SKARP-1 binding to a substrate or to bind to C/SKARP-1 can also be determined. Determining the ability of the test compound to modulate C/SKARP-1 binding to a substrate can be accomplished, for example, by coupling the C/SKARP-1 substrate with a radioisotope or enzymatic label such that binding of the C/SKARP-1 substrate to C/SKARP-1 can be determined by detecting the labeled C/SKARP-1 substrate in a complex. Determining the ability of the test compound to bind C/SKARP-1 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to C/SKARP-1 can be determined by detecting the labeled C/SKARP-1 compound in a complex. For example, compounds (e.g., C/SKARP-1 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[1051] It is also within the scope of this invention to determine the ability of a compound (e.g., a C/SKARP-1 substrate) to interact with C/SKARP-1 without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with C/SKARP-1 without the labeling of either the compound or the C/SKARP-1. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and C/SKARP-1.

[1052] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a C/SKARP-1 target molecule (e.g., a C/SKARP-1 substrate) with a test compound and determining the ability of the test compound to modulate (e.g. stimulate or inhibit) the activity of the C/SKARP-1 target molecule. Determining the ability of the test compound to modulate the activity of a C/SKARP-1 target molecule can be accomplished, for example, by determining the ability of the C/SKARP-1 protein to bind to or interact with the C/SKARP-1 target molecule.

[1053] Determining the ability of the C/SKARP-1 protein or a biologically active fragment thereof, to bind to or interact with a C/SKARP-1 target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the C/SKARP-1 protein to bind to or interact with a C/SKARP-1 target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e., intracellular Ca2+, diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response.

[1054] In yet another embodiment, an assay of the present invention is a cell-free assay in which a C/SKARP-1 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the C/SKARP-1 protein or biologically active portion thereof is determined. Preferred biologically active portions of the C/SKARP-1 proteins to be used in assays of the present invention include fragments which participate in interactions with non-C/SKARP-1 molecules, e.g., fragments with high surface probability scores (see, for example, FIG. 23). Binding of the test compound to the C/SKARP-1 protein can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the C/SKARP-1 protein or biologically active portion thereof with a known compound which binds C/SKARP-1 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a C/SKARP-1 protein, wherein determining the ability of the test compound to interact with a C/SKARP-1 protein comprises determining the ability of the test compound to preferentially bind to C/SKARP-1 or biologically active portion thereof as compared to the known compound.

[1055] In another embodiment, the assay is a cell-free assay in which a C/SKARP-1 protein or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the C/SKARP-1 protein or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a C/SKARP-1 protein can be accomplished, for example, by determining the ability of the C/SKARP-1 protein to bind to a C/SKARP-1 target molecule by one of the methods described above for determining direct binding. Determining the ability of the C/SKARP-1 protein to bind to a C/SKARP-1 target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[1056] In an alternative embodiment, determining the ability of the test compound to modulate the activity of a C/SKARP-1 protein can be accomplished by determining the ability of the C/SKARP-1 protein to further modulate the activity of a downstream effector of a C/SKARP-1 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.

[1057] In yet another embodiment, the cell-free assay involves contacting a C/SKARP-1 protein or biologically active portion thereof with a known compound which binds the C/SKARP-1 protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the C/SKARP-1 protein, wherein determining the ability of the test compound to interact with the C/SKARP-1 protein comprises determining the ability of the C/SKARP-1 protein to preferentially bind to or modulate the activity of a C/SKARP-1 target molecule.

[1058] The cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g., C/SKARP-1 proteins or biologically active portions thereof ). In the case of cell-free assays in which a membrane-bound form of an isolated protein is used it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the isolated protein is maintained in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100, Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)n, 3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

[1059] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either C/SKARP-1 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a C/SKARP-1 protein, or interaction of a C/SKARP-1 protein with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/C/SKARP-1 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or C/SKARP-1 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of C/SKARP-1 binding or activity determined using standard techniques.

[1060] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a C/SKARP-1 protein or a C/SKARP-1 target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated C/SKARP-1 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with C/SKARP-1 protein or target molecules but which do not interfere with binding of the C/SKARP-1 protein to its target molecule can be derivatized to the wells of the plate, and unbound target or C/SKARP-1 protein trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the C/SKARP-1 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the C/SKARP-1 protein or target molecule.

[1061] In another embodiment, modulators of C/SKARP-1 expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of C/SKARP-1 mRNA or protein in the cell is determined. The level of expression of C/SKARP-1 mRNA or protein in the presence of the candidate compound is compared to the level of expression of C/SKARP-1 mRNA or protein in the absence of the candidate compound. The candidate compound can then be identified as a modulator of C/SKARP-1 expression based on this comparison. For example, when expression of C/SKARP-1 mRNA or protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of C/SKARP-1 mRNA or protein expression. Alternatively, when expression of C/SKARP-1 mRNA or protein is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of C/SKARP-1 mRNA or protein expression. The level of C/SKARP-1 mRNA or protein expression in the cells can be determined by methods described herein for detecting C/SKARP-1 mRNA or protein.

[1062] In yet another aspect of the invention, the C/SKARP-1 proteins can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent WO94/10300), to identify other proteins, which bind to or interact with C/SKARP-1 (“C/SKARP-1-binding proteins” or “C/SKARP-1-bp”) and are involved in C/SKARP-1 activity. Such C/SKARP-1-binding proteins are also likely to be involved in the propagation of signals by the C/SKARP-1 proteins or C/SKARP-1 targets as, for example, downstream elements of a C/SKARP-1-mediated signaling pathway. Alternatively, such C/SKARP-1-binding proteins are likely to be C/SKARP-1 inhibitors.

[1063] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a C/SKARP-1 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a C/SKARP-1-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the C/SKARP-1 protein.

[1064] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a C/SKARP-1 protein can be confirmed in vivo, e.g., in an animal such as an animal model for a cardiovascular disorder.

[1065] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a C/SKARP-1 modulating agent, an antisense C/SKARP-1 nucleic acid molecule, a C/SKARP-1-specific antibody, or a C/SKARP-1-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[1066] B. Detection Assays

[1067] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[1068] 1. Chromosome Mapping

[1069] Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the C/SKARP-1 nucleotide sequences, described herein, can be used to map the location of the C/SKARP-1 genes on a chromosome. The mapping of the C/SKARP-1 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

[1070] Briefly, C/SKARP-1 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the C/SKARP-1 nucleotide sequences. Computer analysis of the C/SKARP-1 sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the C/SKARP-1 sequences will yield an amplified fragment.

[1071] Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

[1072] PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the C/SKARP-1 nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a C/SKARP-1 sequence to its chromosome include in situ hybridization (described in Fan, Y. et al (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

[1073] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

[1074] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[1075] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[1076] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the C/SKARP-1 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[1077] 2. Tissue Typing

[1078] The C/SKARP-1 sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[1079] Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the C/SKARP-1 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[1080] Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The C/SKARP-1 nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:14 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:16 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[1081] If a panel of reagents from C/SKARP-1 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[1082] 3. Use of Partial C/SKARP-1 Sequences in Forensic Biology

[1083] DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[1084] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:14 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the C/SKARP-1 nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:14 having a length of at least 20 bases, preferably at least 30 bases.

[1085] The C/SKARP-1 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such C/SKARP-1 probes can be used to identify tissue by species and/or by organ type.

[1086] In a similar fashion, these reagents, e.g., C/SKARP-1 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[1087] C. Predictive Medicine:

[1088] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining C/SKARP-1 protein and/or nucleic acid expression as well as C/SKARP-1 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted C/SKARP-1 expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with C/SKARP-1 protein, nucleic acid expression or activity. For example, mutations in a C/SKARP-1 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with C/SKARP-1 protein, nucleic acid expression or activity.

[1089] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of C/SKARP-1 in clinical trials.

[1090] These and other agents are described in further detail in the following sections.

[1091] 1. Diagnostic Assays An exemplary method for detecting the presence or absence of C/SKARP-1 protein, polypeptide or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting C/SKARP-1 protein, polypeptide or nucleic acid (e.g., mRNA, genomic DNA) that encodes C/SKARP-1 protein such that the presence of C/SKARP-1 protein, polypeptide or nucleic acid is detected in the biological sample. In another aspect, the present invention provides a method for detecting the presence of C/SKARP-1 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of C/SKARP-1 activity such that the presence of C/SKARP-1 activity is detected in the biological sample. A preferred agent for detecting C/SKARP-1 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to C/SKARP-1 mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length C/SKARP-1 nucleic acid, such as the nucleic acid of SEQ ID NO:14 or 16, or the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to C/SKARP-1 mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[1092] A preferred agent for detecting C/SKARP-1 protein is an antibody capable of binding to C/SKARP-1 protein, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect C/SKARP-1 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of C/SKARP-1 mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of C/SKARP-1 protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of C/SKARP-1 genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of C/SKARP-1 protein include introducing into a subject a labeled anti-C/SKARP-1 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[1093] The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a C/SKARP-1 protein; (ii) aberrant expression of a gene encoding a C/SKARP-1 protein; (iii) mis-regulation of the gene; and (iv) aberrant post-translational modification of a C/SKARP-1 protein, wherein a wild-type form of the gene encodes a protein with a C/SKARP-1 activity. “Misexpression or aberrant expression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes, but is not limited to, expression at non-wild type levels (e.g., over or under expression); a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed (e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage); a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene (e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus).

[1094] In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.

[1095] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting C/SKARP-1 protein, mRNA, or genomic DNA, such that the presence of C/SKARP-1 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of C/SKARP-1 protein, mRNA or genomic DNA in the control sample with the presence of C/SKARP-1 protein, mRNA or genomic DNA in the test sample.

[1096] The invention also encompasses kits for detecting the presence of C/SKARP-1 in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting C/SKARP-1 protein or mRNA in a biological sample; means for determining the amount of C/SKARP-1 in the sample; and means for comparing the amount of C/SKARP-1 in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect C/SKARP-1 protein or nucleic acid.

[1097] 2. Prognostic Assays The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted C/SKARP-1 expression or activity. As used herein, the term “aberrant” includes a C/SKARP-1 expression or activity which deviates from the wild type C/SKARP-1 expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant C/SKARP-1 expression or activity is intended to include the cases in which a mutation in the C/SKARP-1 gene causes the C/SKARP-1 gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional C/SKARP-1 protein or a protein which does not function in a wild-type fashion, e.g., a protein which does not interact with a C/SKARP-1 ligand or one which interacts with a non-C/SKARP-1 ligand. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response such as proliferation or differentiation. For example, the term unwanted includes a C/SKARP-1 expression or activity which is undesirable in a subject.

[1098] The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in C/SKARP-1 protein activity or nucleic acid expression, such as a cardiovascular disorder. Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in C/SKARP-1 protein activity or nucleic acid expression, such as a cardiovascular disorder. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted C/SKARP-1 expression or activity in which a test sample is obtained from a subject and C/SKARP-1 protein or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of C/SKARP-1 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted C/SKARP-1 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue (e.g., cardiac or skeletal muscle tissue).

[1099] Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted C/SKARP-1 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a cardiovascular disorder. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted C/SKARP-1 expression or activity in which a test sample is obtained and C/SKARP-1 protein or nucleic acid expression or activity is detected (e.g., wherein the abundance of C/SKARP-1 protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted C/SKARP-1 expression or activity).

[1100] The methods of the invention can also be used to detect genetic alterations in a C/SKARP-1 gene, thereby determnining if a subject with the altered gene is at risk for a disorder characterized by misregulation in C/SKARP-1 protein activity or nucleic acid expression, such as a cardiovascular disorder. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a C/SKARP-1-protein, or the mis-expression of the C/SKARP-1 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a C/SKARP-1 gene; 2) an addition of one or more nucleotides to a C/SKARP-1 gene; 3) a substitution of one or more nucleotides of a C/SKARP-1 gene, 4) a chromosomal rearrangement of a C/SKARP-1 gene; 5) an alteration in the level of a messenger RNA transcript of a C/SKARP-1 gene, 6) aberrant modification of a C/SKARP-1 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a C/SKARP-1 gene, 8) a non-wild type level of a C/SKARP-1-protein, 9) allelic loss of a C/SKARP-1 gene, and 10) inappropriate post-translational modification of a C/SKARP-1-protein. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in a C/SKARP-1 gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.

[1101] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in the C/SKARP-1-gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a C/SKARP-1 gene under conditions such that hybridization and amplification of the C/SKARP-1-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[1102] Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[1103] In an alternative embodiment, mutations in a C/SKARP-1 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[1104] In other embodiments, genetic mutations in C/SKARP-1 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in C/SKARP-1 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[1105] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the C/SKARP-1 gene and detect mutations by comparing the sequence of the sample C/SKARP-1 with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

[1106] Other methods for detecting mutations in the C/SKARP-1 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type C/SKARP-1 sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

[1107] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in C/SKARP-1 cDNAs obtained from samples of cells. For example, the muty enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a C/SKARP-1 sequence, e.g., a wild-type C/SKARP-1 sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[1108] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in C/SKARP-1 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control C/SKARP-1 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[1109] In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[1110] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[1111] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[1112] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a C/SKARP-1 gene.

[1113] Furthermore, any cell type or tissue in which C/SKARP-1 is expressed may be utilized in the prognostic assays described herein.

[1114] 3. Monitoring of Effects during Clinical Trials

[1115] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a C/SKARP-1 protein (e.g., the modulation signaling pathways associated with cellular growth and differentiation) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase C/SKARP-1 gene expression, protein levels, or upregulate C/SKARP-1 activity, can be monitored in clinical trials of subjects exhibiting decreased C/SKARP-1 gene expression, protein levels, or downregulated C/SKARP-1 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease C/SKARP-1 gene expression, protein levels, or downregulate C/SKARP-1 activity, can be monitored in clinical trials of subjects exhibiting increased C/SKARP-1 gene expression, protein levels, or upregulated C/SKARP-1 activity. In such clinical trials, the expression or activity of a C/SKARP-1 gene, and preferably, other genes that have been implicated in, for example, a C/SKARP-1-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[1116] For example, and not by way of limitation, genes, including C/SKARP-1, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates C/SKARP-1 activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on C/SKARP-1-associated disorders (e.g., disorders characterized by deregulated cellular growth or differentiation), for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of C/SKARP-1 and other genes implicated in the C/SKARP-1-associated disorder, respectively. The levels of gene expression (e.g., a gene expression pattern) can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one of the methods as described herein, or by measuring the levels of activity of C/SKARP-1 or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.

[1117] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a C/SKARP-1 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the C/SKARP-1 protein, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the C/SKARP-1 protein, mRNA, or genomic DNA in the pre-administration sample with the C/SKARP-1 protein, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of C/SKARP-1 to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of C/SKARP-1 to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, C/SKARP-1 expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.

[1118] D. Methods of Treatment:

[1119] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted C/SKARP-1 expression or activity. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”.) Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the C/SKARP-1 molecules of the present invention or C/SKARP-1 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[1120] Treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

[1121] A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[1122] 1. Prophylactic Methods

[1123] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted C/SKARP-1 expression or activity, by administering to the subject a C/SKARP-1 or an agent which modulates C/SKARP-1 expression or at least one C/SKARP-1 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted C/SKARP-1 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the C/SKARP-1 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of C/SKARP-1 aberrancy, for example, a C/SKARP-1, C/SKARP-1 agonist or C/SKARP-1 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[1124] 2. Therapeutic Methods

[1125] Another aspect of the invention pertains to methods of modulating C/SKARP-1 expression or activity for therapeutic purposes (e.g., for treating subjects having a cardiovascular disease or disorder, for example, congestive heart failure or cardiomyopathy). Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell capable of expressing C/SKARP-1 with an agent that modulates one or more of the activities of C/SKARP-1 protein activity associated with the cell, such that C/SKARP-1 activity in the cell is modulated. An agent that modulates C/SKARP-1 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a C/SKARP-1 protein (e.g., a C/SKARP-1 substrate), a C/SKARP-1 antibody, a C/SKARP-1 agonist or antagonist, a peptidomimetic of a C/SKARP-1 agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more C/SKARP-1 activities. Examples of such stimulatory agents include active C/SKARP-1 protein and a nucleic acid molecule encoding C/SKARP-1 that has been introduced into the cell. In another embodiment, the agent inhibits one or more C/SKARP-1 activities. Examples of such inhibitory agents include antisense C/SKARP-1 nucleic acid molecules, anti-C/SKARP-1 antibodies, and C/SKARP-1 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a C/SKARP-1 protein or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) C/SKARP-1 expression or activity. In another embodiment, the method involves administering a C/SKARP-1 protein or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted C/SKARP-1 expression or activity.

[1126] Stimulation of C/SKARP-1 activity is desirable in situations in which C/SKARP-1 is abnormally downregulated and/or in which increased C/SKARP-1 activity is likely to have a beneficial effect. For example, stimulation of C/SKARP-1 activity is desirable in situations in which a C/SKARP-1 is downregulated and/or in which increased C/SKARP-1 activity is likely to have a beneficial effect. Likewise, inhibition of C/SKARP-1 activity is desirable in situations in which C/SKARP-1 is abnormally upregulated and/or in which decreased C/SKARP-1 activity is likely to have a beneficial effect.

[1127] 3. Pharmacogenomics

[1128] The C/SKARP-1 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on C/SKARP-1 activity (e.g., C/SKARP-1 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) C/SKARP-1-associated disorders (e.g., cardiovascular disorders) associated with aberrant or unwanted C/SKARP-1 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharnacogenomics studies in determining whether to administer a C/SKARP-1 molecule or C/SKARP-1 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a C/SKARP-1 molecule or C/SKARP-1 modulator.

[1129] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[1130] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[1131] Alternatively, a method termed the “candidate gene approach”, can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drugs target is known (e.g., a C/SKARP-1 protein of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[1132] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

[1133] Alternatively, a method termed the “gene expression profiling”, can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a C/SKARP-1 molecule or C/SKARP-1 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[1134] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a C/SKARP-1 molecule or C/SKARP-1 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[1135] 4. Use of C/SKARP-1 Molecules as Surrogate Markers

[1136] The C/SKARP-1 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the C/SKARP-1 molecules of the invention may be detected, and may be correlated with one or 25 more biological states in vivo. For example, the C/SKARP-1 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[1137] The C/SKARP-1 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharnacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharmacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a C/SKARP-1 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-C/SKARP-1 antibodies may be employed in an immune-based detection system for a C/SKARP-1 protein marker, or C/SKARP-1-specific radiolabeled probes may be used to detect a C/SKARP-1 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharmacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[1138] The C/SKARP-1 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or protein (e.g., C/SKARP-1 protein or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in C/SKARP-1 DNA may correlate C/SKARP-1 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[1139] VI. Electronic Apparatus Readable Media and Arrays

[1140] Electronic apparatus readable media comprising C/SKARP-1 sequence information is also provided. As used herein, “C/SKARP-1 sequence information” refers to any nucleotide and/or amino acid sequence information particular to the C/SKARP-1 molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like. Moreover, information “related to” said C/SKARP-1 sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantitative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon C/SKARP-1 sequence information of the present invention.

[1141] As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

[1142] As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the C/SKARP-1 sequence information.

[1143] A variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium. For example, the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of dataprocessor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the C/SKARP-1 sequence information.

[1144] By providing C/SKARP-1 sequence information in readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

[1145] The present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder, wherein the method comprises the steps of determining C/SKARP-1 sequence information associated with the subject and based on the C/SKARP-1 sequence information, determining whether the subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[1146] The present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a disease associated with a C/SKARP-1 wherein the method comprises the steps of determining C/SKARP-1 sequence information associated with the subject, and based on the C/SKARP-1 sequence information, determining whether the subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. The method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.

[1147] The present invention also provides in a network, a method for determining whether a subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder associated with C/SKARP-1, said method comprising the steps of receiving C/SKARP-1 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to C/SKARP-1 and/or a C/SKARP-1-associated disease or disorder, and based on one or more of the phenotypic information, the C/SKARP-1 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1148] The present invention also provides a business method for determining whether a subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder, said method comprising the steps of receiving information related to C/SKARP-1 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to C/SKARP-1 and/or related to a C/SKARP-1-associated disease or disorder, and based on one or more of the phenotypic information, the C/SKARP-1 information, and the acquired information, determining whether the subject has a C/SKARP-1-associated disease or disorder or a pre-disposition to a C/SKARP-1-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1149] The invention also includes an array comprising a C/SKARP-1 sequence of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be C/SKARP-1. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

[1150] In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[1151] In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a C/SKARP-1-associated disease or disorder, progression of C/SKARP-1-associated disease or disorder, and processes, such a cellular transformation associated with the C/SKARP-1-associated disease or disorder.

[1152] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of C/SKARP-1 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[1153] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including C/SKARP-1) that could serve as a molecular target for diagnosis or therapeutic intervention.

[1154] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.

EXAMPLES

Example 1

[1155] Identification and Characterization of Human C/SKARP-1 cDNA

[1156] In this example, the identification and characterization of the gene encoding human C/SKARP-1 (also referred to as clone Fbh33358) is described.

[1157] Isolation of the Human C/SKARP-1 cDNA The invention is based, at least in part, on the discovery of genes encoding novel members of the acetyltransferase family.

[1158] The nucleotide sequences encoding the human C/SKARP-1 protein is shown in FIGS. 21A-B and is set forth as SEQ ID NO:14. The C/SKARP-1 protein encoded by this nucleic acid comprises about 323 amino acids and has the amino acid sequence shown in FIGS. 21A-B and set forth as SEQ ID NO:15. The C/SKARP-1 coding region (open reading frame) of SEQ ID NO:14 is set forth as SEQ ID NO:16. Clone Fbh33358 comprising the human C/SKARP-1 cDNA was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______, and assigned Accession No. ______.

[1159] Analysis of the Human C/SKARP-1 Molecules

[1160] A search was performed against the HMM database resulting in the identification of six ankyrin repeats (i.e., an ankyrin repeat domain) in the amino acid sequence of human C/SKARP-1 (SEQ ID NO:15) at about residues 64-259 (score: 103.3) of SEQ ID NO:15. Six “ankyrin domains” (“Ank domain”) were identified in the amino acid sequence of C/SKARP-1 (SEQ ID NO:15) at about residues 64-96 (score: 17.3); at about residues 97-129 (score: 24.7); at about residues 130-162 (score: 16.4); at about residues 165-194 (score: 12.0); at about residues 195-227 (score: 20.7); and at about residues 229-259 (score: 27.3).

[1161] C/SKARP-1 also includes potential casein kinase II phosphorylation sites, for example, from about amino acid residues 101-104, 239-242, 263-266, and 272-275 of SEQ ID NO:15. A potential tyrosine kinase phosphorylation site is found, for example, from about amino acid residues 50-56 of SEQ ID NO:15. Potential N-myristoylation sites are found, for example, from about amino acid residues 58-63, 88-93, 108-113, 121-126, and 142-147 of SEQ ID NO:15. Dileucine motifs are found, for example, from about amino acid residues 26-27, 34-35, 78-79, 117-118, 150-151, 182-183,215-216, 246-247, 278-279, and 279-280 of SEQ ID NO:15. A potential signal peptide is found, for example, within the first 70 amino acids (amino acid 1 to amino acid 70), of SEQ ID NO:15

[1162] Further domain motifs were identified by using the amino acid sequence of C/SKARP-1 (SEQ ID NO:15) to search through the ProDom database. Numerous matches against protein domains described as “ankyrin repeat chromosome XV reading frame”, “ankyrin precursor kinase domain signal inhibitor EGF-like”, “ankyrin protein cytoskeleton alternative splicing phosphorylation UNC-44 multigene”, “F22G12.4 protein”, “F34D10.6 protein”, “hypothetical 57.7 kD protein”, “COL-O putative RNA helicase A”, and “mouse BAC library complete BAC-284H12 12P13”, and the like were identified.

[1163] Tissue Distribution of C/SKARP-1 mRNA

[1164] This example describes the tissue distribution of C/SKARP-1 mRNA, as determined by RT-PCR, and as may be determined By Northern blot analysis.

[1165] Various cDNA libraries were analyzed by RT-PCR using a human C/SKARP-specific probe. From this analysis it was determined that C/SKARP-1 mRNA was expressed predominantly in heart libraries, from both normal and congestive heart failure samples. C/SKARP-1 mRNA was found to a lesser extent in melanocytes and esophagus (see FIGS. 22A-D).

[1166] Northern blot hybridizations with the various RNA samples would be performed under standard conditions and washed under stringent conditions, i.e., 0.2×SSC at 65° C. The DNA probe was radioactively labeled with 32P-dCTP (using the Prime-It kit (Stratagene, La Jolla, Calif.) according to the instructions of the supplier). Filters containing human tissue mRNA (MultiTissue Northern I and MultiTissue Northern II from Clontech, Palo Alto, Ccalif.) were probed in ExpressHyb hybridization solution (Clontech) and washed at high stringency according to manufacturer's recommendations.

Example 2

[1167] Expression of Recombinant C/SKARP-1 Protein in Bacterial Cells

[1168] In this example, C/SKARP-1 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, C/SKARP-1 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-C/SKARP-1 fusion protein in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

Example 3

[1169] Expression of Recombinant C/SKARP-1 Protein in Cos Cells

[1170] To express the C/SKARP-1 gene in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire C/SKARP-1 protein and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant protein under the control of the CMV promoter.

[1171] To construct the plasmid, the C/SKARP-1 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the C/SKARP-1 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the C/SKARP-1 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the C/SKARP-1 gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5a, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[1172] COS cells are subsequently transfected with the C/SKARP-1-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of the C/SKARP-1 polypeptide is detected by radiolabeling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[1173] Alternatively, DNA containing the C/SKARP-1 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the C/SKARP-1 polypeptide is detected by radiolabeling and immunoprecipitation using a C/SKARP-1 specific monoclonal antibody.

Example 4

[1174] Tissue Distribution of Human C/SKARP-1 mRNA Using Taqman™ Analysis

[1175] This example describes the tissue distribution of human C/SKARP-1 mRNA in a variety of cells and tissues, as determined using the TaqMan™ procedure. The Taqman™ procedure is a quantitative, reverse transcription PCR-based approach for detecting mRNA. The RT-PCR reaction exploits the 5′ nuclease activity of AmpliTaq GoId™ DNA Polymerase to cleave a TaqMan™ probe during PCR. Briefly, cDNA was generated from the samples of interest, e.g., various human tissue samples, and used as the starting material for PCR amplification. In addition to the 5′ and 3′ gene-specific primers, a gene-specific oligonucleotide probe (complementary to the region being amplified) was included in the reaction (i.e., the Taqman™ probe). The TaqMan™ probe includes the oligonucleotide with a fluorescent reporter dye covalently linked to the 5′ end of the probe (such as FAM (6-carboxyfluorescein), TET (6-carboxy-4,7,2′, 7′-tetrachlorofluorescein), JOE (6-carboxy-4,5-dichloro-2,7-dimethoxyfluorescein), or VIC) and a quencher dye (TAMRA (6-carboxy-N,N,N′,N′-tetramethylrhodamine) at the 3′ end of the probe.

[1176] During the PCR reaction, cleavage of the probe separates the reporter dye and the quencher dye, resulting in increased fluorescence of the reporter. Accumulation of PCR products is detected directly by monitoring the increase in fluorescence of the reporter dye. When the probe is intact, the proximity of the reporter dye to the quencher dye results in suppression of the reporter fluorescence. During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The 5′-3′ nucleolytic activity of the AmpliTaq™ Gold DNA Polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, and polymerization of the strand continues. The 3′ end of the probe is blocked to prevent extension of the probe during PCR. This process occurs in every cycle and does not interfere with the exponential accumulation of product. RNA was prepared using the trizol method and treated with DNase to remove contaminating genomic DNA. cDNA was synthesized using standard techniques. Mock cDNA synthesis in the absence of reverse transcriptase resulted in samples with no detectable PCR amplification of the control gene confirms efficient removal of genomic DNA contamination.

[1177] Strong expression of C/SKARP-1 mRNA was detected in normal skeletal muscle tissue (set forth in Table 1). In addition, C/SKARP-1 expression was elevated in chronic heart failure tissue as compared with normal heart tissue.

2TABLE 1Human C/SKARP-1 Taqman DataTissue TypeMeanβ 2 MeanCtExpressionArtery normal39.6222.1617.470Aorta diseased35.3222.213.120Vein normal4020.0819.920Coronary SMC4020.5219.480HUVEC38.4320.9417.490Hemangioma36.0519.4816.570Heart normal25.6120.55.1129.0564Heart CHF25.0620.794.2751.8325Kidney38.119.5618.550Skeletal Muscle25.6321.574.0560.1622Adipose normal39.6320.5119.110Pancreas3721.7815.220primary osteoblasts4020.219.80Osteoclasts (diff)39.517.322.20Skin normal35.9422.0413.90Spinal cord normal39.8920.7719.120Brain Cortex normal38.0821.6616.410Brain Hypothalamus normal39.6722.2517.420Nerve29.7321.638.113.6195DRG (Dorsal Root Ganglion)38.4521.317.160Breast normal39.7220.6119.10Breast tumor37.3220.4716.850Ovary normal39.2219.4519.770Ovary Tumor38.8418.4320.410Prostate Normal39.0319.2119.820Prostate Tumor39.7319.9519.790Salivary glands38.8419.2419.60Colon normal37.7918.5219.270ColonTumor32.6321.1611.480.3513Lung normal35.818.0817.720Lung tumor29.5320.319.221.6769Lung COPD36.1618.2517.910Colon IBD37.6217.4120.210Liver normal39.8919.8220.070Liver fibrosis38.0420.4417.60Spleen normal37.4718.2719.20Tonsil normal35.0818.2716.810Lymph node normal38.4119.7918.630Small intestine normal39.9519.6620.30Macrophages4016.8423.160Synovium4019.5520.450BM-MNC4018.521.50Activated PBMC39.7817.5922.180Neutrophils4017.7822.220Megakaryocytes4018.2521.750Erythroid38.5920.4518.150positive control30.0120.189.841.0949

[1178] V. 32529, A Novel Human Guanine Nucleotide Exchange Factor Family Member and Uses Thereof

Background of the Invention

[1179] The G protein superfamily (e.g., heterotrimeric and small G proteins) encompasses a diverse array of proteins which regulate a complex range of biological processes, including the regulation of protein synthesis, cellular trafficking (e.g., vesicular and nuclear transport), regulation of the cell cycle, growth, differentiation, apoptosis, and cytoskeletal rearrangements (Cerione et al. (1996) Curr. Op. Cell Biol. 8:216-222; Cherfils et al. (1999) Trends Biochem. Sci. 24:306-311). The common motif among this important family of proteins is the presence of a GTP-binding domain (Alberts et al. (1994) Molecular Biology of the Cell, Garland Publishing, Inc., New York, N.Y. pp. 206-207, 641). These proteins act as molecular switches that can cycle between active (GTP-bound) and inactive (GDP-bound) states (Boume et al. (1990) Nature, 348:125-132). In the active state, G proteins are able to interact with a broad range of effector molecules. These effector molecules constitute components of a variety of signaling cascades. Upon hydrolysis of bound GTP, the G protein switches to the inactive state, a step that is facilitated by GTPase activating proteins (GAPs) (Scheffzek et al. (1998) Trends Biochem Sci. 23:257-262; Gamblin and Smerdon (1998) Curr. Opinion in Struct. Biol. 8:195-201).

[1180] Activation of G proteins is mediated by the exchange of GDP for GTP. Dissociation of GDP from the inactive small G protein is facilitated by a class of proteins known as guanine nucleotide exchange factors (GEFs). The small G protein is then able to bind GTP and undergo conformational changes which allow it to interact with effector molecules.

[1181] GEFs consist of four families based on sequence similarity among family members and on selectivity of small G protein activated by the GEF, including GEFs of Ran, ARF, Ras, and Rho (also known as the Dbl homology (DH) domain-containing GEFs). GEF family members all contain a GEF homology domain amino terminal to a pleckstrin homology (PH) domain, and most contain other functional domains commonly found in signaling molecules (Cerione et al. and Cherfils et al., supra). For example, the GEF family members Dbs and Vav both have Src homology (SH3) domains at their carboxyl termini (Whitehead et al. (1995) Oncogene 10:713-721).

[1182] Many GEF family members have been identified to date including Dbl, Ost, Tiam-1, Ect-2, Vav, Lbc, FGD1, Dbs, Lfc, Tim, Brc, Abr, Sos, and Ras GEF. These proteins are found in various tissues including adrenal gland, brain, gonad, heart, keratinocyte, kidney, liver, lung, mammary epithelial, myeloid, pancreas, placenta, spleen, skeletal muscle, testis, and fetal brain and heart, and in diffuse B-cell lymphomas, osteosarcomas, T-lymphoma cells, and myeloid leukemias. The Sos protein is ubiquitous (Cerione et al., supra).

[1183] It is the regulated cycling between active and inactive states of G proteins that allows for proper transduction of many vital cellular signals. Indeed, the regulation of GTP/GDP levels in the cell by small G proteins and their accessory GEF molecules, has been implicated in a number of diseases, including oncogenesis and metastasis, faciogenital dysplasia, chronic myelogenous, and leukemia (Cerione et al., supra).

Summary of the Invention

[1184] The present invention is based, at least in part, on the discovery of novel guanine nucleotide exchange factor family members, referred to herein as “guanine nucleotide exchange factor-32529” or “GEF32529” nucleic acid and polypeptide molecules. The GEF32529 nucleic acid and polypeptide molecules of the present invention are useful as modulating agents in regulating a variety of cellular processes, e.g., cell signaling, tumor inhibition (e.g., growth, differentiation, and apoptosis), cytoskeletal organization (e.g., cell morphology), and cellular trafficking. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding GEF32529 polypeptides or biologically active portions thereof, as well as nucleic acid fragments suitable as primers or hybridization probes for the detection of GEF32529-encoding nucleic acids.

[1185] In one embodiment, the invention features an isolated nucleic acid molecule that includes the nucleotide sequence set forth in SEQ ID NO:17 or 19. In another embodiment, the invention features an isolated nucleic acid molecule that encodes a polypeptide including the amino acid sequence set forth in SEQ ID NO:18. In another embodiment, the invention features an isolated nucleic acid molecule that includes the nucleotide sequence contained in the plasmid deposited with ATCC® as Accession Number ______.

[1186] In still other embodiments, the invention features isolated nucleic acid molecules including nucleotide sequences that are substantially identical (e.g., 60% identical) to the nucleotide sequence set forth as SEQ ID NO:17 or 19. The invention further features isolated nucleic acid molecules including at least 30 contiguous nucleotides of the nucleotide sequence set forth as SEQ ID NO:17 or 19. In another embodiment, the invention features isolated nucleic acid molecules which encode a polypeptide including an amino acid sequence that is substantially identical (e.g., 60% identical) to the amino acid sequence set forth as SEQ ID NO:18. The present invention also features nucleic acid molecules which encode allelic variants of the polypeptide having the amino acid sequence set forth as SEQ ID NO:18. In addition to isolated nucleic acid molecules encoding full-length polypeptides, the present invention also features nucleic acid molecules which encode fragments, for example, biologically active or antigenic fragments, of the full-length polypeptides of the present invention (e.g., fragments including at least 10 contiguous amino acid residues of the amino acid sequence of SEQ ID NO:18). In still other embodiments, the invention features nucleic acid molecules that are complementary to, antisense to, or hybridize under stringent conditions to the isolated nucleic acid molecules described herein.

[1187] In a related aspect, the invention provides vectors including the isolated nucleic acid molecules described herein (e.g., GEF32529-encoding nucleic acid molecules). Such vectors can optionally include nucleotide sequences encoding heterologous polypeptides. Also featured are host cells including such vectors (e.g., host cells including vectors suitable for producing GEF32529 nucleic acid molecules and polypeptides).

[1188] In another aspect, the invention features isolated GEF32529 polypeptides and/or biologically active or antigenic fragments thereof. Exemplary embodiments feature a polypeptide including the amino acid sequence set forth as SEQ ID NO:18, a polypeptide including an amino acid sequence at least 60% identical to the amino acid sequence set forth as SEQ ID NO:18, a polypeptide encoded by a nucleic acid molecule including a nucleotide sequence at least 60% identical to the nucleotide sequence set forth as SEQ ID NO:17 or 19. Also featured are fragments of the full-length polypeptides described herein (e.g., fragments including at least 10 contiguous amino acid residues of the sequence set forth as SEQ ID NO:18) as well as allelic variants of the polypeptide having the amino acid sequence set forth as SEQ ID NO:18.

[1189] The GEF32529 polypeptides and/or biologically active or antigenic fragments thereof, are useful, for example, as reagents or targets in assays applicable to treatment and/or diagnosis of GEF32529 mediated or related disorders. In one embodiment, a GEF32529 polypeptide or fragment thereof, has a GEF32529 activity. In another embodiment, a GEF32529 polypeptide or fragment thereof, has a GEF domain, a signal sequence, and optionally, has a GEF32529 activity. In a related aspect, the invention features antibodies (e.g., antibodies which specifically bind to any one of the polypeptides described herein) as well as fusion polypeptides including all or a fragment of a polypeptide described herein.

[1190] The present invention further features methods for detecting GEF32529 polypeptides and/or GEF32529 nucleic acid molecules, such methods featuring, for example, a probe, primer or antibody described herein. Also featured are kits for the detection of GEF32529 polypeptides and/or GEF32529 nucleic acid molecules. In a related aspect, the invention features methods for identifying compounds which bind to and/or modulate the activity of a GEF32529 polypeptide or GEF32529 nucleic acid molecule described herein. Further featured are methods for modulating a GEF32529 activity.

[1191] Other features and advantages of the invention will be apparent from the following detailed description and claims.

DETAILED DESCRIPTION OF THE INVENTION

[1192] The present invention is based, at least in part, on the discovery of novel molecules, referred to herein as “guanine nucleotide exchange factor-32529” or “GEF32529” nucleic acid and polypeptide molecules, which are novel members of the guanine nucleotide exchange factor family (e.g., the RhoGEF family). These novel molecules are capable of, for example, modulating small G protein mediated activity (e.g., dissociating GDP from a small G protein, for example a Rho/Rac-mediated activity) in a cell, e.g., an adrenal gland, brain, gonad, heart, keratinocyte, kidney, liver, lung, mammary epithelial, myeloid, pancreas, placenta, spleen, skeletal muscle, testis, fetal brain and heart, diffuse B-cell lymphoma, osteosarcoma, T-lymphoma cell, or myeloid leukemia cell. These novel molecules thus, may play a role in or function in a variety of cellular processes, e.g., regulating signal transduction, regulating tumor inhibition, regulating cytoskeletal organization, and/or regulating cellular trafficking. Thus, the GEF32529 molecules of the present invention provide novel diagnostic targets and therapeutic agents to control GEF associated disorders.

[1193] As used herein, the term “GEF associated disorder” or “RhoGEF associated disorder” or “Rho/RacGEF associated disorder” includes disorders, diseases, or conditions which are characterized by aberrant, e.g., upregulated or downregulated, GDP dissociation from small G proteins. Examples of such disorders include cancer, inflammation, diabetes, and pathogenic invasion of host cells. Other examples of GEF associated disorders are described herein.

[1194] The term “family” when referring to the polypeptide and nucleic acid molecules of the invention is intended to mean two or more polypeptides or nucleic acid molecules having a common structural domain or motif and having sufficient amino acid or nucleotide sequence homology as defined herein. Such family members can be naturally or non-naturally occurring and can be from either the same or different species. For example, a family can contain a first polypeptide of human origin, as well as other, distinct polypeptides of human origin or alternatively, can contain homologues of non-human origin, e.g., monkey polypeptides. Members of a family may also have common functional characteristics.

[1195] In one embodiment, a GEF32529 molecule of the present invention is identified based on the presence of at least one “GEF domain” or “RhoGEF domain” or “Rho/RacGEF domain” As used herein, the term “GEF domain” or “RhoGEF domain” or “Rho/RacGEF domain” includes a protein domain having at least about 80-220 amino acid residues and a bit score of at least 15 when compared against a GEF Hidden Markov Model (HMM in PFAM). Preferably, a GEF domain or RhoGEF domain or Rho/RacGEF domain includes a polypeptide having an amino acid sequence of about 100-200, 110-190, 120-180, or more preferably, about 179 amino acid residues and a bit score of at least 20, 30, 40, 50, or more preferably, 64.5. To identify the presence of a GEF domain in a GEF32529 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the PFAM HMM database). A GEF domain HMM (referred to also as RhoGEF) has been assigned the PFAM Accession PF00621 (http://genome.wustl.edu/Pfam/html). A search was performed against the HMM database resulting in the identification of a GEF domain in the amino acid sequence of human GEF32529 (SEQ ID NO:18) at about residues 380-559 of SEQ ID NO:18. The results of the search are set forth in FIGS. 26A-C.

[1196] Preferably a “GEF domain” or “RhoGEF domain” or “Rho/RacGEF domain” has a guanine nucleotide exchange or release activity. Accordingly, identifying the presence of a “GEF domain” or “RhoGEF domain” or “Rho/RacGEF domain” can include isolating a fragment of a GEF32529 molecule (e.g., a GEF32529 polypeptide) and assaying for the ability of the fragment to exchange or release a guanine nucleotide (e.g., GDP) from a guanine nucleotide bound substrate.

[1197] In another embodiment, a GEF32529 molecule of the present invention is identified based on the presence of at least one “PH domain.” As used herein, the term “PH domain” includes a protein domain having at least about 70-170 amino acid residues and a bit score of at least 10 when compared against a PH Hidden Markov Model (HMM in PFAM). Preferably, a PH domain includes a polypeptide having an amino acid sequence of about 50-150, 60-140, 70-130, 80-120, or more preferably, about 111 amino acid residues and a bit score of at least 15, 20, 25, 30, or more preferably, 33. To identify the presence of a PH domain in a GEF32529 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the PFAM HMM database). A PFAM PH domain HMM has been assigned the PFAM Accession PF00169. A search was performed against the PFAM HMM database resulting in the identification of a PH domain in the amino acid sequence of human GEF32529 (SEQ ID NO:18) at about residues 593-704 of SEQ ID NO:18. The results of the search are set forth in FIGS. 26A-C.

[1198] Preferably a “PH domain” has a “PH domain activity,” for example, the ability to bind inositol lipids (e.g., phosphatidylinositol lipids), regulate membrane anchoring (e.g., anchoring of the host protein, i.e., the protein containing the domain, to a cellular membrane), modulate enzymatic activity of the host protein (e.g., modulate the activity of adjacent nucleotide exchange domains), target the host protein to a correct subcellular location, and/or respond to upstream signals. Accordingly, identifying the presence of a “PH domain” can include isolating a fragment of a GEF32529 molecule (e.g., a GEF32529 polypeptide) and assaying for the ability of the fragment to exhibit one of the aforementioned PH domain activities.

[1199] In another embodiment, a GEF32529 molecule of the present invention is identified based on the presence of at least one “SH3 domain.” As used herein, the term “SH3 domain” includes a protein domain having at least about 5-100 amino acid residues and a bit score of at least 10 when compared against an SH3 Hidden Markov Model (HMM in PFAM). Preferably, an SH3 domain includes a polypeptide having an amino acid sequence of about 10-90, 20-80, 30-70, 40-60, or more preferably, about 50 amino acid residues and a bit score of at least 15, 20, 25, 30, or more preferably, 33. To identify the presence of an SH3 domain in a GEF32529 protein, and make the determination that a protein of interest has a particular profile, the amino acid sequence of the protein may be searched against a database of known protein domains (e.g., the PFAM HMM database). A PFAM SH3 domain HMM has been assigned the PFAM Accession PF00018. A search was performed against the HMM database resulting in the identification of an SH3 domain in the amino acid sequence of human GEF32529 (SEQ ID NO:18) at about residues 724-774 of SEQ ID NO:18. The results of the search are set forth in FIGS. 26A-C.

[1200] Preferably an “SH3 domain” has an “SH3 domain activity,” for example, the ability to bind peptides (e.g., proline-rich peptides), regulate signal transduction (e.g., linking signals transmitted from tyrosine kinases at the plasma membrane to effector proteins), and/or modulate cytoskeletal organization (e.g., mediate binding of cytoskeletal proteins to other proteins). Accordingly, identifying the presence of an “SH3 domain” can include isolating a fragment of a GEF32529 molecule (e.g., a GEF32529 polypeptide) and assaying for the ability of the fragment to exhibit one of the aforementioned SH3 domain activities.

[1201] A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28:405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al.(1994) J. Mol. Biol. 235:1501-1531; and Stultz et al.(1993) Protein Sci. 2:305-314, the contents of which are incorporated herein by reference.

[1202] In a preferred embodiment, the GEF32529 molecules of the invention include at least one GEF domain, and/or at least one PH domain, and/or at least one SH3 domain.

[1203] Isolated polypeptides of the present invention, preferably GEF32529 polypeptides, have an amino acid sequence sufficiently identical to the amino acid sequence of SEQ ID NO:18 or are encoded by a nucleotide sequence sufficiently identical to SEQ ID NO:17 or 19. As used herein, the term “sufficiently identical” refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains or motifs and/or a common functional activity. For example, amino acid or nucleotide sequences which share common structural domains having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity across the amino acid sequences of the domains and contain at least one and preferably two structural domains or motifs, are defined herein as sufficiently identical. Furthermore, amino acid or nucleotide sequences which share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homology or identity and share a common functional activity are defined herein as sufficiently identical.

[1204] In a preferred embodiment, a GEF32529 polypeptide includes at least one or more of the following domains: a GEF domain, a PH domain, and/or an SH3 domain, and has an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous or identical to the amino acid sequence of SEQ ID NO:18, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In yet another preferred embodiment, a GEF32529 polypeptide includes at least one or more of the following domains: a GEF domain, a PH domain, and/or an SH3 domain, and is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17 or 19. In another preferred embodiment, a GEF32529 polypeptide includes at least one or more of the following domains: a GEF domain, a PH domain, and/or an SH3 domain, and has a GEF32529 activity.

[1205] As used interchangeably herein, a “GEF32529 activity”, “biological activity of GEF32529” or “functional activity of GEF32529”, refers to an activity exerted by a GEF32529 polypeptide or nucleic acid molecule, for example, in a GEF32529 expressing cell or tissue, or on a GEF32529 target or substrate (e.g., on a GEF32529 binding partner or on a GEF32529 polypeptide, for example, an allosteric activity within the host polypeptide), as determined in vivo, or in vitro, according to standard techniques. In one embodiment, a GEF32529 activity is a direct activity, such as association with or enzymatic modification of a GEF32529-target molecule. As used herein, a “target molecule” or “binding partner” is a molecule with which a GEF32529 polypeptide binds or interacts in nature, such that GEF32529-mediated function is achieved. A GEF32529 target molecule can be a non-GEF32529 molecule or a GEF32529 polypeptide or polypeptide of the present invention. In an exemplary embodiment, a GEF32529 target molecule is a GEF32529 substrate (e.g., a GEF family domain ligand, for example, GDP-bound to a small G protein). Alternatively, a GEF32529 activity is an indirect activity, such as a cellular signaling activity mediated by interaction of the GEF32529 polypeptide with a GEF32529 substrate or binding partner. The biological activities of GEF32529 are described herein. For example, the GEF32529 polypeptides of the present invention can have one or more of the following activities: (1) association with a GEF32529 substrate or binding partner (e.g., a GDP-bound small G protein, for example, a Ras-like or Rho/Rac-like small G protein); (2) dissociation of GDP from a GEF32529 substrate or binding partner (e.g., a GDP-bound small G protein); (3) destabilization of a GDP-bound small G protein; (4) stabilization of a nucleotide-free small G protein, and (5) activation of a GEF32529 substrate or binding partner; (6) modulation of signal transduction (e.g., signal transduction cascades involving small GTP-binding proteins); (7) control of cell morphology; (8) modulation of adhesion and/or motility of cells; (9) mediation of cytoskeletal organization or reorganization; (10) modulation of cellular trafficking (e.g., vesicular transport); and (11) modulation of tumor inhibition.

[1206] Accordingly, another embodiment of the invention features isolated GEF32529 polypeptides and polypeptides having a GEF32529 activity. Preferred polypeptides are GEF32529 polypeptides having at least one or more of the following domains: a GEF domain, a PH domain, and/or an SH3 domain, and, preferably, a GEF32529 activity.

[1207] Additional preferred polypeptides have one or more of the following domains: a GEF domain, a PH domain, and/or an SH3 domain, and are, preferably, encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17 or 19.

[1208] The nucleotide sequence of the isolated human GEF32529 cDNA and the predicted amino acid sequence of the human GEF32529 polypeptide are shown in FIGS. 24A-E and in SEQ ID NOs:17 and 18, respectively. A plasmid containing the nucleotide sequence encoding human GEF32529 was deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______ and assigned Accession Number ______. This deposit will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure. This deposit was made merely as a convenience for those of skill in the art and is not an admission that a deposit is required under 35 U.S.C. §112.

[1209] The human GEF32529 gene, which is approximately 3075 nucleotides in length, encodes a polypeptide which is approximately 802 amino acid residues in length.

[1210] Various aspects of the invention are described in further detail in the following subsections:

[1211] I. Isolated Nucleic Acid Molecules

[1212] One aspect of the invention pertains to isolated nucleic acid molecules that encode GEF32529 polypeptides or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify GEF32529-encoding nucleic acid molecules (e.g., GEF32529 mRNA) and fragments for use as PCR primers for the amplification or mutation of GEF32529 nucleic acid molecules. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

[1213] The term “isolated nucleic acid molecule” includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. For example, with regard to genomic DNA, the term “isolated” includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated GEF32529 nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

[1214] A nucleic acid molecule of the present invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. Using all or a portion of the nucleic acid sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, as a hybridization probe, GEF32529 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[1215] Moreover, a nucleic acid molecule encompassing all or a portion of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[1216] A nucleic acid of the invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to GEF32529 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[1217] In one embodiment, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO:17. The sequence of SEQ ID NO:17 corresponds to the human GEF32529 cDNA. This cDNA comprises sequences encoding the human GEF32529 polypeptide (i.e., “the coding region”, from nucleotides 186-2595) as well as 5′ untranslated sequences (nucleotides 1-185) and 3′ untranslated sequences (nucleotides 2596-3075). Alternatively, the nucleic acid molecule can comprise only the coding region of SEQ ID NO:17 (e.g., nucleotides 186-2595, corresponding to SEQ ID NO:19). Accordingly, in another embodiment, the isolated nucleic acid molecule comprises SEQ ID NO:19 and nucleotides 1-185 and 2596-3075 of SEQ ID NO:17. In yet another embodiment, the nucleic acid molecule consists of the nucleotide sequence set forth as SEQ ID NO:17 or 19.

[1218] In still another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement of the nucleotide sequence shown in SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion of any of these nucleotide sequences. A nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, such that it can hybridize to the nucleotide sequence shown in SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, thereby forming a stable duplex.

[1219] In still another preferred embodiment, an isolated nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to the nucleotide sequence shown in SEQ ID NO:17 or 19 (e.g., to the entire length of the nucleotide sequence), or to the nucleotide sequence (e.g., the entire length of the nucleotide sequence) of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or to a portion or complement of any of these nucleotide sequences. In one embodiment, a nucleic acid molecule of the present invention comprises a nucleotide sequence which is at least (or no greater than) 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-1250, 1250-1500, 1500-1700, 1700-1950, 1950-2200, 2200-2450, 2450-2700, 2700-3000 or more nucleotides in length and hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[1220] Moreover, the nucleic acid molecule of the invention can comprise only a portion of the nucleic acid sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, for example, a fragment which can be used as a probe or primer or a fragment encoding a portion of a GEF32529 polypeptide, e.g., a biologically active portion of a GEF32529 polypeptide. The nucleotide sequence determined from the cloning of the GEF32529 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other GEF32529 family members, as well as GEF32529 homologues from other species. The probe/primer typically comprises substantially purified oligonucleotide. The probe/primer (e.g., oligonucleotide) typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12 or 15, preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, or 100 or more consecutive nucleotides of a sense sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, of an anti-sense sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or of a naturally occurring allelic variant or mutant of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______.

[1221] Exemplary probes or primers are at least (or no greater than) 12 or 15, 20 or 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more nucleotides in length and/or comprise consecutive nucleotides of an isolated nucleic acid molecule described herein. Also included within the scope of the present invention are probes or primers comprising contiguous or consecutive nucleotides of an isolated nucleic acid molecule described herein, but for the difference of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases within the probe or primer sequence. Probes based on the GEF32529 nucleotide sequences can be used to detect (e.g., specifically detect) transcripts or genomic sequences encoding the same or homologous polypeptides. In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor. In another embodiment a set of primers is provided, e.g., primers suitable for use in a PCR, which can be used to amplify a selected region of a GEF32529 sequence, e.g., a domain, region, site or other sequence described herein. The primers should be at least 5, 10, or 50 base pairs in length and less than 100, or less than 200, base pairs in length. The primers should be identical, or differs by no greater than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 bases when compared to a sequence disclosed herein or to the sequence of a naturally occurring variant. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a GEF32529 polypeptide, such as by measuring a level of a GEF32529-encoding nucleic acid in a sample of cells from a subject e.g., detecting GEF32529 mRNA levels or determining whether a genomic GEF32529 gene has been mutated or deleted.

[1222] A nucleic acid fragment encoding a “biologically active portion of a GEF32529 polypeptide” can be prepared by isolating a portion of the nucleotide sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, which encodes a polypeptide having a GEF32529 biological activity (the biological activities of the GEF32529 polypeptides are described herein), expressing the encoded portion of the GEF32529 polypeptide (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of the GEF32529 polypeptide. In an exemplary embodiment, the nucleic acid molecule is at least 50-100, 100-250, 250-500, 500-700, 700-1000, 1000-1250, 1250-1500, 1500-1700, 1700-1950, 1950-2200, 2200-2450, 2450-2700, 2700-3000 or more nucleotides in length and encodes a polypeptide having a GEF32529 activity (as described herein).

[1223] The invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. Such differences can be due to due to degeneracy of the genetic code, thus resulting in a nucleic acid which encodes the same GEF32529 polypeptides as those encoded by the nucleotide sequence shown in SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a polypeptide having an amino acid sequence which differs by at least 1, but no greater than 5, 10, 20, 50 or 100 amino acid residues from the amino acid sequence shown in SEQ ID NO:18, or the amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number ______. In yet another embodiment, the nucleic acid molecule encodes the amino acid sequence of human GEF32529. If an alignment is needed for this comparison, the sequences should be aligned for maximum homology.

[1224] Nucleic acid variants can be naturally occurring, such as allelic variants (same locus), homologues (different locus), and orthologues (different organism) or can be non-naturally occurring. Non-naturally occurring variants can be made by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms. The variants can contain nucleotide substitutions, deletions, inversions and insertions. Variation can occur in either or both the coding and non-coding regions. The variations can produce both conservative and non-conservative amino acid substitutions (as compared in the encoded product).

[1225] Allelic variants result, for example, from DNA sequence polymorphisms within a population (e.g., the human population) that lead to changes in the amino acid sequences of the GEF32529 polypeptides. Such genetic polymorphisms in the GEF32529 genes may exist among individuals within a population due to natural allelic variation. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a GEF32529 polypeptide, preferably a mammalian GEF32529 polypeptide, and can further include non-coding regulatory sequences, and introns.

[1226] Accordingly, in one embodiment, the invention features isolated nucleic acid molecules which encode a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence of SEQ ID NO:18, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with ATCC as Accession Number ______, wherein the nucleic acid molecule hybridizes to a complement of a nucleic acid molecule comprising SEQ ID NO:17 or 19, for example, under stringent hybridization conditions.

[1227] Allelic variants of human GEF32529 include both functional and non-functional GEF32529 polypeptides. Functional allelic variants are naturally occurring amino acid sequence variants of the human GEF32529 polypeptide that maintain the ability to bind a GEF32529 ligand or substrate and/or modulate GDP dissociation or signal transduction. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:18, or substitution, deletion or insertion of non-critical residues in non-critical regions of the polypeptide.

[1228] Non-functional allelic variants are naturally occurring amino acid sequence variants of the human GEF32529 polypeptide that do not have the ability to mediate nucleoside hydrolysis. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO:18, or a substitution, insertion or deletion in critical residues or critical regions.

[1229] The present invention further provides non-human orthologues (e.g., non-human orthologues of the human GEF32529 polypeptide). Orthologues of the human GEF32529 polypeptides are polypeptides that are isolated from non-human organisms and possess the same GEF32529 ligand binding and/or modulation of membrane excitation mechanisms of the human GEF32529 polypeptide. Orthologues of the human GEF32529 polypeptide can readily be identified as comprising an amino acid sequence that is substantially identical to SEQ ID NO:18.

[1230] Moreover, nucleic acid molecules encoding other GEF32529 family members and, thus, which have a nucleotide sequence which differs from the GEF32529 sequences of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ are intended to be within the scope of the invention. For example, another GEF32529 cDNA can be identified based on the nucleotide sequence of human GEF32529. Moreover, nucleic acid molecules encoding GEF32529 polypeptides from different species, and which, thus, have a nucleotide sequence which differs from the GEF32529 sequences of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number are intended to be within the scope of the invention. For example, a mouse GEF32529 cDNA can be identified based on the nucleotide sequence of a human GEF32529.

[1231] Nucleic acid molecules corresponding to natural allelic variants and homologues of the GEF32529 cDNAs of the invention can be isolated based on their homology to the GEF32529 nucleic acids disclosed herein using the cDNAs disclosed herein, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the GEF32529 cDNAs of the invention can further be isolated by mapping to the same chromosome or locus as the GEF32529 gene.

[1232] Orthologues, homologues and allelic variants can be identified using methods known in the art (e.g., by hybridization to an isolated nucleic acid molecule of the present invention, for example, under stringent hybridization conditions). In one embodiment, an isolated nucleic acid molecule of the invention is at least 15, 20, 25, 30 or more nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. In other embodiment, the nucleic acid is at least 100,100-150, 150-200, 200-250, 250-300,300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 900-950, 950-1000, 1000-1050, 1050-1070, 1070-1100, 1100-1150, 1150-1200, 1200-1250, 1250-1300, 1300-1350, 1350-1400, 1400-1450, 1450-1500, 1500-1550, 1550-1600, 1600-1650, 1650-1700, 1700-1950, 1950-2200, 2200-2450, 2450-2700, 2700-3000 or more nucleotides in length.

[1233] As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences that are significantly identical or homologous to each other remain hybridized to each other. Preferably, the conditions are such that sequences at least about 70%, more preferably at least about 80%, even more preferably at least about 85% or 90% identical to each other remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons, Inc. (1995), sections 2, 4 and 6. Additional stringent conditions can be found in Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), chapters 7, 9 and 11. A preferred, non-limiting example of stringent hybridization conditions includes hybridization in 4Xsodium chloride/sodium citrate (SSC), at about 65-70° C. (or hybridization in 4×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 1×SSC, at about 65-70° C. A preferred, non-limiting example of highly stringent hybridization conditions includes hybridization in 1×SSC, at about 65-70° C. (or hybridization in 1×SSC plus 50% formamide at about 42-50° C.) followed by one or more washes in 0.3×SSC, at about 65-70° C. A preferred, non-limiting example of reduced stringency hybridization conditions includes hybridization in 4×SSC, at about 50-60° C. (or alternatively hybridization in 6×SSC plus 50% formamide at about 40-45° C.) followed by one or more washes in 2×SSC, at about 50-60° C. Ranges intermediate to the above-recited values, e.g., at 65-70° C. or at 42-50° C. are also intended to be encompassed by the present invention. SSPE (1×SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes each after hybridization is complete. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and 49 base pairs in length, Tm(° C.)=81.5+16.6(log10[Na+])+0.41 (% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165 M). It will also be recognized by the skilled practitioner that additional reagents may be added to hybridization and/or wash buffers to decrease non-specific hybridization of nucleic acid molecules to membranes, for example, nitrocellulose or nylon membranes, including but not limited to blocking agents (e.g., BSA or salmon or herring sperm carrier DNA), detergents (e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like. When using nylon membranes, in particular, an additional preferred, non-limiting example of stringent hybridization conditions is hybridization in 0.25-0.5 M NaH2PO4, 7% SDS at about 65° C., followed by one or more washes at 0.02 M NaH2PO4, 1% SDS at 65° C., see e.g., Church and Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995, (or, alternatively, 0.2×SSC, 1% SDS).

[1234] Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:17 or 19 and corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural polypeptide).

[1235] In addition to naturally-occurring allelic variants of the GEF32529 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, thereby leading to changes in the amino acid sequence of the encoded GEF32529 polypeptides, without altering the functional ability of the GEF32529 polypeptides. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues can be made in the sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of GEF32529 (e.g., the sequence of SEQ ID NO:18) without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. For example, amino acid residues that are conserved among the GEF32529 polypeptides of the present invention, e.g., those present in a GEF domain, are predicted to be particularly unamenable to alteration. Furthermore, additional amino acid residues that are conserved between the GEF32529 polypeptides of the present invention and other members of the GEF32529 family are not likely to be amenable to alteration.

[1236] Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding GEF32529 polypeptides that contain changes in amino acid residues that are not essential for activity. Such GEF32529 polypeptides differ in amino acid sequence from SEQ ID NO:18, yet retain biological activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:18 (e.g., to the entire length of SEQ ID NO:18).

[1237] An isolated nucleic acid molecule encoding a GEF32529 polypeptide identical to the polypeptide of SEQ ID NO:18, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded polypeptide. Mutations can be introduced into SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______ by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a GEF32529 polypeptide is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a GEF32529 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for GEF32529 biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______, the encoded polypeptide can be expressed recombinantly and the activity of the polypeptide can be determined.

[1238] In a preferred embodiment, a mutant GEF32529 polypeptide can be assayed for the ability to (i) associate with a GEF32529 substrate or binding partner (e.g., a GDP-bound small G protein, for example, a Ras-like or Rho/Rac-like small G protein); (ii) dissociate GDP from a GEF32529 substrate or binding partner (e.g., a GDP-bound small G protein); (iii) destabilize a GDP-bound small G protein; (iv) stabilize a nucleotide-free small G protein, and (v) activate a GEF32529 substrate or binding partner. In another example, a mutant GEF32529 polypeptide can be assayed for the ability to: (1) modulate signal transduction (e.g., signal transduction cascades involving small GTP-binding proteins); (2) control cell morphology; (3) modulate adhesion and/or motility of cells; (4) mediate cytoskeletal organization or reorganization; (5) modulate cellular trafficking (e.g., vesicular transport); and (6) modulate tumor inhibition.

[1239] In addition to the nucleic acid molecules encoding GEF32529 polypeptides described above, another aspect of the invention pertains to isolated nucleic acid molecules which are antisense thereto. In an exemplary embodiment, the invention provides an isolated nucleic acid molecule which is antisense to a GEF32529 nucleic acid molecule (e.g., is antisense to the coding strand of a GEF32529 nucleic acid molecule). An “antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a polypeptide, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire GEF32529 coding strand, or to only a portion thereof. In one embodiment, an antisense nucleic acid molecule is antisense to a “coding region” of the coding strand of a nucleotide sequence encoding GEF32529. The term “coding region” refers to the region of the nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of human GEF32529 corresponds to SEQ ID NO:19). In another embodiment, the antisense nucleic acid molecule is antisense to a “noncoding region” of the coding strand of a nucleotide sequence encoding GEF32529. The term “noncoding region” refers to 5′ and 3′ sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

[1240] Given the coding strand sequences encoding GEF32529 disclosed herein (e.g., SEQ ID NO:19), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of GEF32529 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion of the coding or noncoding region of GEF32529 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of GEF32529 mRNA (e.g., between the −10 and +10 regions of the start site of a gene nucleotide sequence). An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).

[1241] The antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a GEF32529 polypeptide to thereby inhibit expression of the polypeptide, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix. An example of a route of administration of antisense nucleic acid molecules of the invention include direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, for systemic administration, antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.

[1242] In yet another embodiment, the antisense nucleic acid molecule of the invention is an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).

[1243] In still another embodiment, an antisense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave GEF32529 mRNA transcripts to thereby inhibit translation of GEF32529 mRNA. A ribozyme having specificity for a GEF32529-encoding nucleic acid can be designed based upon the nucleotide sequence of a GEF32529 cDNA disclosed herein (i.e., SEQ ID NO:17 or 19, or the nucleotide sequence of the DNA insert of the plasmid deposited with ATCC as Accession Number ______). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a GEF32529-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, GEF32529 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

[1244] Alternatively, GEF32529 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the GEF32529 (e.g., the GEF32529 promoter and/or enhancers) to form triple helical structures that prevent transcription of the GEF32529 gene in target cells. See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36; and Maher, L. J. (1992) Bioassays 14(12):807-15.

[1245] In yet another embodiment, the GEF32529 nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms “peptide nucleic acids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs has been shown to allow for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. Proc. Natl. Acad. Sci. 93: 14670-675.

[1246] PNAs of GEF32529 nucleic acid molecules can be used in therapeutic and diagnostic applications. For example, PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication. PNAs of GEF32529 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as ‘artificial restriction enzymes’ when used in combination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

[1247] In another embodiment, PNAs of GEF32529 can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art. For example, PNA-DNA chimeras of GEF32529 nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes, (e.g., RNase H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P. J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5′ PNA segment and a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively, chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNA segment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).

[1248] In other embodiments, the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCT Publication No. W088/09810) or the blood-brain barrier (see, e.g., PCT Publication No. W089/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).

[1249] Alternatively, the expression characteristics of an endogenous GEF32529 gene within a cell line or microorganism may be modified by inserting a heterologous DNA regulatory element into the genome of a stable cell line or cloned microorganism such that the inserted regulatory element is operatively linked with the endogenous GEF32529 gene. For example, an endogenous GEF32529 gene which is normally “transcriptionally silent”, i.e., a GEF32529 gene which is normally not expressed, or is expressed only at very low levels in a cell line or microorganism, may be activated by inserting a regulatory element which is capable of promoting the expression of a normally expressed gene product in that cell line or microorganism. Alternatively, a transcriptionally silent, endogenous GEF32529 gene may be activated by insertion of a promiscuous regulatory element that works across cell types.

[1250] A heterologous regulatory element may be inserted into a stable cell line or cloned microorganism, such that it is operatively linked with an endogenous GEF32529 gene, using techniques, such as targeted homologous recombination, which are well known to those of skill in the art, and described, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publication No. WO 91/06667, published May 16, 1991.

[1251] II. Isolated GEF32529 Polypeptides and Anti-GEF32529 Antibodies

[1252] One aspect of the invention pertains to isolated GEF32529 or recombinant polypeptides, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-GEF32529 antibodies. In one embodiment, native GEF32529 polypeptides can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, GEF32529 polypeptides are produced by recombinant DNA techniques. Alternative to recombinant expression, a GEF32529 polypeptide or polypeptide can be synthesized chemically using standard peptide synthesis techniques.

[1253] An “isolated” or “purified” polypeptide or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the GEF32529 polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. The language “substantially free of cellular material” includes preparations of GEF32529 polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced. In one embodiment, the language “substantially free of cellular material” includes preparations of GEF32529 polypeptide having less than about 30% (by dry weight) of non-GEF32529 polypeptide (also referred to herein as a “contaminating protein”), more preferably less than about 20% of non-GEF32529 polypeptide, still more preferably less than about 10% of non-GEF32529 polypeptide, and most preferably less than about 5% non-GEF32529 polypeptide. When the GEF32529 polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[1254] The language “substantially free of chemical precursors or other chemicals” includes preparations of GEF32529 polypeptide in which the polypeptide is separated from chemical precursors or other chemicals which are involved in the synthesis of the polypeptide. In one embodiment, the language “substantially free of chemical precursors or other chemicals” includes preparations of GEF32529 polypeptide having less than about 30% (by dry weight) of chemical precursors or non-GEF32529 chemicals, more preferably less than about 20% chemical precursors or non-GEF32529 chemicals, still more preferably less than about 10% chemical precursors or non-GEF32529 chemicals, and most preferably less than about 5% chemical precursors or non-GEF32529 chemicals.

[1255] As used herein, a “biologically active portion” of a GEF32529 polypeptide includes a fragment of a GEF32529 polypeptide which participates in an interaction between a GEF32529 molecule and a non-GEF32529 molecule. Biologically active portions of a GEF32529 polypeptide include peptides comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the GEF32529 polypeptide, e.g., the amino acid sequence shown in SEQ ID NO:18, which include less amino acids than the full length GEF32529 polypeptides, and exhibit at least one activity of a GEF32529 polypeptide. Typically, biologically active portions comprise a domain or motif with at least one activity of the GEF32529 polypeptide, e.g., dissociating GDP from a small G protein. A biologically active portion of a GEF32529 polypeptide can be a polypeptide which is, for example, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775 or 800 or more amino acids in length. Biologically active portions of a GEF32529 polypeptide can be used as targets for developing agents which modulate a GEF32529 mediated activity, e.g., dissociating GDP from a small G protein.

[1256] In one embodiment, a biologically active portion of a GEF32529 polypeptide comprises at least one GEF domain. It is to be understood that a preferred biologically active portion of a GEF32529 polypeptide of the present invention comprises at least one or more of the following domains: a GEF domain, a PH domain, and/or an SH3 domain. Moreover, other biologically active portions, in which other regions of the polypeptide are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native GEF32529 polypeptide.

[1257] Another aspect of the invention features fragments of the polypeptide having the amino acid sequence of SEQ ID NO:18, for example, for use as immunogens. In one embodiment, a fragment comprises at least 5 amino acids (e.g., contiguous or consecutive amino acids) of the amino acid sequence of SEQ ID NO:18, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number _____. In another embodiment, a fragment comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids (e.g., contiguous or consecutive amino acids) of the amino acid sequence of SEQ ID NO:18, or an amino acid sequence encoded by the DNA insert of the plasmid deposited with the ATCC as Accession Number ______.

[1258] In a preferred embodiment, a GEF32529 polypeptide has an amino acid sequence shown in SEQ ID NO:18. In other embodiments, the GEF32529 polypeptide is substantially identical to SEQ ID NO:18, and retains the functional activity of the polypeptide of SEQ ID NO:18, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above. In another embodiment, the GEF32529 polypeptide is a polypeptide which comprises an amino acid sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:18.

[1259] In another embodiment, the invention features a GEF32529 polypeptide which is encoded by a nucleic acid molecule consisting of a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identical to a nucleotide sequence of SEQ ID NO:17 or 19, or a complement thereof. This invention further features a GEF32529 polypeptide which is encoded by a nucleic acid molecule consisting of a nucleotide sequence which hybridizes under stringent hybridization conditions to a complement of a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:17 or 19, or a complement thereof.

[1260] To determine the percent identity of two amino acid sequences or of two nucleic acid 20 sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% of the length of the reference sequence (e.g., when aligning a second sequence to the GEF32529 amino acid sequence of SEQ ID NO:18 having 802 amino acid residues, at least 241, preferably at least 321, more preferably at least 401, more preferably at least 481, even more preferably at least 561, and even more preferably at least 642 or 722 or more amino 30 acid residues are aligned). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[1261] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A preferred, non-limiting example of parameters to be used in conjunction with the GAP program include a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[1262] In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0 or version 2.0U), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[1263] The nucleic acid and polypeptide sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to GEF32529 nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=100, wordlength=3, and a Blosum62 matrix to obtain amino acid sequences homologous to GEF32529 polypeptide molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[1264] The invention also provides GEF32529 chimeric or fusion proteins. As used herein, a GEF32529 “chimeric protein” or “fusion protein” comprises a GEF32529 polypeptide operatively linked to a non-GEF32529 polypeptide. A “GEF32529 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to GEF32529, whereas a “non-GEF32529 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to a polypeptide which is not substantially homologous to the GEF32529 polypeptide, e.g., a polypeptide which is different from the GEF32529 polypeptide and which is derived from the same or a different organism. Within a GEF32529 fusion protein the GEF32529 polypeptide can correspond to all or a portion of a GEF32529 polypeptide. In a preferred embodiment, a GEF32529 fusion protein comprises at least one biologically active portion of a GEF32529 polypeptide. In another preferred embodiment, a GEF32529 fusion protein comprises at least two biologically active portions of a GEF32529 polypeptide. Within the fusion protein, the term “operatively linked” is intended to indicate that the GEF32529 polypeptide and the non-GEF32529 polypeptide are fused in-frame to each other. The non-GEF32529 polypeptide can be fused to the N-terminus or C-terminus of the GEF32529 polypeptide.

[1265] For example, in one embodiment, the fusion protein is a GST-GEF32529 fusion protein in which the GEF32529 sequences are fused to the C-terminus of the GST sequences. Such fusion proteins can facilitate the purification of recombinant GEF32529.

[1266] In another embodiment, the fusion protein is a GEF32529 polypeptide containing a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian host cells), expression and/or secretion of GEF32529 can be increased through the use of a heterologous signal sequence.

[1267] The GEF32529 fusion proteins of the invention can be incorporated into pharmaceutical compositions and administered to a subject in vivo. The GEF32529 fusion proteins can be used to affect the bioavailability of a GEF32529 substrate. Use of GEF32529 fusion proteins may be useful therapeutically for the treatment of disorders caused by, for example, (i) aberrant modification or mutation of a gene encoding a GEF32529 polypeptide; (ii) mis-regulation of the GEF32529 gene; and (iii) aberrant post-translational modification of a GEF32529 polypeptide.

[1268] Moreover, the GEF32529-fusion proteins of the invention can be used as immunogens to produce anti-GEF32529 antibodies in a subject, to purify GEF32529 ligands and in screening assays to identify molecules which inhibit the interaction of GEF32529 with a GEF32529 substrate.

[1269] Preferably, a GEF32529 chimeric or fusion protein of the invention is produced by standard recombinant DNA techniques. For example, DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide). A GEF32529-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the GEF32529 polypeptide.

[1270] The present invention also pertains to variants of the GEF32529 polypeptides which function as either GEF32529 agonists (mimetics) or as GEF32529 antagonists. Variants of the GEF32529 polypeptides can be generated by mutagenesis, e.g., discrete point mutation or truncation of a GEF32529 polypeptide. An agonist of the GEF32529 polypeptides can retain substantially the same, or a subset, of the biological activities of the naturally occurring form of a GEF32529 polypeptide. An antagonist of a GEF32529 polypeptide can inhibit one or more of the activities of the naturally occurring form of the GEF32529 polypeptide by, for example, competitively modulating a GEF32529-mediated activity of a GEF32529 polypeptide. Thus, specific biological effects can be elicited by treatment with a variant of limited function. In one embodiment, treatment of a subject with a variant having a subset of the biological activities of the naturally occurring form of the polypeptide has fewer side effects in a subject relative to treatment with the naturally occurring form of the GEF32529 polypeptide.

[1271] In one embodiment, variants of a GEF32529 polypeptide which function as either GEF32529 agonists (mimetics) or as GEF32529 antagonists can be identified by screening combinatorial libraries of mutants, e.g., truncation mutants, of a GEF32529 polypeptide for GEF32529 polypeptide agonist or antagonist activity. In one embodiment, a variegated library of GEF32529 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library. A variegated library of GEF32529 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential GEF32529 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of GEF32529 sequences therein. There are a variety of methods which can be used to produce libraries of potential GEF32529 variants from a degenerate oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can be performed in an automatic DNA synthesizer, and the synthetic gene then ligated into an appropriate expression vector. Use of a degenerate set of genes allows for the provision, in one mixture, of all of the sequences encoding the desired set of potential GEF32529 sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

[1272] In addition, libraries of fragments of a GEF32529 polypeptide coding sequence can be used to generate a variegated population of GEF32529 fragments for screening and subsequent selection of variants of a GEF32529 polypeptide. In one embodiment, a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a GEF32529 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes of the GEF32529 polypeptide.

[1273] Several techniques are known in the art for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property. Such techniques are adaptable for rapid screening of the gene libraries generated by the combinatorial mutagenesis of GEF32529 polypeptides. The most widely used techniques, which are amenable to high through-put analysis, for screening large gene libraries typically include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the combinatorial genes under conditions in which detection of a desired activity facilitates isolation of the vector encoding the gene whose product was detected. Recursive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify GEF32529 variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering 6(3):327-33 1).

[1274] In one embodiment, cell based assays can be exploited to analyze a variegated GEF32529 library. For example, a library of expression vectors can be transfected into a cell line, e.g., an endothelial cell line, which ordinarily responds to GEF32529 in a particular GEF32529 substrate-dependent manner. The transfected cells are then contacted with GEF32529 and the effect of expression of the mutant on signaling by the GEF32529 substrate can be detected, e.g., by monitoring intracellular GDP concentrations. Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of signaling by the GEF32529 substrate, and the individual clones further characterized.

[1275] An isolated GEF32529 polypeptide, or a portion or fragment thereof, can be used as an immunogen to generate antibodies that bind GEF32529 using standard techniques for polyclonal and monoclonal antibody preparation. A full-length GEF32529 polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments of GEF32529 for use as immunogens. The antigenic peptide of GEF32529 comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:18 and encompasses an epitope of GEF32529 such that an antibody raised against the peptide forms a specific immune complex with GEF32529. Preferably, the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.

[1276] Preferred epitopes encompassed by the antigenic peptide are regions of GEF32529 that are located on the surface of the polypeptide, e.g., hydrophilic regions, as well as regions with high antigenicity (see, for example, FIG. 25).

[1277] A GEF32529 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for example, recombinantly expressed GEF32529 polypeptide or a chemically synthesized GEF32529 polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic GEF32529 preparation induces a polyclonal anti-GEF32529 antibody response.

[1278] Accordingly, another aspect of the invention pertains to anti-GEF32529 antibodies. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as GEF32529. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab′)2 fragments which can be generated by treating the antibody with an enzyme such as pepsin. The invention provides polyclonal and monoclonal antibodies that bind GEF32529. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of GEF32529. A monoclonal antibody composition thus typically displays a single binding affinity for a particular GEF32529 polypeptide with which it immunoreacts.

[1279] Polyclonal anti-GEF32529 antibodies can be prepared as described above by immunizing a suitable subject with a GEF32529 immunogen. The anti-GEF32529 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized GEF32529. If desired, the antibody molecules directed against GEF32529 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the anti-GEF32529 antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al. (1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75), the more recent human B cell hybridoma technique (Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) or trioma techniques. The technology for producing monoclonal antibody hybridomas is well known (see generally R. H. Kenneth, in Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lemer (1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typically a myeloma) is fused to lymphocytes (typically splenocytes) from a mammal immunized with a GEF32529 immunogen as described above, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds GEF32529.

[1280] Any of the many well known protocols used for fusing lymphocytes and immortalized cell lines can be applied for the purpose of generating an anti-GEF32529 monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature 266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, cited supra). Moreover, the ordinarily skilled worker will appreciate that there are many variations of such methods which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a number of myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind GEF32529, e.g., using a standard ELISA assay.

[1281] Alternative to preparing monoclonal antibody-secreting hybridomas, a monoclonal anti-GEF32529 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with GEF32529 to thereby isolate immunoglobulin library members that bind GEF32529. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). Additionally, examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT 10 International Publication WO 93/01288; McCafferty et al. PCT International Publication No. WO 92/01047; Garrard et al. PCT International Publication No. WO 92/09690; Ladner et al. PCT International Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology 9:1369-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J. Mol. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nucleic Acids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

[1282] Additionally, recombinant anti-GEF32529 antibodies, such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in 25 Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application 125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst. 80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al. (1986) Biotechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyen et al. (1988) Science 239:1534; and Beidler et al. (1988) J. Immunol. 141:4053-4060.

[1283] An anti-GEF32529 antibody (e.g., monoclonal antibody) can be used to isolate GEF32529 by standard techniques, such as affinity chromatography or immunoprecipitation. An anti-GEF32529 antibody can facilitate the purification of natural GEF32529 from cells and of recombinantly produced GEF32529 expressed in host cells. Moreover, an anti-GEF32529 antibody can be used to detect GEF32529 polypeptide (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression of the GEF32529 polypeptide. Anti-GEF32529 antibodies can be used diagnostically to monitor polypeptide levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidinbiotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S or 3H.

[1284] III. Recombinant Expression Vectors and Host Cells

[1285] Another aspect of the invention pertains to vectors, for example recombinant expression vectors, containing a nucleic acid containing a GEF32529 nucleic acid molecule or vectors containing a nucleic acid molecule which encodes a GEF32529 polypeptide (or a portion thereof). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[1286] The recombinant expression vectors of the invention comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cells and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of polypeptide desired, and the like. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., GEF32529 polypeptides, mutant forms of GEF32529 polypeptides, fusion proteins, and the like).

[1287] Accordingly, an exemplary embodiment provides a method for producing a polypeptide, preferably a GEF32529 polypeptide, by culturing in a suitable medium a host cell of the invention (e.g., a mammalian host cell such as a non-human mammalian cell) containing a recombinant expression vector, such that the polypeptide is produced.

[1288] The recombinant expression vectors of the invention can be designed for expression of GEF32529 polypeptides in prokaryotic or eukaryotic cells. For example, GEF32529 polypeptides can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[1289] Expression of proteins in prokaryotes is most often carried out in E. coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.

[1290] Purified fusion proteins can be utilized in GEF32529 activity assays, (e.g., direct assays or competitive assays described in detail below), or to generate antibodies specific for GEF32529 polypeptides, for example. In a preferred embodiment, a GEF32529 fusion protein expressed in a retroviral expression vector of the present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology of the subject recipient is then examined after sufficient time has passed (e.g., six (6) weeks).

[1291] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89). Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target gene expression from the pET 11d vector relies on transcription from a T7 gn10-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident prophage harboring a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter.

[1292] One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 119-128). Another strategy is to alter the nucleic acid sequence of the nucleic acid to be inserted into an expression vector so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

[1293] In another embodiment, the GEF32529 expression vector is a yeast expression vector. Examples of vectors for expression in yeast S. cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (Invitrogen Corporation, San Diego, Calif.).

[1294] Alternatively, GEF32529 polypeptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

[1295] In yet another embodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, B. (1987) Nature 329:840) and pMT2PC (Kaufinan et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40. For other suitable expression systems for both prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[1296] In another embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and immunoglobulins (Baneji et al. (1983) Cell 33:729-740; Queen and Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985) Science 230:912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the α-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).

[1297] The invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to GEF32529 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced. For a discussion of the regulation of gene expression using antisense genes see Weintraub, H. et al., Antisense RNA as a molecular tool for genetic analysis, Reviews—Trends in Genetics, Vol. 1(1) 1986.

[1298] Another aspect of the invention pertains to host cells into which a GEF32529 nucleic acid molecule of the invention is introduced, e.g., a GEF32529 nucleic acid molecule within a vector (e.g., a recombinant expression vector) or a GEF32529 nucleic acid molecule containing sequences which allow it to homologously recombine into a specific site of the host cell's genome. The terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[1299] A host cell can be any prokaryotic or eukaryotic cell. For example, a GEF32529 polypeptide can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells). Other suitable host cells are known to those skilled in the art.

[1300] Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989), and other laboratory manuals.

[1301] For stable transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. In order to identify and select these integrants, a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Preferred selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding a GEF32529 polypeptide or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).

[1302] A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) a GEF32529 polypeptide. Accordingly, the invention further provides methods for producing a GEF32529 polypeptide using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding a GEF32529 polypeptide has been introduced) in a suitable medium such that a GEF32529 polypeptide is produced. In another embodiment, the method further comprises isolating a GEF32529 polypeptide from the medium or the host cell.

[1303] The host cells of the invention can also be used to produce non-human transgenic animals. For example, in one embodiment, a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which GEF32529-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous GEF32529 sequences have been introduced into their genome or homologous recombinant animals in which endogenous GEF32529 sequences have been altered. Such animals are useful for studying the function and/or activity of a GEF32529 and for identifying and/or evaluating modulators of GEF32529 activity. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more of the cells of the animal includes a transgene. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, and the like. A transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal. As used herein, a “homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous GEF32529 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.

[1304] A transgenic animal of the invention can be created by introducing a GEF32529-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal. The GEF32529 cDNA sequence of SEQ ID NO:17 can be introduced as a transgene into the genome of a non-human animal. Alternatively, a nonhuman homologue of a human GEF32529 gene, such as a mouse or rat GEF32529 gene, can be used as a transgene. Alternatively, a GEF32529 gene homologue, such as another GEF32529 family member, can be isolated based on hybridization to the GEF32529 cDNA sequences of SEQ ID NO:17 or 19, or the DNA insert of the plasmid deposited with ATCC as Accession Number______ (described further in subsection I above) and used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene. A tissue-specific regulatory sequence(s) can be operably linked to a GEF32529 transgene to direct expression of a GEF32529 polypeptide to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art and are described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals. A transgenic founder animal can be identified based upon the presence of a GEF32529 transgene in its genome and/or expression of GEF32529 mRNA in tissues or cells of the animals. A transgenic founder animal can then be used to breed additional animals carrying the transgene. Moreover, transgenic animals carrying a transgene encoding a GEF32529 polypeptide can further be bred to other transgenic animals carrying other transgenes.

[1305] To create a homologous recombinant animal, a vector is prepared which contains at least a portion of a GEF32529 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the GEF32529 gene. The GEF32529 gene can be a human gene (e.g., the cDNA of SEQ ID NO:19), but more preferably, is a non-human homologue of a human GEF32529 gene (e.g., a cDNA isolated by stringent hybridization with the nucleotide sequence of SEQ ID NO:17). For example, a mouse GEF32529 gene can be used to construct a homologous recombination nucleic acid molecule, e.g., a vector, suitable for altering an endogenous GEF32529 gene in the mouse genome. In a preferred embodiment, the homologous recombination nucleic acid molecule is designed such that, upon homologous recombination, the endogenous GEF32529 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a “knock out” vector). Alternatively, the homologous recombination nucleic acid molecule can be designed such that, upon homologous recombination, the endogenous GEF32529 gene is mutated or otherwise altered but still encodes functional polypeptide (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous GEF32529 polypeptide). In the homologous recombination nucleic acid molecule, the altered portion of the GEF32529 gene is flanked at its 5′ and 3′ ends by additional nucleic acid sequence of the GEF32529 gene to allow for homologous recombination to occur between the exogenous GEF32529 gene carried by the homologous recombination nucleic acid molecule and an endogenous GEF32529 gene in a cell, e.g., an embryonic stem cell. The additional flanking GEF32529 nucleic acid sequence is of sufficient length for successful homologous recombination with the endogenous gene. Typically, several kilobases of flanking DNA (both at the 5′ and 3′ ends) are included in the homologous recombination nucleic acid molecule (see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for a description of homologous recombination vectors). The homologous recombination nucleic acid molecule is introduced into a cell, e.g., an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced GEF32529 gene has homologously recombined with the endogenous GEF32529 gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915). The selected cells can then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term. Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination nucleic acid molecules, e.g., vectors, or homologous recombinant animals are described further in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829 and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec et al.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO 93/04169 by Bems et al.

[1306] In another embodiment, transgenic non-human animals can be produced which contain selected systems which allow for regulated expression of the transgene. One example of such a system is the cre/loxP recombinase system of bacteriophage P1. For a description of the cre/loxP recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad. Sci. USA 89:6232-6236. Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be provided through the construction of “double” transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.

[1307] Clones of the non-human transgenic animals described herein can also be produced according to the methods described in Wilmut, I. et al (1997) Nature 385:810-813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, from the transgenic animal can be isolated and induced to exit the growth cycle and enter GO phase. The quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated. The reconstructed oocyte is then cultured such that it develops to morula or blastocyte and then transferred to pseudopregnant female foster animal. The offspring borne of this female foster animal will be a clone of the animal from which the cell, e.g., the somatic cell, is isolated.

[1308] IV. Pharmaceutical Compositions

[1309] The GEF32529 nucleic acid molecules, fragments of GEF32529 polypeptides, and anti-GEF32529 antibodies (also referred to herein as “active compounds”) of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the nucleic acid molecule, polypeptide, or antibody and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[1310] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[1311] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[1312] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a fragment of a GEF32529 polypeptide or an anti-GEF32529 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[1313] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[1314] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[1315] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[1316] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[1317] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[1318] It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[1319] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[1320] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[1321] As defined herein, a therapeutically effective amount of polypeptide (i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a polypeptide or antibody can include a single treatment or, preferably, can include a series of treatments.

[1322] In a preferred example, a subject is treated with antibody or polypeptide in the range of between about 0.1 to 20 mg/kg body weight, one time per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of antibody or polypeptide used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein.

[1323] The present invention encompasses agents which modulate expression or activity. An agent may, for example, be a small molecule. For example, such small molecules include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, organic or inorganic compounds (i.e.,. including heteroorganic and organometallic compounds) having a molecular weight less than about 10,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds. It is understood that appropriate doses of small molecule agents depends upon a number of factors within the ken of the ordinarily skilled physician, veterinarian, or researcher. The dose(s) of the small molecule will vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the small molecule to have upon the nucleic acid or polypeptide of the invention.

[1324] Exemplary doses include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram. It is furthermore understood that appropriate doses of a small molecule depend upon the potency of the small molecule with respect to the expression or activity to be modulated. Such appropriate doses may be determined using the assays described herein. When one or more of these small molecules is to be administered to an animal (e.g., a human) in order to modulate expression or activity of a polypeptide or nucleic acid of the invention, a physician, veterinarian, or researcher may, for example, prescribe a relatively low dose at first, subsequently increasing the dose until an appropriate response is obtained. In addition, it is understood that the specific dose level for any particular animal subject will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, gender, and diet of the subject, the time of administration, the route of administration, the rate of excretion, any drug combination, and the degree of expression or activity to be modulated.

[1325] Further, an antibody (or fragment thereof) may be conjugated to a therapeutic moiety such as a cytotoxin, a therapeutic agent or a radioactive metal ion. A cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologues thereof Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine and vinblastine).

[1326] The conjugates of the invention can be used for modifying a given biological response, the drug moiety is not to be construed as limited to classical chemical therapeutic agents. For example, the drug moiety may be a protein or polypeptide possessing a desired biological activity. Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator; or, biological response modifiers such as, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.

[1327] Techniques for conjugating such therapeutic moiety to antibodies are well known, see, e.g., Amon et al., “Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”, in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

[1328] The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Pat. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

[1329] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[1330] V. Uses and Methods of the Invention

[1331] The nucleic acid molecules, proteins, protein homologues, protein fragments, GEF32529 modulators, and antibodies described herein can be used in one or more of the following methods: a) screening assays; b) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and c) methods of treatment (e.g., therapeutic and prophylactic). As described herein, a GEF32529 polypeptide of the invention has one or more of the following activities: (i) association with a GEF32529 substrate or binding partner (e.g., a GDP-bound small G protein, for example, a Ras-like or Rho/Rac-like small G protein); (ii) dissociation of GDP from a GEF32529 substrate or binding partner (e.g., a GDP-bound small G protein); (iii) destabilization of a GDP-bound small G protein; (iv) stabilization of a nucleotide-free small G protein, and (v) activation of a GEF32529 substrate or binding partner. In another example, a GEF32529 activity is at least one or more of the following activities: (1) modulation of signal transduction (e.g., signal transduction cascades involving small GTP-binding proteins); (2) control of cell morphology; (3) modulation of adhesion and/or motility of cells; (4) mediation of cytoskeletal organization or reorganization; (5) modulation of cellular trafficking (e.g., vesicular transport); and (6) modulation of tumor inhibition.

[1332] The isolated nucleic acid molecules of the invention can be used, for example, to express GEF32529 polypeptide (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect GEF32529 mRNA (e.g., in a biological sample) or a genetic alteration in a GEF32529 gene, and to modulate GEF32529 activity, as described further below. The GEF32529 polypeptides can be used to treat disorders characterized by insufficient or excessive production of a GEF32529 substrate or production of GEF32529 inhibitors (i.e., a “GEF32529 associated,” “GEF associated” or “Rho/Rac GEF associated” disorder). As used herein, the term “GEF associated disorder” includes disorders, diseases, or conditions which are characterized by aberrant, e.g., upregulated or downregulated, GDP dissociation from small G proteins. Examples of such disorders include cancer, inflammation, diabetes, and pathogenic invasion of host cells. Other examples are cardiovascular disorders, e.g., arteriosclerosis, ischemia reperfusion injury, restenosis, arterial inflammation, vascular wall remodeling, ventricular remodeling, rapid ventricular pacing, coronary microembolism, tachycardia, bradycardia, pressure overload, aortic bending, coronary artery ligation, vascular heart disease, atrial fibrillation, long-QT syndrome, congestive heart failure, sinus node dysfunction, angina, heart failure, hypertension, atrial fibrillation, atrial flutter, dilated cardiomyopathy, idiopathic cardiomyopathy, myocardial infarction, coronary artery disease, coronary artery spasm, or arrhythmia.

[1333] In another example, the activity of a GEF32529 molecule of the present invention is an oncogenic or metastatic activity. As such, GEF32529 molecules are particularly useful in screening for modulators of oncogenesis and/or metastasis, the modulators further being useful in the prophylactic and/or therapeutic methods described herein.

[1334] Other examples of GEF associated disorders include disorders of the central nervous system, e.g., cystic fibrosis, type 1 neurofibromatosis, cognitive and neurodegenerative disorders, examples of which include, but are not limited to, Alzheimer's disease, dementias related to Alzheimer's disease (such as Pick's disease), Parkinson's and other Lewy diffuse body diseases, senile dementia, Huntington's disease, Gilles de la Tourette's syndrome, multiple sclerosis, amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy, and Creutzfeldt-Jakob disease; autonomic function disorders such as hypertension and sleep disorders, and neuropsychiatric disorders, such as depression, schizophrenia, schizoaffective disorder, korsakoff's psychosis, mania, anxiety disorders, or phobic disorders; learning or memory disorders, e.g., amnesia or age-related memory loss, attention deficit disorder, dysthymic disorder, major depressive disorder, mania, obsessive-compulsive disorder, psychoactive substance use disorders, anxiety, phobias, panic disorder, as well as bipolar affective disorder, e.g., severe bipolar affective (mood) disorder (BP-1), and bipolar affective neurological disorders, e.g., migraine and obesity. Further GEF-related disorders include, for example, those listed in the American Psychiatric Association's Diagnostic and Statistical manual of Mental Disorders (DSM), the most current version of which is incorporated herein by reference in its entirety.

[1335] Still other examples of GEF associated disorders include cellular proliferation, growth, differentiation, or migration disorders. Cellular proliferation, growth, differentiation, or migration disorders include those disorders that affect cell proliferation, growth, differentiation, or migration processes. As used herein, a “cellular proliferation, growth, differentiation, or migration process” is a process by which a cell increases in number, size or content, by which a cell develops a specialized set of characteristics which differ from that of other cells, or by which a cell moves closer to or further from a particular location or stimulus. Such disorders include cancer, e.g., carcinoma, sarcoma, or leukemia; tumor angiogenesis and metastasis; skeletal dysplasia; hepatic disorders; and hematopoietic and/or myeloproliferative disorders.

[1336] Still other examples of GEF associated disorders include disorders of the immune system, such as Wiskott-Aldrich syndrome, viral infection, autoimmune disorders or immune deficiency disorders, e.g., congenital X-linked infantile hypogammaglobulinemia, transient hypogammaglobulinemia, common variable immunodeficiency, selective IgA deficiency, chronic mucocutaneous candidiasis, or severe combined immunodeficiency. Other examples of GEF-related disorders include congenital malfornalities, including facio-genital dysplasia; and skin disorders, including microphthalmia with linear skin defects syndrome.

[1337] In addition, the GEF32529 polypeptides can be used to screen for naturally occurring GEF32529 substrates, to screen for drugs or compounds which modulate GEF32529 activity, as well as to treat disorders characterized by insufficient or excessive production of GEF32529 polypeptide or production of GEF32529 polypeptide forms which have decreased, aberrant or unwanted activity compared to GEF32529 wild type polypeptide (e.g., nucleoside hydrolysis disorders (such as cell permeabilization, cell necrosis or apoptosis, triggering of second messengers, cell proliferation, cell motility, or signal transduction disorders)). Moreover, the anti-GEF32529 antibodies of the invention can be used to detect and isolate GEF32529 polypeptides, to regulate the bioavailability of GEF32529 polypeptides, and modulate GEF32529 activity.

[1338] A. Screening Assays:

[1339] The invention provides a method (also referred to herein as a “screening assay”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) which bind to GEF32529 polypeptides, have a stimulatory or inhibitory effect on, for example, GEF32529 expression or GEF32529 activity, or have a stimulatory or inhibitory effect on, for example, the expression or activity of GEF32529 substrate.

[1340] In one embodiment, the invention provides assays for screening candidate or test compounds which are substrates of a GEF32529 polypeptide or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a GEF32529 polypeptide or polypeptide or biologically active portion thereof. The test compounds of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the ‘one-bead one-compound’ library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

[1341] Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422; Zuckennann et al. (1994). J. Med. Chem. 37:2678; Cho etal (1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; and in Gallop et al. (1994) J. Med. Chem. 37:1233.

[1342] Libraries of compounds may be presented in solution (e.g., Houghten (1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (Ladner U.S. Pat. No. 5,223,409), spores (Ladner USP '409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science 249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladner supra.).

[1343] In one embodiment, an assay is a cell-based assay in which a cell which expresses a GEF32529 polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate GEF32529 activity is determined. Determining the ability of the test compound to modulate GEF32529 activity can be accomplished by monitoring, for example, intracellular GDP concentrations. The cell, for example, can be of mammalian origin, e.g., a heart, placenta, lung, liver, skeletal muscle, thymus, kidney, pancreas, testis, ovary, prostate, colon, or brain cell.

[1344] The ability of the test compound to modulate GEF32529 binding to a substrate or to bind to GEF32529 can also be determined. Determining the ability of the test compound to modulate GEF32529 binding to a substrate can be accomplished, for example, by coupling the GEF32529 substrate with a radioisotope or enzymatic label such that binding of the GEF32529 substrate to GEF32529 can be determined by detecting the labeled GEF32529 substrate in a complex. Alternatively, GEF32529 could be coupled with a radioisotope or enzymatic label to monitor the ability of a test compound to modulate GEF32529 binding to a GEF32529 substrate in a complex. Determining the ability of the test compound to bind GEF32529 can be accomplished, for example, by coupling the compound with a radioisotope or enzymatic label such that binding of the compound to GEF32529 can be determined by detecting the labeled GEF32529 compound in a complex. For example, compounds (e.g., GEF32529 substrates) can be labeled with 125I, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting. Alternatively, compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.

[1345] It is also within the scope of this invention to determine the ability of a compound (e.g., a GEF32529 substrate) to interact with GEF32529 without the labeling of any of the interactants. For example, a microphysiometer can be used to detect the interaction of a compound with GEF32529 without the labeling of either the compound or the GEF32529. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a “microphysiometer” (e.g., Cytosensor) is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a compound and GEF32529.

[1346] In another embodiment, an assay is a cell-based assay comprising contacting a cell expressing a GEF32529 target molecule (e.g., a GEF32529 substrate) with a test compound and determining the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GEF32529 target molecule. Determining the ability of the test compound to modulate the activity of a GEF32529 target molecule can be accomplished, for example, by determining the ability of the GEF32529 polypeptide to bind to or interact with the GEF32529 target molecule.

[1347] Determining the ability of the GEF32529 polypeptide, or a biologically active fragment thereof, to bind to or interact with a GEF32529 target molecule can be accomplished by one of the methods described above for determining direct binding. In a preferred embodiment, determining the ability of the GEF32529 polypeptide to bind to or interact with a GEF32529 target molecule can be accomplished by determining the activity of the target molecule. For example, the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (i.e., intracellular Ca2+, diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target using an appropriate substrate, detecting the induction of a reporter gene (comprising a target-responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a target-regulated cellular response.

[1348] In yet another embodiment, an assay of the present invention is a cell-free assay in which a GEF32529 polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to bind to the GEF32529 polypeptide or biologically active portion thereof is determined. Preferred biologically active portions of the GEF32529 polypeptides to be used in assays of the present invention include fragments which participate in interactions with non-GEF32529 molecules, e.g., fragments with high surface probability scores (see, for example, FIG. 25). Binding of the test compound to the GEF32529 polypeptide can be determined either directly or indirectly as described above. In a preferred embodiment, the assay includes contacting the GEF32529 polypeptide or biologically active portion thereof with a known compound which binds GEF32529 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with a GEF32529 polypeptide, wherein determining the ability of the test compound to interact with a GEF32529 polypeptide comprises determining the ability of the test compound to preferentially bind to GEF32529 or biologically active portion thereof as compared to the known compound.

[1349] In another embodiment, the assay is a cell-free assay in which a GEF32529 polypeptide or biologically active portion thereof is contacted with a test compound and the ability of the test compound to modulate (e.g., stimulate or inhibit) the activity of the GEF32529 polypeptide or biologically active portion thereof is determined. Determining the ability of the test compound to modulate the activity of a GEF32529 polypeptide can be accomplished, for example, by determining the ability of the GEF32529 polypeptide to bind to a GEF32529 target molecule by one of the methods described above for determining direct binding. Determining the ability of the GEF32529 polypeptide to bind to a GEF32529 target molecule can also be accomplished using a technology such as real-time Biomolecular Interaction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991) Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705. As used herein, “BIA” is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

[1350] In an alternative embodiment, determining the ability of the test compound to modulate the activity of a GEF32529 polypeptide can be accomplished by determining the ability of the GEF32529 polypeptide to further modulate the activity of a downstream effector of a GEF32529 target molecule. For example, the activity of the effector molecule on an appropriate target can be determined or the binding of the effector to an appropriate target can be determined as previously described.

[1351] In yet another embodiment, the cell-free assay involves contacting a GEF32529 polypeptide or biologically active portion thereof with a known compound which binds the GEF32529 polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the GEF32529 polypeptide, wherein determining the ability of the test compound to interact with the GEF32529 polypeptide comprises determining the ability of the GEF32529 polypeptide to preferentially bind to or modulate the activity of a GEF32529 target molecule.

[1352] In more than one embodiment of the above assay methods of the present invention, it may be desirable to immobilize either GEF32529 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate automation of the assay. Binding of a test compound to a GEF32529 polypeptide, or interaction of a GEF32529 polypeptide with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and micro-centrifuge tubes. In one embodiment, a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix. For example, glutathione-S-transferase/GEF32529 fusion proteins or glutathione-S-transferase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized micrometer plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or GEF32529 polypeptide, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or micrometer plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of GEF32529 binding or activity determined using standard techniques.

[1353] Other techniques for immobilizing proteins on matrices can also be used in the screening assays of the invention. For example, either a GEF32529 polypeptide or a GEF32529 target molecule can be immobilized utilizing conjugation of biotin and streptavidin. Biotinylated GEF32529 polypeptide or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical). Alternatively, antibodies reactive with GEF32529 polypeptide or target molecules but which do not interfere with binding of the GEF32529 polypeptide to its target molecule can be derivatized to the wells of the plate, and unbound target or GEF32529 polypeptide trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described above for the GST-immobilized complexes, include immunodetection of complexes using antibodies reactive with the GEF32529 polypeptide or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the GEF32529 polypeptide or target molecule.

[1354] In another embodiment, modulators of GEF32529 expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of GEF32529 mRNA or polypeptide in the cell is determined. The level of expression of GEF32529 mRNA or polypeptide in the presence of the candidate compound is compared to the level of expression of GEF32529 mRNA or polypeptide in the absence of the candidate compound. The candidate compound can then be identified as a modulator of GEF32529 expression based on this comparison. For example, when expression of GEF32529 mRNA or polypeptide is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GEF32529 mRNA or polypeptide expression. Alternatively, when expression of GEF32529 mRNA or polypeptide is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GEF32529 mRNA or polypeptide expression. The level of GEF32529 mRNA or polypeptide expression in the cells can be determined by methods described herein for detecting GEF32529 mRNA or polypeptide.

[1355] In yet another aspect of the invention, the GEF32529 polypeptides can be used as “bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and Brent W094/10300), to identify other proteins, which bind to or interact with GEF32529 (“GEF32529-binding proteins” or “GEF32529-bp”) and are involved in GEF32529 activity. Such GEF32529-binding proteins are also likely to be involved in the propagation of signals by the GEF32529 polypeptides or GEF32529 targets as, for example, downstream elements of a GEF32529-mediated signaling pathway. Alternatively, such GEF32529-binding proteins are likely to be GEF32529 inhibitors.

[1356] The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a GEF32529 polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4). In the other construct, a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor. If the “bait” and the “prey” proteins are able to interact, in vivo, forming a GEF32529-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the GEF32529 polypeptide.

[1357] In another aspect, the invention pertains to a combination of two or more of the assays described herein. For example, a modulating agent can be identified using a cell-based or a cell free assay, and the ability of the agent to modulate the activity of a GEF32529 polypeptide can be confirmed in vivo, e.g., in an animal such as an animal model for cellular transformation and/or tumorigenesis.

[1358] This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model. For example, an agent identified as described herein (e.g., a GEF32529 modulating agent, an antisense GEF32529 nucleic acid molecule, a GEF32529-specific antibody, or a GEF32529-binding partner) can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent. Furthermore, this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.

[1359] B. Detection Assays

[1360] Portions or fragments of the cDNA sequences identified herein (and the corresponding complete gene sequences) can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue typing); and (iii) aid in forensic identification of a biological sample. These applications are described in the subsections below.

[1361] 1. Chromosome Mapping

[1362] Once the sequence (or a portion of the sequence) of a gene has been isolated, this sequence can be used to map the location of the gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments of the GEF32529 nucleotide sequences, described herein, can be used to map the location of the GEF32529 genes on a chromosome. The mapping of the GEF32529 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.

[1363] Briefly, GEF32529 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the GEF32529 nucleotide sequences. Computer analysis of the GEF32529 sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the GEF32529 sequences will yield an amplified fragment.

[1364] Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells). As hybrids of human and mouse cells grow and divide, they gradually lose human chromosomes in random order, but retain the mouse chromosomes. By using media in which mouse cells cannot grow, because they lack a particular enzyme, but human cells can, the one human chromosome that contains the gene encoding the needed enzyme, will be retained. By using various media, panels of hybrid cell lines can be established. Each cell line in a panel contains either a single human chromosome or a small number of human chromosomes, and a full set of mouse chromosomes, allowing easy mapping of individual genes to specific human chromosomes. (D'Eustachio P. et al. (1983) Science 220:919-924). Somatic cell hybrids containing only fragments of human chromosomes can also be produced by using human chromosomes with translocations and deletions.

[1365] PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the GEF32529 nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a GEF32529 sequence to its chromosome include in situ hybridization (described in Fan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screening with labeled flow-sorted chromosomes, and pre-selection by hybridization to chromosome specific cDNA libraries.

[1366] Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step. Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle. The chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually. The FISH technique can be used with a DNA sequence as short as 500 or 600 bases. However, clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. Preferably 1,000 bases, and more preferably 2,000 bases will suffice to get good results at a reasonable amount of time. For a review of this technique, see Verma et al., Human Chromosomes: A Manual of Basic Techniques (Pergamon Press, New York 1988).

[1367] Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions of the genes actually are preferred for mapping purposes. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.

[1368] Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. (Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man, available on-line through Johns Hopkins University Welch Medical Library). The relationship between a gene and a disease, mapped to the same chromosomal region, can then be identified through linkage analysis (co-inheritance of physically adjacent genes), described in, for example, Egeland, J. et al. (1987) Nature, 325:783-787.

[1369] Moreover, differences in the DNA sequences between individuals affected and unaffected with a disease associated with the GEF32529 gene, can be determined. If a mutation is observed in some or all of the affected individuals but not in any unaffected individuals, then the mutation is likely to be the causative agent of the particular disease. Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several individuals can be performed to confirm the presence of a mutation and to distinguish mutations from polymorphisms.

[1370] 2. Tissue Typing

[1371] The GEF32529 sequences of the present invention can also be used to identify individuals from minute biological samples. The United States military, for example, is considering the use of restriction fragment length polymorphism (RFLP) for identification of its personnel. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of “Dog Tags” which can be lost, switched, or stolen, making positive identification difficult. The sequences of the present invention are useful as additional DNA markers for RFLP (described in U.S. Pat. No. 5,272,057).

[1372] Furthermore, the sequences of the present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome. Thus, the GEF32529 nucleotide sequences described herein can be used to prepare two PCR primers from the 5′ and 3′ ends of the sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it.

[1373] Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the present invention can be used to obtain such identification sequences from individuals and from tissue. The GEF32529 nucleotide sequences of the invention uniquely represent portions of the human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases. Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals. The noncoding sequences of SEQ ID NO:17 can comfortably provide positive individual identification with a panel of perhaps 10 to 1,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO:19 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

[1374] If a panel of reagents from GEF32529 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual. Using the unique identification database, positive identification of the individual, living or dead, can be made from extremely small tissue samples.

[1375] 3. Use of GEF32529 Sequences in Forensic Biology

[1376] DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a perpetrator of a crime. To make such an identification, PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification of the origin of the biological sample.

[1377] The sequences of the present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another “identification marker” (i.e. another DNA sequence that is unique to a particular individual). As mentioned above, actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments. Sequences targeted to noncoding regions of SEQ ID NO:17 are particularly appropriate for this use as greater numbers of polymorphisms occur in the noncoding regions, making it easier to differentiate individuals using this technique. Examples of polynucleotide reagents include the GEF32529 nucleotide sequences or portions thereof, e.g., fragments derived from the noncoding regions of SEQ ID NO:17 having a length of at least 20 bases, preferably at least 30 bases.

[1378] The GEF32529 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of such GEF32529 probes can be used to identify tissue by species and/or by organ type.

[1379] In a similar fashion, these reagents, e.g., GEF32529 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture).

[1380] C. Predictive Medicine:

[1381] The present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trials are used for prognostic (predictive) purposes to thereby treat an individual prophylactically. Accordingly, one aspect of the present invention relates to diagnostic assays for determining GEF32529 polypeptide and/or nucleic acid expression as well as GEF32529 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant or unwanted GEF32529 expression or activity. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with GEF32529 polypeptide, nucleic acid expression or activity. For example, mutations in a GEF32529 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive purpose to thereby prophylactically treat an individual prior to the onset of a disorder characterized by or associated with GEF32529 polypeptide, nucleic acid expression or activity.

[1382] Another aspect of the invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of GEF32529 in clinical trials.

[1383] These and other agents are described in further detail in the following sections.

[1384] 1. Diagnostic Assays An exemplary method for detecting the presence or absence of GEF32529 polypeptide or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting GEF32529 polypeptide or nucleic acid (e.g., mRNA, or genomic DNA) that encodes GEF32529 polypeptide such that the presence of GEF32529 polypeptide or nucleic acid is detected in the biological sample. In another aspect, the present invention provides a method for detecting the presence of GEF32529 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of GEF32529 activity such that the presence of GEF32529 activity is detected in the biological sample. A preferred agent for detecting GEF32529 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to GEF32529 mRNA or genomic DNA. The nucleic acid probe can be, for example, the GEF32529 nucleic acid set forth in SEQ ID NO:17 or 19, or the DNA insert of the plasmid deposited with ATCC as Accession Number ______, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to GEF32529 mRNA or genomic DNA. Other suitable probes for use in the diagnostic assays of the invention are described herein.

[1385] A preferred agent for detecting GEF32529 polypeptide is an antibody capable of binding to GEF32529 polypeptide, preferably an antibody with a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab′)2) can be used. The term “labeled”, with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin. The term “biological sample” is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect GEF32529 mRNA, polypeptide, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of GEF32529 mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of GEF32529 polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. In vitro techniques for detection of GEF32529 genomic DNA include Southern hybridizations. Furthermore, in vivo techniques for detection of GEF32529 polypeptide include introducing into a subject a labeled anti-GEF32529 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

[1386] The present invention also provides diagnostic assays for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a GEF32529 polypeptide; (ii) aberrant expression of a gene encoding a GEF32529 polypeptide; (iii) mis-regulation of the gene; and (iv) aberrant post-translational modification of a GEF32529 polypeptide, wherein a wild-type form of the gene encodes a polypeptide with a GEF32529 activity. “Misexpression or aberrant expression”, as used herein, refers to a non-wild type pattern of gene expression, at the RNA or protein level. It includes, but is not limited to, expression at non-wild type levels (e.g., over or under expression); a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed (e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage); a pattern of expression that differs from wild type in terms of decreased expression (as compared with wild type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the splicing size, amino acid sequence, post-transitional modification, or biological activity of the expressed polypeptide; a pattern of expression that differs from wild type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene (e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus).

[1387] In one embodiment, the biological sample contains protein molecules from the test subject. Alternatively, the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject. A preferred biological sample is a serum sample isolated by conventional means from a subject.

[1388] In another embodiment, the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting GEF32529 polypeptide, mRNA, or genomic DNA, such that the presence of GEF32529 polypeptide, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of GEF32529 polypeptide, mRNA or genomic DNA in the control sample with the presence of GEF32529 polypeptide, mRNA or genomic DNA in the test sample.

[1389] The invention also encompasses kits for detecting the presence of GEF32529 in a biological sample. For example, the kit can comprise a labeled compound or agent capable of detecting GEF32529 polypeptide or mRNA in a biological sample; means for determining the amount of GEF32529 in the sample; and means for comparing the amount of GEF32529 in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect GEF32529 polypeptide or nucleic acid.

[1390] 2. Prognostic Assays

[1391] The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant or unwanted GEF32529 expression or activity. As used herein, the term “aberrant” includes a GEF32529 expression or activity which deviates from the wild type GEF32529 expression or activity. Aberrant expression or activity includes increased or decreased expression or activity, as well as expression or activity which does not follow the wild type developmental pattern of expression or the subcellular pattern of expression. For example, aberrant GEF32529 expression or activity is intended to include the cases in which a mutation in the GEF32529 gene causes the GEF32529 gene to be under-expressed or over-expressed and situations in which such mutations result in a non-functional GEF32529 polypeptide or a polypeptide which does not function in a wild-type fashion, e.g., a polypeptide which does not interact with a GEF32529 substrate, e.g., a non-GEF subunit or ligand, or one which interacts with a non-GEF32529 substrate, e.g. a non-GEF subunit or ligand. As used herein, the term “unwanted” includes an unwanted phenomenon involved in a biological response, such as cellular proliferation. For example, the term unwanted includes a GEF32529 expression or activity which is undesirable in a subject.

[1392] The assays described herein, such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with a misregulation in GEF32529 polypeptide activity or nucleic acid expression, such as a GDP dissociation disorder (e.g., a cell signaling, tumor inhibition, cytoskeletal organization, or cellular trafficking disorder). Alternatively, the prognostic assays can be utilized to identify a subject having or at risk for developing a disorder associated with a misregulation in GEF32529 polypeptide activity or nucleic acid expression, such as a GDP dissociation disorder, or a cell signaling, tumor inhibition, cytoskeletal organization, or cellular trafficking disorder. Thus, the present invention provides a method for identifying a disease or disorder associated with aberrant or unwanted GEF32529 expression or activity in which a test sample is obtained from a subject and GEF32529 polypeptide or nucleic acid (e.g., mRNA or genomic DNA) is detected, wherein the presence of GEF32529 polypeptide or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant or unwanted GEF32529 expression or activity. As used herein, a “test sample” refers to a biological sample obtained from a subject of interest. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.

[1393] Furthermore, the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate) to treat a disease or disorder associated with aberrant or unwanted GEF32529 expression or activity. For example, such methods can be used to determine whether a subject can be effectively treated with an agent for a GDP dissociation disorder, or a cell signaling, tumor inhibition, cytoskeletal organization, or cellular trafficking disorder. Thus, the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant or unwanted GEF32529 expression or activity in which a test sample is obtained and GEF32529 polypeptide or nucleic acid expression or activity is detected (e.g., wherein the abundance of GEF32529 polypeptide or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant or unwanted GEF32529 expression or activity).

[1394] The methods of the invention can also be used to detect genetic alterations in a GEF32529 gene, thereby determining if a subject with the altered gene is at risk for a disorder characterized by misregulation in GEF32529 polypeptide activity or nucleic acid expression, such as a GDP dissociation disorder, or a cell signaling, tumor inhibition, cytoskeletal organization, or cellular trafficking disorder. In preferred embodiments, the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a GEF32529-polypeptide, or the mis-expression of the GEF32529 gene. For example, such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from a GEF32529 gene; 2) an addition of one or more nucleotides to a GEF32529 gene; 3) a substitution of one or more nucleotides of a GEF32529 gene, 4) a chromosomal rearrangement of a GEF32529 gene; 5) an alteration in the level of a messenger RNA transcript of a GEF32529 gene, 6) aberrant modification of a GEF32529 gene, such as of the methylation pattern of the genomic DNA, 7) the presence of a non-wild type splicing pattern of a messenger RNA transcript of a GEF32529 gene, 8) a non-wild type level of a GEF32529-polypeptide, 9) allelic loss of a GEF32529 gene, and 10) inappropriate post-translational modification of a GEF32529-polypeptide. As described herein, there are a large number of assays known in the art which can be used for detecting alterations in a GEF32529 gene. A preferred biological sample is a tissue or serum sample isolated by conventional means from a subject.

[1395] In certain embodiments, detection of the alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the latter of which can be particularly useful for detecting point mutations in the GEF32529-gene (see Abravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method can include the steps of collecting a sample of cells from a subject, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells of the sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a GEF32529 gene under conditions such that hybridization and amplification of the GEF32529-gene (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

[1396] Alternative amplification methods include: self sustained sequence replication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.

[1397] In an alternative embodiment, mutations in a GEF32529 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA. Moreover, the use of sequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531) can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

[1398] In other embodiments, genetic mutations in GEF32529 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M. T. et al. (1996) Human Mutation 7: 244-255; Kozal, M. J. et al. (1996) Nature Medicine 2: 753-759). For example, genetic mutations in GEF32529 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M. T. et al. supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.

[1399] In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence the GEF32529 gene and detect mutations by comparing the sequence of the sample GEF32529 with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on techniques developed by Maxam and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol. 38:147-159).

[1400] Other methods for detecting mutations in the GEF32529 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242). In general, the art technique of “mismatch cleavage” starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type GEF32529 sequence with potentially mutant RNA or DNA obtained from a tissue sample. The double-stranded duplexes are treated with an agent which cleaves single-stranded regions of the duplex such as which will exist due to basepair mismatches between the control and sample strands. For instance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically digesting the mismatched regions. In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion of the mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) Methods Enzymol. 217:286-295. In a preferred embodiment, the control DNA or RNA can be labeled for detection.

[1401] In still another embodiment, the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called “DNA mismatch repair” enzymes) in defined systems for detecting and mapping point mutations in GEF32529 cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662). According to an exemplary embodiment, a probe based on a GEF32529 sequence, e.g., a wild-type GEF32529 sequence, is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like. See, for example, U.S. Pat. No. 5,459,039.

[1402] In other embodiments, alterations in electrophoretic mobility will be used to identify mutations in GEF32529 genes. For example, single strand conformation polymorphism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments of sample and control GEF32529 nucleic acids will be denatured and allowed to renature. The secondary structure of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a change in sequence. In a preferred embodiment, the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

[1403] In yet another embodiment the movement of mutant or wild-type fragments in polyacrylamide gels containing a gradient of denaturant is assayed using denaturing gradient gel electrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. In a further embodiment, a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).

[1404] Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci. USA 86:6230). Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

[1405] Alternatively, allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention. Oligonucleotides used as primers for specific amplification may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization) (Gibbs et al. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prossner (1993) Tibtech 11:238). In addition it may be desirable to introduce a novel restriction site in the region of the mutation to create cleavage-based detection (Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certain embodiments amplification may also be performed using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In such cases, ligation will occur only if there is a perfect match at the 3′ end of the 5′ sequence making it possible to detect the presence of a known mutation at a specific site by looking for the presence or absence of amplification.

[1406] The methods described herein may be performed, for example, by utilizing pre-packaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a GEF32529 gene.

[1407] Furthermore, any cell type or tissue in which GEF32529 is expressed may be utilized in the prognostic assays described herein.

[1408] 3. Monitoring of Effects during Clinical Trials

[1409] Monitoring the influence of agents (e.g., drugs) on the expression or activity of a GEF32529 polypeptide (e.g., the modulation of membrane excitability) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay as described herein to increase GEF32529 gene expression, polypeptide levels, or upregulate GEF32529 activity, can be monitored in clinical trials of subjects exhibiting decreased GEF32529 gene expression, polypeptide levels, or downregulated GEF32529 activity. Alternatively, the effectiveness of an agent determined by a screening assay to decrease GEF32529 gene expression, polypeptide levels, or downregulate GEF32529 activity, can be monitored in clinical trials of subjects exhibiting increased GEF32529 gene expression, polypeptide levels, or upregulated GEF32529 activity. In such clinical trials, the expression or activity of a GEF32529 gene, and preferably, other genes that have been implicated in, for example, a GEF32529-associated disorder can be used as a “read out” or markers of the phenotype of a particular cell.

[1410] For example, and not by way of limitation, genes, including GEF32529, that are modulated in cells by treatment with an agent (e.g., compound, drug or small molecule) which modulates GEF32529 activity (e.g., identified in a screening assay as described herein) can be identified. Thus, to study the effect of agents on GEF32529-associated disorders (e.g., disorders characterized by deregulated GEF activity), for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of GEF32529 and other genes implicated in the GEF32529-associated disorder, respectively. The levels of gene expression (e.g., a gene expression pattern) can be quantified by northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of polypeptide produced, by one of the methods as described herein, or by measuring the levels of activity of GEF32529 or other genes. In this way, the gene expression pattern can serve as a marker, indicative of the physiological response of the cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment of the individual with the agent.

[1411] In a preferred embodiment, the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drug candidate identified by the screening assays described herein) including the steps of (i) obtaining a pre-administration sample from a subject prior to administration of the agent; (ii) detecting the level of expression of a GEF32529 polypeptide, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post-administration samples from the subject; (iv) detecting the level of expression or activity of the GEF32529 polypeptide, mRNA, or genomic DNA in the post-administration samples; (v) comparing the level of expression or activity of the GEF32529 polypeptide, mRNA, or genomic DNA in the pre-administration sample with the GEF32529 polypeptide, mRNA, or genomic DNA in the post administration sample or samples; and (vi) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of GEF32529 to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of GEF32529 to lower levels than detected, i.e. to decrease the effectiveness of the agent. According to such an embodiment, GEF32529 expression or activity may be used as an indicator of the effectiveness of an agent, even in the absence of an observable phenotypic response.

[1412] D. Methods of Treatment:

[1413] The present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant or unwanted GEF32529 expression or activity, e.g. a GEF associated or GEF related disorder, for example, a cell signaling, tumor inhibition, cytoskeletal organization, or cellular trafficking disorder. With regards to both prophylactic and therapeutic methods of treatment, such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. “Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”). Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the GEF32529 molecules of the present invention or GEF32529 modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

[1414] Treatment is defined as the application or administration of a therapeutic agent to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has a disease, a symptom of disease or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease, the symptoms of disease or the predisposition toward disease.

[1415] A therapeutic agent includes, but is not limited to, small molecules, peptides, antibodies, ribozymes and antisense oligonucleotides.

[1416] I. Prophylactic Methods

[1417] In one aspect, the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant or unwanted GEF32529 expression or activity, by administering to the subject a GEF32529 or an agent which modulates GEF32529 expression or at least one GEF32529 activity. Subjects at risk for a disease which is caused or contributed to by aberrant or unwanted GEF32529 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the GEF32529 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of GEF32529 aberrancy, for example, a GEF32529, GEF32529 agonist or GEF32529 antagonist agent can be used for treating the subject. The appropriate agent can be determined based on screening assays described herein.

[1418] 2. Therapeutic Methods

[1419] Another aspect of the invention pertains to methods of modulating GEF32529 expression or activity for therapeutic purposes. Accordingly, in an exemplary embodiment, the modulatory method of the invention involves contacting a cell capable of expressing GEF32529 with an agent that modulates one or more of the activities of GEF32529 polypeptide activity associated with the cell, such that GEF32529 activity in the cell is modulated. An agent that modulates GEF32529 polypeptide activity can be an agent as described herein, such as a nucleic acid or a polypeptide, a naturally-occurring target molecule of a GEF32529 polypeptide (e.g., a GEF32529 substrate), a GEF32529 antibody, a GEF32529 agonist or antagonist, a peptidomimetic of a GEF32529 agonist or antagonist, or other small molecule. In one embodiment, the agent stimulates one or more GEF32529 activities. Examples of such stimulatory agents include active GEF32529 polypeptide and a nucleic acid molecule encoding GEF32529 that has been introduced into the cell. In another embodiment, the agent inhibits one or more GEF32529 activities. Examples of such inhibitory agents include antisense GEF32529 nucleic acid molecules, anti-GEF32529 antibodies, and GEF32529 inhibitors. These modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant or unwanted expression or activity of a GEF32529 polypeptide or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) GEF32529 expression or activity. In another embodiment, the method involves administering a GEF32529 polypeptide or nucleic acid molecule as therapy to compensate for reduced, aberrant, or unwanted GEF32529 expression or activity.

[1420] Stimulation of GEF32529 activity is desirable in situations in which GEF32529 is abnormally downregulated and/or in which increased GEF32529 activity is likely to have a beneficial effect. Likewise, inhibition of GEF32529 activity is desirable in situations in which GEF32529 is abnormally upregulated and/or in which decreased GEF32529 activity is likely to have a beneficial effect.

[1421] 3. Pharmacogenomics

[1422] The GEF32529 molecules of the present invention, as well as agents, or modulators which have a stimulatory or inhibitory effect on GEF32529 activity (e.g., GEF32529 gene expression) as identified by a screening assay described herein can be administered to individuals to treat (prophylactically or therapeutically) GEF32529-associated disorders (e.g., proliferative disorders) associated with aberrant or unwanted GEF32529 activity. In conjunction with such treatment, pharmacogenomics (i.e., the study of the relationship between an individual's genotype and that individual's response to a foreign compound or drug) may be considered. Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer a GEF32529 molecule or GEF32529 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with a GEF32529 molecule or GEF32529 modulator.

[1423] Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See, for example, Eichelbaum, M. et al. (1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M. W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drug action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymorphisms. For example, glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

[1424] One pharmacogenomics approach to identifying genes that predict drug response, known as “a genome-wide association”, relies primarily on a high-resolution map of the human genome consisting of already known gene-related markers (e.g., a “bi-allelic” gene marker map which consists of 60,000-100,000 polymorphic or variable sites on the human genome, each of which has two variants.) Such a high-resolution genetic map can be compared to a map of the genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect. Alternatively, such a high resolution map can be generated from a combination of some ten-million known single nucleotide polymorphisms (SNPs) in the human genome. As used herein, a “SNP” is a common alteration that occurs in a single nucleotide base in a stretch of DNA. For example, a SNP may occur once per every 1000 bases of DNA. A SNP may be involved in a disease process, however, the vast majority may not be disease-associated. Given a genetic map based on the occurrence of such SNPs, individuals can be grouped into genetic categories depending on a particular pattern of SNPs in their individual genome. In such a manner, treatment regimens can be tailored to groups of genetically similar individuals, taking into account traits that may be common among such genetically similar individuals.

[1425] Alternatively, a method termed the “candidate gene approach”, can be utilized to identify genes that predict drug response. According to this method, if a gene that encodes a drug's target is known (e.g., a GEF32529 polypeptide of the present invention), all common variants of that gene can be fairly easily identified in the population and it can be determined if having one version of the gene versus another is associated with a particular drug response.

[1426] As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymorphisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an explanation as to why some patients do not obtain the expected drug effects or show exaggerated drug response and serious toxicity after taking the standard and safe dose of a drug. These polymorphisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the gene coding for CYP2D6 is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C 19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.

[1427] Alternatively, a method termed the “gene expression profiling”, can be utilized to identify genes that predict drug response. For example, the gene expression of an animal dosed with a drug (e.g., a GEF32529 molecule or GEF32529 modulator of the present invention) can give an indication whether gene pathways related to toxicity have been turned on.

[1428] Information generated from more than one of the above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a GEF32529 molecule or GEF32529 modulator, such as a modulator identified by one of the exemplary screening assays described herein.

[1429] 4. Use of GEF32529 Molecules as Surrogate Markers

[1430] The GEF32529 molecules of the invention are also useful as markers of disorders or disease states, as markers for precursors of disease states, as markers for predisposition of disease states, as markers of drug activity, or as markers of the pharmacogenomic profile of a subject. Using the methods described herein, the presence, absence and/or quantity of the GEF32529 molecules of the invention may be detected, and may be correlated with one or more biological states in vivo. For example, the GEF32529 molecules of the invention may serve as surrogate markers for one or more disorders or disease states or for conditions leading up to disease states. As used herein, a “surrogate marker” is an objective biochemical marker which correlates with the absence or presence of a disease or disorder, or with the progression of a disease or disorder (e.g., with the presence or absence of a tumor). The presence or quantity of such markers is independent of the disease. Therefore, these markers may serve to indicate whether a particular course of treatment is effective in lessening a disease state or disorder. Surrogate markers are of particular use when the presence or extent of a disease state or disorder is difficult to assess through standard methodologies (e.g., early stage tumors), or when an assessment of disease progression is desired before a potentially dangerous clinical endpoint is reached (e.g., an assessment of cardiovascular disease may be made using cholesterol levels as a surrogate marker, and an analysis of HIV infection may be made using HIV RNA levels as a surrogate marker, well in advance of the undesirable clinical outcomes of myocardial infarction or fully-developed AIDS). Examples of the use of surrogate markers in the art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264; and James (1994) AIDS Treatment News Archive 209.

[1431] The GEF32529 molecules of the invention are also useful as pharmacodynamic markers. As used herein, a “pharmacodynamic marker” is an objective biochemical marker which correlates specifically with drug effects. The presence or quantity of a pharnacodynamic marker is not related to the disease state or disorder for which the drug is being administered; therefore, the presence or quantity of the marker is indicative of the presence or activity of the drug in a subject. For example, a pharmacodynamic marker may be indicative of the concentration of the drug in a biological tissue, in that the marker is either expressed or transcribed or not expressed or transcribed in that tissue in relationship to the level of the drug. In this fashion, the distribution or uptake of the drug may be monitored by the pharmacodynamic marker. Similarly, the presence or quantity of the pharmacodynamic marker may be related to the presence or quantity of the metabolic product of a drug, such that the presence or quantity of the marker is indicative of the relative breakdown rate of the drug in vivo. Pharmacodynamic markers are of particular use in increasing the sensitivity of detection of drug effects, particularly when the drug is administered in low doses. Since even a small amount of a drug may be sufficient to activate multiple rounds of marker (e.g., a GEF32529 marker) transcription or expression, the amplified marker may be in a quantity which is more readily detectable than the drug itself. Also, the marker may be more easily detected due to the nature of the marker itself; for example, using the methods described herein, anti-GEF32529 antibodies may be employed in an immune-based detection system for a GEF32529 polypeptide marker, or GEF32529-specific radiolabeled probes may be used to detect a GEF32529 mRNA marker. Furthermore, the use of a pharmacodynamic marker may offer mechanism-based prediction of risk due to drug treatment beyond the range of possible direct observations. Examples of the use of pharnacodynamic markers in the art include: Matsuda et al. U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90: 229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3: S21-S24; and Nicolau (1999) Am, J. Health-Syst. Pharm. 56 Suppl. 3: S16-S20.

[1432] The GEF32529 molecules of the invention are also useful as pharmacogenomic markers. As used herein, a “pharmacogenomic marker” is an objective biochemical marker which correlates with a specific clinical drug response or susceptibility in a subject (see, e.g., McLeod et al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence or quantity of the pharmacogenomic marker is related to the predicted response of the subject to a specific drug or class of drugs prior to administration of the drug. By assessing the presence or quantity of one or more pharmacogenomic markers in a subject, a drug therapy which is most appropriate for the subject, or which is predicted to have a greater degree of success, may be selected. For example, based on the presence or quantity of RNA, or polypeptide (e.g., GEF32529 polypeptide or RNA) for specific tumor markers in a subject, a drug or course of treatment may be selected that is optimized for the treatment of the specific tumor likely to be present in the subject. Similarly, the presence or absence of a specific sequence mutation in GEF32529 DNA may correlate GEF32529 drug response. The use of pharmacogenomic markers therefore permits the application of the most appropriate treatment for each subject without having to administer the therapy.

[1433] VI. Electronic Apparatus Readable Media and Arrays

[1434] Electronic apparatus readable media comprising GEF32529 sequence information is also provided. As used herein, “GEF32529 sequence information” refers to any nucleotide and/or amino acid sequence information particular to the GEF32529 molecules of the present invention, including but not limited to full-length nucleotide and/or amino acid sequences, partial nucleotide and/or amino acid sequences, polymorphic sequences including single nucleotide polymorphisms (SNPs), epitope sequences, and the like. Moreover, information “related to” said GEF32529 sequence information includes detection of the presence or absence of a sequence (e.g., detection of expression of a sequence, fragment, polymorphism, etc.), determination of the level of a sequence (e.g., detection of a level of expression, for example, a quantitative detection), detection of a reactivity to a sequence (e.g., detection of protein expression and/or levels, for example, using a sequence-specific antibody), and the like. As used herein, “electronic apparatus readable media” refers to any suitable medium for storing, holding or containing data or information that can be read and accessed directly by an electronic apparatus. Such media can include, but are not limited to: magnetic storage media, such as floppy discs, hard disc storage medium, and magnetic tape; optical storage media such as compact disc; electronic storage media such as RAM, ROM, EPROM, EEPROM and the like; general hard disks and hybrids of these categories such as magnetic/optical storage media. The medium is adapted or configured for having recorded thereon GEF32529 sequence information of the present invention.

[1435] As used herein, the term “electronic apparatus” is intended to include any suitable computing or processing apparatus or other device configured or adapted for storing data or information. Examples of electronic apparatus suitable for use with the present invention include stand-alone computing apparatus; networks, including a local area network (LAN), a wide area network (WAN) Internet, Intranet, and Extranet; electronic appliances such as a personal digital assistants (PDAs), cellular phone, pager and the like; and local and distributed processing systems.

[1436] As used herein, “recorded” refers to a process for storing or encoding information on the electronic apparatus readable medium. Those skilled in the art can readily adopt any of the presently known methods for recording information on known media to generate manufactures comprising the GEF32529 sequence information.

[1437] A variety of software programs and formats can be used to store the sequence information on the electronic apparatus readable medium. For example, the sequence information can be represented in a word processing text file, formatted in commercially-available software such as WordPerfect and Microsoft Word, or represented in the form of an ASCII file, stored in a database application, such as DB2, Sybase, Oracle, or the like, as well as in other forms. Any number of dataprocessor structuring formats (e.g., text file or database) may be employed in order to obtain or create a medium having recorded thereon the GEF32529 sequence information.

[1438] By providing GEF32529 sequence information in readable form, one can routinely access the sequence information for a variety of purposes. For example, one skilled in the art can use the sequence information in readable form to compare a target sequence or target structural motif with the sequence information stored within the data storage means. Search means are used to identify fragments or regions of the sequences of the invention which match a particular target sequence or target motif.

[1439] The present invention therefore provides a medium for holding instructions for performing a method for determining whether a subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder, wherein the method comprises the steps of determining GEF32529 sequence information associated with the subject and based on the GEF32529 sequence information, determining whether the subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder and/or recommending a particular treatment for the disease, disorder or pre-disease condition.

[1440] The present invention further provides in an electronic system and/or in a network, a method for determining whether a subject has a GEF32529-associated disease or disorder or a pre-disposition to a disease associated with a GEF32529 wherein the method comprises the steps of determining GEF32529 sequence information associated with the subject, and based on the GEF32529 sequence information, determining whether the subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder, and/or recommending a particular treatment for the disease, disorder or pre-disease condition. The method may further comprise the step of receiving phenotypic information associated with the subject and/or acquiring from a network phenotypic information associated with the subject.

[1441] The present invention also provides in a network, a method for determining whether a subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder associated with GEF32529, said method comprising the steps of receiving GEF32529 sequence information from the subject and/or information related thereto, receiving phenotypic information associated with the subject, acquiring information from the network corresponding to GEF32529 and/or a GEF32529-associated disease or disorder, and based on one or more of the phenotypic information, the GEF32529 information (e.g., sequence information and/or information related thereto), and the acquired information, determining whether the subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1442] The present invention also provides a business method for determining whether a subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder, said method comprising the steps of receiving information related to GEF32529 (e.g., sequence information and/or information related thereto), receiving phenotypic information associated with the subject, acquiring information from the network related to GEF32529 and/or related to a GEF32529-associated disease or disorder, and based on one or more of the phenotypic information, the GEF32529 information, and the acquired information, determining whether the subject has a GEF32529-associated disease or disorder or a pre-disposition to a GEF32529-associated disease or disorder. The method may further comprise the step of recommending a particular treatment for the disease, disorder or pre-disease condition.

[1443] The invention also includes an array comprising a GEF32529 sequence of the present invention. The array can be used to assay expression of one or more genes in the array. In one embodiment, the array can be used to assay gene expression in a tissue to ascertain tissue specificity of genes in the array. In this manner, up to about 7600 genes can be simultaneously assayed for expression, one of which can be GEF32529. This allows a profile to be developed showing a battery of genes specifically expressed in one or more tissues.

[1444] In addition to such qualitative determination, the invention allows the quantitation of gene expression. Thus, not only tissue specificity, but also the level of expression of a battery of genes in the tissue is ascertainable. Thus, genes can be grouped on the basis of their tissue expression per se and level of expression in that tissue. This is useful, for example, in ascertaining the relationship of gene expression between or among tissues. Thus, one tissue can be perturbed and the effect on gene expression in a second tissue can be determined. In this context, the effect of one cell type on another cell type in response to a biological stimulus can be determined. Such a determination is useful, for example, to know the effect of cell-cell interaction at the level of gene expression. If an agent is administered therapeutically to treat one cell type but has an undesirable effect on another cell type, the invention provides an assay to determine the molecular basis of the undesirable effect and thus provides the opportunity to co-administer a counteracting agent or otherwise treat the undesired effect. Similarly, even within a single cell type, undesirable biological effects can be determined at the molecular level. Thus, the effects of an agent on expression of other than the target gene can be ascertained and counteracted.

[1445] In another embodiment, the array can be used to monitor the time course of expression of one or more genes in the array. This can occur in various biological contexts, as disclosed herein, for example development of a GEF32529-associated disease or disorder, progression of GEF32529-associated disease or disorder, and processes, such a cellular transformation associated with the GEF32529-associated disease or disorder.

[1446] The array is also useful for ascertaining the effect of the expression of a gene on the expression of other genes in the same cell or in different cells (e.g., ascertaining the effect of GEF32529 expression on the expression of other genes). This provides, for example, for a selection of alternate molecular targets for therapeutic intervention if the ultimate or downstream target cannot be regulated.

[1447] The array is also useful for ascertaining differential expression patterns of one or more genes in normal and abnormal cells. This provides a battery of genes (e.g., including GEF32529) that could serve as a molecular target for diagnosis or therapeutic intervention.

[1448] This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are incorporated herein by reference.

EXAMPLES

Example 1

[1449] Identification and Characterization of Human GEF32529 cDNA

[1450] In this example, the identification and characterization of the gene encoding human GEF32529 (clone 32529) is described.

[1451] Isolation of the Human GEF32529 cDNA

[1452] The invention is based, at least in part, on the discovery of a human gene encoding a novel polypeptide, referred to herein as human GEF32529. The entire sequence of the human clone 32529 was determined and found to contain an open reading frame termed human “GEF32529. ” The nucleotide sequence of the human GEF32529 gene is set forth in FIGS. 24A-E and in the Sequence Listing as SEQ ID NO:17. The amino acid sequence of the human GEF32529 expression product is set forth in FIGS. 24A-E and in the Sequence Listing as SEQ ID NO:18. The GEF32529 polypeptide comprises about 802 amino acids. The coding region (open reading frame) of SEQ ID NO:17 is set forth as SEQ ID NO:19. Clone 32529, comprising the coding region of human GEF32529, was deposited with the American Type Culture Collection (ATCC®), 10801 University Boulevard, Manassas, Va. 20110-2209, on ______, and assigned Accession No. ______.

[1453] Analysis of the Human GEF32529 Molecules

[1454] A search using the polypeptide sequence of SEQ ID NO:18 was performed against the HMM database in PFAM (FIGS. 26A-C) resulting in the identification of a GEF domain in the amino acid sequence of human GEF32529 at about residues 380-559 of SEQ ID NO:18 (score=64.5), a potential PGAM domain in the amino acid sequence of human GEF32529 at about residues 592-598 of SEQ ID NO:18 (score=5.2), a PH domain in the amino acid sequence of human GEF32529 at about residues 593-704 of SEQ ID NO:18 (score=33.0), and a SH3 domain in the amino acid sequence of human GEF32529 at about residues 724-774 of SEQ ID NO:18 (score=29.7).

[1455] A search using the polypeptide sequence of SEQ ID NO:18 was performed against the HMM database in SMART (FIGS. 26A-C), a database of HMMs which has been revised and updated by Applicant, confirming the identification of the GEF domain in the amino acid sequence of human GEF32529 (e.g., at about residues 380-559 of SEQ ID NO:18 (score=158.4)), the PH domain in the amino acid sequence of human GEF32529 (e.g., at about residues 593-706 of SEQ ID NO:18 (score=32.9)), and the SH3 domain in the amino acid sequence of human GEF32529 (e.g., at about residues 718-775 of SEQ ID NO:18 (score=47.6)).

[1456] The amino acid sequence of human GEF32529 was analyzed using the program PSORT (http://www.psort.nibb.ac.p) to predict the localization of the proteins within the cell. This program assesses the presence of different targeting and localization amino acid sequences within the query sequence. The results of the analyses show that human GEF32529 may be localized to the nucleus or to the cytoplasm.

[1457] Searches of the amino acid sequence of human GEF32529 were further performed against the Prosite database. These searches resulted in the identification in the amino acid sequence of human GEF32529 of a potential N-glycosylation site, a potential glycosaminoglycan attachment site, a number of potential cAMP- and cGMP-dependent protein kinase phosphorylation sites, a number of potential protein kinase C phosphorylation sites, a number of potential casein kinase II phosphorylation sites, and a number of potential N-myristoylation sites.

[1458] A MEMSAT analysis of the polypeptide sequence of SEQ ID NO:18 was also performed, predicting a possible transmembrane domain in the amino acid sequence of human GEF32529 (SEQ ID NO:18) at about residues 390-406.

[1459] Further hits were identified by using the amino acid sequence of GEF32529 (SEQ ID NO:18) to search the ProDom database. Numerous matches against proteins and/or protein domains described as “TIM oncogene guanine nucleotide neuroblastoma factor exchange”, “neuroblastoma”, “KIAA0915”, “BCDNA:GH03693 K07D4.7”, “TIM guanine nucleotide oncogene factor exchange”, “factor releasing guanine-nucleotide exchange proto-oncogene domain binding phorbol-ester”, “polymerase subunit gamma III DNA”, “receptor dopamine family polymorphism G-protein D4 D2C multigene. coupled repeat”, “Rho exchange CG1225 nucleotide factor guanine”, “FRGA”, “early immediate transcription factor response activated ETR101 growth inducible cyclohexamide-induced”, “CG10555”, “transporter ABC”, “QCCE-12673 brain cDNA”, “membrane”, “element transposable TN4556 transposon”, “kinase serine/threonine serine/threonine-protein”, “CG5606”, “cell trophinin-associated repeat adhesion tastin trophinin-assisting”, “UL71”, “calcium binding”, and the like were identified.

[1460] Tissue Distribution of human GEF32529 mRNA

[1461] This example describes the tissue distribution of human GEF32529 mRNA, as may be determined by in situ analysis using oligonucleotide probes based on the human GEF32529 sequence.

[1462] For in situ analysis, various tissues, e.g. tissues obtained from brain, are first frozen on dry ice. Ten-micrometer-thick sections of the tissues are postfixed with 4% formaldehyde in DEPC treated 1×phosphate- buffered saline at room temperature for 10 minutes before being rinsed twice in DEPC 1× phosphate-buffered saline and once in 0.1 M triethanolamine-HCl (pH 8.0). Following incubation in 0.25% acetic anhydride-0.1 M triethanolamine-HC1 for 10 minutes, sections are rinsed in DEPC 2×SSC (1×SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Tissue is then dehydrated through a series of ethanol washes, incubated in 100% chloroform for 5 minutes, and then rinsed in 100% ethanol for 1 minute and 95% ethanol for 1 minute and allowed to air dry.

[1463] Hybridizations are performed with 35S-radiolabeled (5×107 cpm/ml) cRNA probes. Probes are incubated in the presence of a solution containing 600 mM NaCl, 10 mM Tris (pH 7.5), 1 mM EDTA, 0.01% sheared salmon sperm DNA, 0.01% yeast tRNA, 0.05% yeast total RNA type X1, 1×Denhardt's solution, 50% formamide, 10% dextran sulfate, 100 mM dithiothreitol, 0.1% sodium dodecyl sulfate (SDS), and 0.1% sodium thiosulfate for 18 hours at 55° C.

[1464] After hybridization, slides are washed with 2×SSC. Sections are then sequentially incubated at 37° C. in TNE (a solution containing 10 mM Tris-HCl (pH 7.6), 500 mM NaCl, and 1 mM EDTA), for 10 minutes, in TNE with 10 μg of RNase A per ml for 30 minutes, and finally in TNE for 10 minutes. Slides are then rinsed with 2×SSC at room temperature, washed with 2×SSC at 50° C. for 1 hour, washed with 0.2×SSC at 55° C. for 1 hour, and 0.2×SSC at 60° C. for 1 hour. Sections are then dehydrated rapidly through serial ethanol-0.3 M sodium acetate concentrations before being air dried and exposed to Kodak Biomax MR scientific imaging film for 24 hours and subsequently dipped in NB-2 photoemulsion and exposed at 4° C. for 7 days before being developed and counter stained.

EXAMPLE 2

[1465] Expression of Recombinant GEF32529 Polypeptide in Bacterial Cells

[1466] In this example, human GEF32529 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized. Specifically, GEF32529 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199. Expression of the GST-GEF32529 fusion polypeptide in PEB199 is induced with IPTG. The recombinant fusion polypeptide is purified from crude bacterial lysates of the induced PEB199 strain by affinity chromatography on glutathione beads. Using polyacrylamide gel electrophoretic analysis of the polypeptide purified from the bacterial lysates, the molecular weight of the resultant fusion polypeptide is determined.

EXAMPLE 3

[1467] Expression of Recombinant GEF32529 Polypeptide in Cos Cells

[1468] To express the human GEF32529 gene in COS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego, Calif.) is used. This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site. A DNA fragment encoding the entire GEF32529 polypeptide and an HA tag (Wilson et al. (1984) Cell 37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment is cloned into the polylinker region of the vector, thereby placing the expression of the recombinant polypeptide under the control of the CMV promoter.

[1469] To construct the plasmid, the human GEF32529 DNA sequence is amplified by PCR using two primers. The 5′ primer contains the restriction site of interest followed by approximately twenty nucleotides of the GEF32529 coding sequence starting from the initiation codon; the 3′ end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag or FLAG tag and the last 20 nucleotides of the GEF32529 coding sequence. The PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, Mass.). Preferably the two restriction sites chosen are different so that the GEF32529 gene is inserted in the correct orientation. The ligation mixture is transformed into E. coli cells (strains HB101, DH5α, SURE, available from Stratagene Cloning Systems, La Jolla, Calif., can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence of the correct fragment.

[1470] COS cells are subsequently transfected with the human GEF32529-pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co-precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation. Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. The expression of the IC54420 polypeptide is detected by radiolabeling (35S-methionine or 35S-cysteine available from NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly, the cells are labeled for 8 hours with 35S-methionine (or 35S-cysteine). The culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

[1471] Alternatively, DNA containing the human GEF32529 coding sequence is cloned directly into the polylinker of the pCDNA/Amp vector using the appropriate restriction sites. The resulting plasmid is transfected into COS cells in the manner described above, and the expression of the GEF32529 polypeptide is detected by radiolabeling and immunoprecipitation using a GEF32529-specific monoclonal antibody.

[1472] Equivalents

[1473] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Number	Date	Country
60191859	Mar 2000	US
60193808	Mar 2000	US
60198466	Apr 2000	US
60212222	Jun 2000	US
60218383	Jul 2000	US

	Number	Date	Country
Parent	10160568	May 2002	US
Child	10860777	Jun 2004	US

	Number	Date	Country
Parent	09816860	Mar 2001	US
Child	10160568	May 2002	US
Parent	09823950	Mar 2001	US
Child	10860777	Jun 2004	US
Parent	09838529	Apr 2001	US
Child	10160568		US
Parent	09884870	Jun 2001	US
Child	10160568		US
Parent	09907495	Jul 2001	US
Child	10160568		US

Novel human 26649, 3259, 57809, 57798, 33358, and 32529 molecules and uses therefor

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