Patent Grant
8148129
The regulation of gene expression by transcription factors is a fundamental aspect of the physiology of all cells, whether prokaryotic or eukaryotic. In eukaryotic organisms, for instance, a variety of transcription factors govern cell growth, differentiation, and death. The appropriate spatial and temporal expression of specific transcription factors governs development. As examples, transcription factors such as Myc and E2F control progression through the cell cycle; homeodomain, paired box, and forkhead transcription factors, among others, are involved in embryonic development; p53 is involved with tumor suppression and cell death; steroid hormone receptors, such as sex hormone, glucocorticoid, mineralocorticoid, and thyroid hormone receptors have pleiotrophic effects on various aspects of physiology.
The aberrant expression of transcription factors can lead to abnormal development and various disease states. The inappropriate expression of proto-oncogenes such as c-Myc through chromosomal translocation can lead to cancers such as Burkitt's lymphoma. The formation of a PML-RARa fusion protein has been shown to be responsible for acute promyelocytic leukemia. Loss of p53 expression results in increased susceptibility to various cancers. The inappropriate expression or loss of expression of heart specific transcription factors such as Tbx1, Tbx5, NRx2.5, Gata4, Sal4, and Eya4 have been shown to result in congenital heart defects.
Improved methods for regulating gene expression by modulating transcription factor function would result in more optimal treatment of many diseases.
One disease which might be approached by modulating transcription factor function is acquired immune deficiency syndrome (AIDS). Human immunodeficiency virus (HIV) has been identified as the etiological agent responsible for AIDS, a fatal disease characterized by destruction of the immune system and the inability to fight off life threatening opportunistic infections. Recent statistics indicate that as many as 33 million people worldwide are infected with the virus. In addition to the large number of individuals already infected, the virus continues to spread. Estimates from 1998 point to close to 6 million new infections in that year alone. In the same year there were approximately 2.5 million deaths associated with HIV and AIDS.
HIV is a member of the class of viruses known as retroviruses. The retroviral genome is composed of RNA, which is converted to DNA by reverse transcription. This retroviral DNA is then stably integrated into a host cell's chromosome and, employing the replication machinery of the host cells, produces new retroviral particles and advances the infection to other cells. HIV appears to have a particular affinity for the human T-4 lymphocyte cell, which plays a vital role in the body's immune system. HIV infection of these white blood cells depletes this white cell population. Eventually, the immune system is rendered inoperative and ineffective against various opportunistic diseases such as, among others, pneumocystic carini pneumonia, Kaposi's sarcoma, and cancer of the lymph system.
There are currently a number of antiviral drugs available to combat the infection. These drugs can be divided into four classes based on the viral protein they target and their mode of action. In particular, one class of such antiviral drugs are competitive inhibitors of the aspartyl protease expressed by HIV. Other agents are nucleoside reverse transcriptase inhibitors that behave as substrate mimics to halt viral cDNA synthesis. A class of non-nucleoside reverse transcriptase inhibitors inhibit the synthesis of viral cDNA via a non-competitive (or uncompetitive) mechanism. Another class are drugs that block viral fusion. Used alone, these drugs show effectiveness in reducing viral replication. However, the effects are only temporary as the virus readily develops resistance to all known agents.
As indicated above, a number of critical points in the HIV life cycle have been identified as possible targets for antiviral drugs including (1) the initial attachment of the virion to the T-4 lymphocyte or macrophage site; (2) the transcription of viral RNA to viral DNA (reverse transcriptase, RT); and (3) the processing of gag-pol protein by HIV protease. An additional, potentially attractive therapeutic target is transcription of the HIV genome. Transcription of the HIV genome is essential for replication of the virus after integration of viral DNA into a host cell chromosome. However, attempts to target HIV transcription have been hampered, in part, by the fact that transcription of the integrated HIV genome utilizes the host cell transcriptional machinery as well as viral transcription factors. Thus, therapies that attempt to target the transcription of the HIV genome may also interfere with transcription of normal host cell genes. Attempts have been made to target specifically HIV transcription by the generation of dominant negative forms of Tat, a virally encoded transcription factor. However, these dominant forms have been shown to have poor activity at inhibiting HIV transcription and viral replication.
Effective new methods to target underexploited aspects of the HIV lifecycle, such as transcription of the HIV genome would be desirable.
The present application demonstrates that potent dominant negative regulators of transcription can be generated by linking a transcription factor to a protein that localizes to the transcriptional machinery.
In one embodiment, a method of regulating transcription of a gene is provided in which a nucleic acid construct is expressed in a cell in an amount sufficient for modulation of transcription, where the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery. In various aspects, the transcription factor protein can be viral transcription factors, nuclear proto-oncogene or oncogene proteins, nuclear tumor suppressor proteins, heart specific transcription factors, and immune system transcription factors. In some further aspects, the viral transcription factors can be HIV-Tat, HPV-E2, HPV-E7, BPV-E2, Adenovirus IVa2, HSV-1 ICP4, EBNA-LP, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, BZLF-1, CMV-IE-1, CMV-IE2, HHSV-8 K bZIP, HBV Hbx, Poxvirus Vaccinia, VETF, HCV NS5A, T-Ag, Adenovirus E1A, Herpesvirus VP16, HTLV Tax, Hepadnavirus X protein, or Baculovirus AcNPV IE-1. In some further aspects, the nuclear proto-oncogene or oncogene proteins can be Abl, Myc, Myb, Rel, Jun, Fos, Spl, Apl, NF-κB, STAT 3 or 5, β-catenin, Notch, GLI, or PML-RARα. In some further aspects, heart specific transcription factors can be Nkx 2, 3, 4, or 5, TBX5, GATA 4, 5, or 6, or MEF2. In some further aspects, the immune cell specific transcription factor can be Ikaros, PU.1, PAX-5, Oct-2, or BOB.1/OBF.1.
In various embodiments, the transcription factor can be a dominant negative transcription factor, or fragment thereof. In further embodiments, the transcription factor can be either a transcriptional activator or repressor. In yet further embodiments, the transcription factor can be an activation domain (AD) fragment of the transcription factor. In yet further embodiments, the transcription factor can be Tat or an activation domain fragment or other fragment of Tat.
In some embodiments, the protein or a fragment thereof that localizes to the transcriptional machinery is a protein with nuclear localization, a component of the transcriptional machinery, or a protein that functions in co-transcriptional processing of RNA. In some aspects, the protein that functions in co-transcriptional processing of RNA is a capping factor, a splicing factor, a polyadenylation factor, an RNA export factor, or a translation factor. In some aspects, the splicing factor is an RS domain containing protein. In yet other aspects, the splicing factor is SF1, U2AF65, or 9G8, and the polyadenylation factor is CstF1.
In some embodiments, the modulation of transcription is inhibition of transcription by at least 25%, or at least 50%, or at least 75%, or at least 95%. In some aspects, the modulation of transcription is by inhibition of transcriptional initiation, or elongation, or termination. In some embodiments, the modulation of transcription is activation of transcription.
In some embodiments, the cell is a T-cell infected with an immunodeficiency virus that can be HIV, FIV, SIV, or BIV. In yet further embodiments, the cell is a cancer cell, heart cell, or immune system cell. In some aspects, the cancer cell is a carcinoma, sarcoma, adenocarcinoma, lymphoma, leukemia, or solid tumors of the kidney, breast, lung, bladder, colon, ovarian, prostate, pancreas, stomach, brain, head and neck, skin, uterine, testicular, glioma, esophagus, or liver. In some aspects, the immune system cell can be a B-cell, T-cell, macrophage, or dendritic cell.
Also included as embodiments are vectors and cells containing the nucleic acids of the embodiments above, as well as, the proteins encoded by these nucleic acids. In further aspects, a composition comprising the nucleic acid construct or protein of the above embodiments and a physiologically acceptable carrier is provided.
In yet further embodiments, a method of regulating transcription of a gene is provided by expressing a nucleic acid construct in a cell in an amount sufficient for modulation of transcription, in which the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a splicing factor or a fragment thereof.
In still further embodiments, a method of inhibiting replication of an immunodeficiency virus by expressing a nucleic acid construct in a cell in an amount sufficient for modulation of viral transcription, in which the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery.
In another embodiment, provided is a method of inhibiting replication of an immunodeficiency virus by expressing in a cell a nucleic acid construct in an amount sufficient for modulation of viral transcription, in which the construct contains a first nucleic acid sequence encoding a Tat protein or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery.
In another embodiment, provided is a method of inhibiting replication of an immunodeficiency virus by expressing in a cell a nucleic acid construct in an amount sufficient for modulation of viral transcription, in which the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a splicing factor or a fragment thereof.
In another embodiment, provided is a method of treating a subject infected with an immunodeficiency virus by administering a nucleic acid construct in an amount sufficient for inhibition of viral transcription, in which the construct contains a first nucleic acid sequence encoding a transcription factor or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery. In some aspects, the treating is with a protein of the embodiments above.
In another embodiment, provided is a method of inhibiting transcription of a HIV genome in a cell by expressing in the cell a nucleic acid construct in an amount sufficient for inhibition of the transcription of the HIV genome, in which the construct contains a first nucleic acid sequence encoding a Tat protein or a fragment thereof linked to a second nucleic acid sequence encoding a U2AF65 protein or a fragment thereof.
In another embodiment, provided is a method of treating a subject with cancer by expressing in the subject a nucleic acid construct in an amount sufficient for modulation of transcription, in which the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery. In some aspects, the treating is with a protein of the embodiments above.
In another embodiment, provided is a method of treating or preventing a disease in a subject by expressing in the subject a nucleic acid construct in an amount sufficient for modulation of transcription, in which the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery, where the disease is viral infection, cancer, heart disease, and inflammation.
In another embodiment, provided is a method of validating a target by expressing a nucleic acid construct in a cell in an amount sufficient for modulation of transcription of the gene for the target, in which the construct contains a first nucleic acid sequence encoding a transcription factor protein or a fragment thereof linked to a second nucleic acid sequence encoding a protein or a fragment thereof that localizes to the transcriptional machinery, where altered expression of the gene for the target provides target confirmation.
The gene product of a dominant negative mutation interferes with the function of a normal, wild-type gene product within the same cell. This usually occurs if the gene product of the dominant negative mutation can still interact with the same elements as the wild-type product, but blocks some aspect of the wild-type protein's function. As an example, in the case of multi-subunit protein complexes, an inactive dominant negative protein can bind to wild-type components of the complex rendering the resulting complex less active or inactive. Genetic engineering has allowed the construction of dominant negative forms of many different types of proteins. In the case of transcription factors, one approach has been to generate transcription factors that lack a gene activation domain but which retain a DNA binding domain. When expressed in cells, such dominant negative proteins are able to bind to their cognate DNA recognition sites thus preventing the binding of a wild type transcription factor and leading to reduced expression of a target gene. However, typically, for dominant negative inhibition to occur, a great excess of dominant negative protein must be expressed in order to effectively out compete the wild-type protein.
A dominant negative approach has previously been used in an attempt to inhibit transcription of the HIV genome and thus viral replication. When a truncated form of Tat, lacking the basic domain, was tested in transient co-transfection experiments, it was found that an 8-30 fold molar excess of the dominant negative Tat over wild-type Tat was required to inhibit the expression of a reporter gene under the control of the HIV-LTR.
The inventors have devised a new method of generating potent dominant negative transcriptional inhibitors for pharmaceutical treatment of diseases, gene therapy, target validation, disease diagnosis, and mechanistic studies of transcription, among other applications. As discussed above, previously described dominant negative transcription factors typically act by competing with other interacting factors or by creating defective oligomers, thus requiring a large excess of inhibitor while providing only a modest amount of inhibition. The inventors have discovered that linking a protein which localizes to the transcriptional machinery to a transcription factor can effectively target and generate high local concentrations of a dominant negative protein, thereby efficiently out-competing wild-type protein when expressed at stoichiometric amounts. In particular, the inventors have made the unexpected finding that fusion of the Tat protein or a fragment thereof, such as the Tat activation domain (Tat AD), to a protein that localizes to the transcriptional machinery, results in a potent inhibitor of transcription of the HIV genome. In particular, when Tat or Tat AD is fused to the splicing factors, SF1 or U2AF65, a potent dominant negative effect is observed. While one embodiment of this invention as described below in the Examples relates to the inhibition of HIV transcription and viral replication, it will be clear to the skilled artisan that the methods of the present invention can be used to generate dominant negative forms of other transcription factors and other classes of proteins.
Dominant Negative Tat
Immediately after HIV infects a cell, the viral RNA is copied into DNA, and the proviral genome is transported to the nucleus where it is integrated into the host genome. Once integrated into the host chromosome, the HIV proviral genome is subject to regulation by a variety of cellular transcription factors, as well as, by virally encoded factors. Among these virally encoded factors, the trans-activator protein (Tat) provides the primary control of HIV transcription.
Transcription of the HIV genome begins at the viral LTR when the host cell RNA polymerase complex binds to the HIV promoter. The HIV LTR, however, is a poor promoter in the absence of Tat. In the absence of Tat, only non-processive (basal) transcription of the HIV genome is observed. However, upon recruitment of Tat to the transcriptional complex at the promoter, transcription of the HIV genome is greatly stimulated. Recruitment of Tat to the HIV promoter is mediated at least in part by the binding of Tat to a short RNA sequence that forms a stem-loop, termed the transactivation-responsive region (TAR), which lies just downstream of the initiation site for transcription. Transcription of TAR by the basal transcriptional machinery to form the TAR RNA stem loop allows Tat to join the complex and stimulate transcription. Upon binding of Tat, it is believed that other cellular factors are recruited to the transcriptional complex that convert the complex into a form that is competent for processive transcript elongation.
In one embodiment of this invention, the inventors have made a fusion of the Tat protein or a fragment thereof, such as the Tat activation domain (Tat AD), to proteins that localize to the transcriptional machinery. When Tat or Tat AD is fused to splicing factors, such as, SF1 or U2AF65, a potent dominant negative effect is observed. Without limiting themselves to any particular mechanism of action, and as explained below in greater detail, the inventors have found that the fused splicing factor proteins act as tethering domains, directing the Tat fusion protein to RNA polymerase at the HIV-1 promoter thus blocking the activity of incoming wild-type Tat proteins. This results in a high local concentration of the inhibiting fusion protein at the site of action.
As used herein, the following terms have the meanings ascribed to them unless specified otherwise.
A “dominant negative” gene product or protein is one that interferes with the function of another gene product or protein. The other gene product affected can be the same or different from the dominant negative protein. Dominant negative gene products can be of many forms, including truncations, full length proteins with point mutations or fragments thereof, or fusions of full length wild type or mutant proteins or fragments thereof with other proteins. The level of inhibition observed can be very low. For example, it may require a large excess of the dominant negative protein compared to the functional protein or proteins involved in a process in order to see an effect. It may be difficult to see effects under normal biological assay conditions.
A “transcription factor” is a protein that regulates transcription. Transcription factors may bind directly to DNA or RNA or may interact with the transcriptional machinery via protein-protein interactions with no direct nucleic acid contact to modulate transcription. Transcription factors in general are reviewed in Barnes and Adcock, Clin. Exp. Allergy 25 Suppl. 2: 46-9 (1995), Roeder, Methods Enzymol. 273: 165-71 (1996), and Brivanlou and Darnell, Science 1 Feb. 2002: 813-818 (2002), among other sources.
A “promoter” is defined as an array of nucleic acid control sequences that direct transcription. As used herein, a promoter typically includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of certain RNA polymerase II type promoters, a TATA element, enhancer, CCAAT box, SP-1 site, etc. As used herein, a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. The promoters often have an element that is responsive to transactivation by a DNA-binding moiety such as a polypeptide, e.g., a nuclear receptor, Gal4, the lac repressor and the like.
A “target site” is the nucleic acid sequence recognized by a transcription factor protein. A single target site typically has about four to about ten or more base pairs. The target site is in any position that allows regulation of gene expression, e.g., adjacent to, up- or downstream of the transcription initiation site; proximal to an enhancer or other transcriptional regulation element such as a repressor (e.g., SP-1 binding sites, hypoxia response elements, nuclear receptor recognition elements, p53 binding sites, etc.), RNA polymerase pause sites; and intron/exon boundaries.
“Linking” or “fusing” as used in this application refers to entities that are directly linked, or linked via an amino acid linker, the size and composition of which can vary, or linked via a chemical linker.
The term “transcriptional machinery” generally refers to the complex of cellular components responsible for making RNA from a DNA template and related co-transcriptional RNA processing. The complex responsible for transcription in a cell is referred to as RNA polymerase. During transcription, a variety of factors join the RNA polymerase complex to effect various aspects of transcription and co-transcriptional RNA processing as described below. In eukaryotic cells, three forms of RNA polymerase exist, termed RNA polymerases I, II, and III. RNA polymerase I synthesizes a pre-rRNA 45S, which matures into 28 S, 18S and 5, 8 S rRNAs which form the major RNA portions of the ribosome. RNA polymerase II synthesizes precursors of mRNAs and most snRNA. Because of the large variety of cellular genes are transcribed by thus polymerase, RNAP II is subject to the highest level of control, requiring a wide range of transcription factors depending on the promoter. RNA polymerase III is responsible for the synthesis of tRNAs, rRNA 5S and other small RNAs found in the nucleus and cytosol. Additionally, other RNA polymerase types are found in mitochondria and chloroplasts.
A 550 kDa complex of 12 subunits, RNAP II is the most intensively studied type of RNA polymerase. A wide range of transcription factors are required for it to bind to its promoters and to begin transcription. In the process of transcription, there are three main stages: (1) initiation, which requires construction of the RNA polymerase complex on the gene's promoter; (2) elongation, during which the RNA transcript is made from the DNA template; (3) and termination, the step at which the formation of the RNA transcript is completed and disassembly of the RNA polymerase complex occurs.
The components of the transcriptional machinery that may be targeted by this invention comprise any factor that is brought into the RNA polymerase complex and can be exemplified by the order in which the TAFs (TBP Associated Factors) attach to form a polymerase complex on a promoter. TBP (TATA Binding Protein) and an attached complex of TAFs, collectively known as TFIID (Transcription Factor for polymerase II D), bind at the TATA box, although not all promoters have the TATA box. TFIIA (three subunits) binds TFIID and DNA, stabilizing the first interactions. TFIIB binds between TFIID and the location of Pol II binding in the near future. TFIIB binds partially sequence specifically, with some preference for BRE. TFIIF and Pol II (two subunits, RAP30 and RAP74, showing some similarity to bacterial sigma factors) enter the complex together. TFIIF helps to speed up the polymerization process. TFIIE enters the complex, and helps to open and close the PolII's ‘Jaw’ like structure, which enables movement down the DNA strand. TFIIE and TFIIH enter concomitantly. Finally TFIIH binds. TFIIH is a large protein complex that contains among others the CDK7/cyclin H kinase complex and a DNA helicase. TFIIH has three functions: it binds specifically to the template strand to ensure that the correct strand of DNA is transcribed and melts or unwinds the DNA (ATP dependently) to separate the two strands using its Helicase activity. It has a kinase activity that phosphorylates the C-terminal domain (CTD) of Pol II at the amino acid serine. This switches the RNA polymerase to start producing RNA, which marks the end of initiation and the start of elongation. Finally it is essential for Nucleotide Excision Repair (NER) of damaged DNA. TFIIH and TFIIE strongly interact with one another. TFIIE affects TFIIH's catalytic activity. Without TFIIE, TFIIH will not unwind the promoter. Mediator then encases all the transcription factors and the Pol II. Mediator interacts with enhancers, areas very far away (upstream or downstream) that help regulate transcription.
A “protein that localizes to the transcriptional machinery” is one that is capable of associating or interacting with the transcriptional machinery as described above or a component thereof. The association or interaction may be non-covalent or covalent and may be reversible or non-reversible. Examples of proteins that localize to the transcriptional machinery include nuclear localized proteins, RNA processing proteins, components of the transcriptional machinery, and proteins involved in co-transcriptional processes. Among the co-transcriptional processes that are subjects of the invention are capping, splicing, polyadenylation, RNA export, translation.
An RS domain containing protein (also referred to in the literature as an SR protein) is a protein with a domain that contains multiple arginine and serine di-peptides (single-letter code RS) and/or serine and arginine di-peptides (single-letter code SR). RS domains are found in a number of cellular proteins, particularly those involved with pre-mRNA splicing and RNA processing events.
A “transcriptional activator” and a “transcriptional repressor” refer to proteins or effector domains of proteins that have the ability to modulate transcription, by binding directly to DNA or RNA or by interacting with the transcriptional machinery via protein-protein interactions with no direct nucleic acid contact. Such proteins include, e.g., transcription factors and co-factors (e.g., KRAB, MAD, ERD, SID, nuclear factor kappa B subunit p65, early growth response factor 1, and nuclear hormone receptors, VP16, VP64), endonucleases, integrases, recombinases, methyltransferases, histone acetyltransferases, histone deacetylases etc. Activators and repressors include co-activators and co-repressors (see, e.g., Utley et al., Nature 394:498-502 (1998)).
The terms “modulating transcription” “inhibiting transcription” and “activating transcription” of a gene refer to the ability of a dominant negative to activate or inhibit transcription of a gene. Activation includes prevention of transcriptional inhibition (i.e., prevention of repression of gene expression) and inhibition includes prevention of transcriptional activation (i.e., prevention of gene activation).
Modulation can be assayed by determining any parameter that is indirectly or directly affected by the expression of the target gene. Such parameters include, e.g., changes in RNA or protein levels, changes in protein activity, changes in product levels, changes in downstream gene expression, changes in reporter gene transcription (luciferase, CAT, 3-galactosidase, β-glucuronidase, GFP (see, e.g., Mistili & Spector, Nature Biotechnology 15:961-964 (1997)); changes in signal transduction, phosphorylation and dephosphorylation, receptor-ligand interactions, second messenger concentrations (e.g., cGMP, cAMP, IP3, and Ca2+), cell growth, and neovascularization. These assays can be in vitro, in vivo, and ex vivo. Such functional effects can be measured by any means known to those skilled in the art, e.g., measurement of RNA or protein levels, measurement of RNA stability, identification of downstream or reporter gene expression, e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, ligand binding assays; changes in intracellular second messengers such as cGMP and inositol triphosphate (IP3); changes in intracellular calcium levels; cytokine release, and the like.
To determine the level of gene expression modulation by a dominant negative construct, cells contacted with nucleic acids encoding dominant negative or dominant negative proteins are compared to control cells which have not received this treatment. Control samples are assigned a relative gene expression activity value of 100%. Modulation/inhibition of gene expression is achieved when the gene expression activity value relative to the control is about 80%, preferably 50% (i.e., 0.5× the activity of the control), more preferably 25%, more preferably 5-0%. Modulation/activation of gene expression is achieved when the gene expression activity value relative to the control is 110%, more preferably 150% (i.e., 1.5× the activity of the control), more preferably 200-500%, more preferably 1000-2000% or more.
The term “heterologous” is a relative term, which when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature. For instance, a nucleic acid that is recombinantly produced typically has two or more sequences from unrelated genes synthetically arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source. The two nucleic acids are thus heterologous to each other in this context. When added to a cell, the recombinant nucleic acids would also be heterologous to the endogenous genes of the cell. Thus, in a chromosome, a heterologous nucleic acid would include an non-native (non-naturally occurring) nucleic acid that has integrated into the chromosome, or a non-native (non-naturally occurring) extrachromosomal nucleic acid. In contrast, a naturally translocated piece of chromosome would not be considered heterologous in the context of this patent application, as it comprises an endogenous nucleic acid sequence that is native to the mutated cell.
Similarly, a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a “fusion protein,” where the two subsequences are encoded by a single nucleic acid sequence). See, e.g., Ausubel, supra, for an introduction to recombinant techniques.
The term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (naturally occurring) form of the cell or express a second copy of a native gene that is otherwise normally or abnormally expressed, under expressed or not expressed at all.
An “expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell, and optionally integration or replication of the expression vector in a host cell. The expression vector can be part of a plasmid, virus, or nucleic acid fragment, of viral or non-viral origin. Typically, the expression vector includes an “expression cassette,” which comprises a nucleic acid to be transcribed operably linked to a promoter. The term expression vector also encompasses naked DNA operably linked to a promoter.
By “host cell” is meant a cell that contains an expression vector or nucleic acid encoding a dominant negative protein of the invention. The host cell typically supports the replication or expression of the expression vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, fungal, protozoal, higher plant, insect, or amphibian cells, or mammalian cells such as CHO, HeLa, 293, COS-1, and the like, e.g., cultured cells (in vitro), explants and primary cultures (in vitro and ex vivo), and cells in vivo.
“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). The term nucleic acid is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and polynucleotide.
The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acid residues is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
“Conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
As to amino acid sequences, one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
The following eight groups each contain amino acids that are conservative substitutions for one another:
1) Alanine (A), Glycine (G);
2) Aspartic acid (D), Glutamic acid (E);
3) Asparagine (N), Glutamine (Q);
4) Arginine (R), Lysine (K);
5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);
7) Serine (S), Threonine (T); and
8) Cysteine (C), Methionine (M)
(see, e.g., Creighton, Proteins (1984)).
The term “substantially identical” indicates that two or more nucleotide sequences share a majority of their sequence. Generally, this will be at least about 90% of their sequence and preferably about 95% of their sequence. Another indication that sequences are substantially identical is if they hybridize to the same nucleotide sequence under stringent conditions (see, e.g., Sambrook and Russell, eds, Molecular Cloning: A Laboratory Manual, 3rd Ed, vols. 1-3, Cold Spring Harbor Laboratory Press, 2001; and Current Protocols in Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc. New York, 1997). Stringent conditions are sequence-dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. (or less) lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm of a DNA duplex is defined as the temperature at which 50% of the nucleotides are paired and corresponds to the midpoint of the spectroscopic hyperchromic absorbance shift during DNA melting. The Tm indicates the transition from double helical to random coil.
Typically, stringent conditions will be those in which the salt concentration is about 0.2×SSC at pH 7 and the temperature is at least about 60° C. For example, a nucleic acid of the invention or fragment thereof can be identified in standard filter hybridizations using the nucleic acids disclosed here under stringent conditions, which for purposes of this disclosure, include at least one wash (usually 2) in 0.2×SSC at a temperature of at least about 60° C., usually about 65° C., sometimes 70° C. for 20 minutes, or equivalent conditions. For PCR, an annealing temperature of about 5° C. below Tm, is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 72° C., e.g., 40° C., 42° C., 45° C., 52° C., 55° C., 57° C., or 62° C., depending on primer length and nucleotide composition or high stringency PCR amplification, a temperature at, or slightly (up to 5° C.) above, primer Tm is typical, although high stringency annealing temperatures can range from about 50° C. to about 72° C., and are often 72° C., depending on the primer and buffer conditions (Ahsen et al., Clin Chem. 47:1956-61, 2001). Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90° C.-95° C. for 30 sec-2 min., an annealing phase lasting 30 sec.-10 min., and an extension phase of about 72° C. for 1-15 min.
The terms “identical” or percent “identity,” in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., at least 70% identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity, over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be “substantially identical.” This definition also refers to the complement of a test sequence. Preferably, the identity exists over a region that is at least about 15, 20 or 25 nucleotides in length, or more preferably over a region that is 50-100 nucleotides in length.
For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 15 to 600, usually about 20 to about 200, more usually about 50 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol. 215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used, with the default parameters described herein, to determine percent sequence identity for the nucleic acids described herein. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=−4 and a comparison of both strands.
The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
“Administering” an expression vector, nucleic acid, protein, or a delivery vehicle to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a protein or nucleic acid can be transported across a cell membrane and preferably into the nucleus of a cell.
A “delivery vehicle” refers to a compound, e.g., a liposome, toxin, or a membrane translocation polypeptide, which is used to administer dominant negative proteins. Delivery vehicles can also be used to administer nucleic acids encoding dominant negative proteins of the invention, e.g., a lipid:nucleic acid complex, an expression vector, a virus, and the like.
Design of Dominant Negative Proteins
The dominant negative proteins of the invention comprise any of a number of possible fusions of a transcription factor or other protein, or fragment thereof, with a protein that is capable of localization to the transcriptional machinery, such as nuclear localized proteins, RNA processing proteins, components of the transcriptional machinery, and proteins involved in co-transcriptional processes. Among the co-transcriptional processes that are subjects of the invention are capping, splicing, polyadenylation, RNA export, translation. The transcription factor can be derived from any of a number of species including, and not limited to, viruses, HIV, bacteria, yeast, Drosophila, C. elegans, Xenopus, mouse, monkey, and human. For human applications, a human TF is generally preferred. One of skill in the art will recognize that a wide variety of transcription factor proteins known in the art may be used in this invention. See Goodrich et al., Cell 84:825-30 (1996), Barnes & Adcock, Clin. Exp. Allergy 25 Suppl. 2:46-9 (1995), and Roeder, Methods Enzymol. 273:165-71 (1996) for general reviews of transcription factors. Databases dedicated to transcription factors are known (see, e.g., Science 269:630 (1995)). Nuclear hormone receptor transcription factors are described in, for example, Rosen et al., J. Med. Chem. 38:4855-74 (1995). The C/EBP family of transcription factors are reviewed in Wedel et al., Immunobiology 193:171-85 (1995). Coactivators and co-repressors that mediate transcription regulation by nuclear hormone receptors are reviewed in, for example, Meier, Eur. J. Endocrinol. 134(2):158-9 (1996); Kaiser et al., Trends Biochem. Sci. 21:342-5 (1996); and Utley et al., Nature 394:498-502 (1998)). GATA transcription factors, which are involved in regulation of hematopoiesis, are described in, for example, Simon, Nat. Genet. 11:9-11 (1995); Weiss et al., Exp. Hematol. 23:99-107. TATA box binding protein (TBP) and its associated TAF polypeptides (which include TAF30, TAF55, TAF80, TAF 110, TAF 150, and TAF250) are described in Goodrich & Tjian, Curr. Opin. Cell Biol. 6:403-9 (1994) and Hurley, Curr. Opin. Struct. Biol. 6:69-75 (1996). The STAT family of transcription factors are reviewed in, for example, Barahmand-Pour et al., Curr. Top. Microbiol. Immunol. 211:121-8 (1996). Transcription factors involved in disease are reviewed in Aso et al., J. Clin. Invest. 97:1561-9 (1996).
As further examples, the transcription factor may be chosen from any of a number of different classes of known transcription factors such as those that contain homeodomains, POU domains, Helix-Loop-Helix (HLH), Zinc Fingers, Leucine Zippers, or Winged Helix, to name but a few of the structural motifs found in transcription factors. Currently, there are about 2000 known transcription factors. See, e.g., Brivanlou and Darnell, Science, 295: 813-818 (2002). Among some of the better known transcription factors include: c-Myc and Max, c-Fos and c-Jun, CREB, c-ErbA, c-Ets, GATA c-Myb, MyoD KF-kB, RAR, and SRF, to name a few.
Among the classes of transcription factors that find use in this invention are viral transcription factors, nuclear proto-oncogene or oncogene proteins, nuclear tumor suppressor proteins, heart specific transcription factors, and immune cell transcription factors. The viral transcription factors useful in the practice of this invention include: HIV-Tat, HPV-E2, HPV-E7, BPV-E2, Adenovirus IVa2, HSV-1 ICP4, EBNA-LP, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, BZLF-1, CMV-IE-1, CMV-IE2, HHSV-8 K bZIP, HBV Hbx, Poxvirus Vaccinia, VETF, HCV NS5A, T-Ag, Adenovirus EIA, Herpesvirus VP16, HTLV Tax, Hepadnavirus X protein, and Baculovirus AcNPV IE-1, among others. The nuclear proto-oncogene or oncogene proteins and nuclear tumor suppressor proteins transcription factors useful in the practice of this invention include: Abl, Myc, Myb, Rel, Jun, Fos, Sp I, Apl, NF-κB, STAT 3 or 5, β-catenin, Notch, GLI, PML-RARα and p53, among others. The heart specific transcription factors useful in the practice of this invention include: Nkx 2, 3, 4, or 5, TBX5, GATA 4, 5, or 6, and MEF2, among others. The immune cell specific transcription factors useful in the practice of this invention include: Ikaros, PU.1, PAX-5, Oct-2, and BOB.1/OBF.1, among others. A non limiting list of transcription factors that may be used in the practice of this invention is provided in Table 3. The transcription factors useful in the practice of this invention can be human as well as derived from yeast or higher eukaryotes such as viruses, HIV, Drosophila, C. elegans, Xenopus, or mouse, among other species.
In the practice of this invention, the transcription factor can be either a transcriptional activator or repressor, examples of which are well known in the art. Non-limiting examples of transcriptional activators and repressors are provided in Table 3.
Proteins that localize to the transcriptional machinery include: components of the transcriptional machinery, nuclear localized proteins, RNA processing proteins, components of the transcriptional machinery, and proteins involved in co-transcriptional processes and RNA processing.
Among the components of the transcriptional machinery that may be used in the practice of this invention are TAFs, CDK7, cyclin H, DNA helicase, unwinding enzymes, transcription factors, among others.
A wide range of proteins have been shown to localize to the nucleus and may be used in the practice of this invention. A non-limiting list of such proteins is provided in Table 1.
Among the co-transcriptional processes and RNA processing activities that are subjects of the invention are capping, splicing, polyadenylation, RNA export, and translation. Accordingly, proteins involved in capping, splicing, polyadenylation, RNA export, and translation may be used in the practice of this invention. Splicing factors represent one particular class of proteins involved in co-transcriptional processing of RNA and are suitable for the practice of this invention. As many as 300 factors are known to comprise the spliceosome. The protein components of spliceosomes are disclosed in Rappsilber, J., Ryder, U., Lamond, A. I., and Mann, M. (2002) Genome Res 12(8), 1231-1245 and Zhou, Z., Licklider, L. J., Gygi, S. P., and Reed, R. (2002) Nature 419(6903), 182-185, among other sources. Many splicing factors useful for the practice of this invention are compiled in Table 2. Particular examples of splicing factors useful in the practice of this invention include SF1, U2AF65, and 9G8.
The RS domain is a structural and functional feature characteristic of many nuclear proteins, particularly splicing factors. A large number of RS domain proteins are known in the art, and many have been identified through a genome-wide survey of RS domain proteins from various species. See Boucher et al., RNA 7:1693-1701 (2001). Among the classes of known RS domain containing proteins that may be used in the practice of the invention are those listed in the table below.
In one embodiment of the invention, HIV Tat protein, or a fragment thereof, can used as the transcription factor in a dominant negative fusion protein as described herein. The human Tat protein is an 86 amino acid protein that is required efficient viral gene expression. The Tat sequence has been subdivided into several distinct regions based on structure and function: a N-terminal activation region (amino acids 1-19), a cysteine-rich domain (amino acids 20-31), a core region (amino acids 32-47), a basic region (amino acids 48-57), and a glutamine-rich region (amino acids 60-76). See Karn, J. (ref). In one particular embodiment, a full length Tat is linked to the splicing factors SF1 or U2AF65. In another embodiment, the Tat activation domain (Tat AD) is linked to the splicing factors SF1 or U2AF65.
Generation of Nucleic Acids Encoding Dominant Negative Proteins.
Dominant negative polypeptides and nucleic acids of the invention can be made using routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)). In addition, essentially any nucleic acid can be custom ordered from any of a variety of commercial sources. Similarly, peptides and antibodies can be custom ordered from any of a variety of commercial sources.
Expression Vectors for Nucleic Acids Encoding Dominant Negative Proteins
A nucleic acid encoding a dominant negative protein is typically cloned into intermediate vectors for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding dominant negative proteins or production of protein. The nucleic acid encoding a dominant negative protein is also typically cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoal cell.
To obtain expression of a cloned gene or nucleic acid, a nucleic acid encoding a dominant negative protein is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994). Bacterial expression systems for expressing a dominant negative protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., Gene 22:229-235 (1983)). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
The promoter used to direct expression of a nucleic acid encoding a dominant negative protein depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of a dominant negative protein. In contrast, when a dominant negative protein is administered in vivo for gene regulation, either a constitutive or an inducible promoter is used, depending on the particular use of the dominant negative protein. In addition, a preferred promoter for administration of a dominant negative protein can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter typically can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, PNAS 89:5547 (1992); Oligino et al., Gene Ther. 5:491-496 (1998); Wang et al., Gene Ther. 4:432-441 (1997); Neering et al., Blood 88:1147-1155 (1996); and Rendahl et al., Nat. Biotechnol. 16:757-761 (1998)).
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the dominant negative protein, and signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.
The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the dominant negative protein, e.g., expression in plants, animals, bacteria, fungus, protozoa etc. (see expression vectors described below). Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available fusion expression systems such as GST and LacZ. A preferred fusion protein is the maltose binding protein, “MBP.” Such fusion proteins are used for purification of the dominant negative protein. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, for monitoring expression, and for monitoring cellular and subcellular localization, e.g., c-myc or FLAG.
Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with a dominant negative protein encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the well known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the protein of choice.
Assays for Determining Regulation of Gene Expression by Dominant Negative Proteins
A variety of assays can be used to determine the level of gene expression regulation by dominant negative proteins. The activity of a particular dominant negative protein can be assessed using a variety of in vitro and in vivo assays, by measuring, e.g., protein or mRNA levels, product levels, enzyme activity, tumor growth; transcriptional activation or repression of a reporter gene such as a fluorescent protein (e.g., GFP); second messenger levels (e.g., cGMP, cAMP, IP3, DAG, Ca2+); cytokine and hormone production levels; and neovascularization, using, e.g., immunoassays (e.g., ELISA and immunohistochemical assays with antibodies), hybridization assays (e.g., RNase protection, northerns, in situ hybridization, oligonucleotide array studies), colorimetric assays, amplification assays, enzyme activity assays, tumor growth assays, phenotypic assays, and the like.
Dominant negative proteins are typically first tested for activity in vitro using cultured cells, e.g., 293 cells, CHO cells, VERO cells, BHK cells, HeLa cells, COS cells, and the like. Preferably, human cells are used. The dominant negative protein is often first tested using a transient expression system with a reporter gene, and then regulation of the target endogenous gene is tested in cells and in animals, both in vivo and ex vivo. The dominant negative protein can be recombinantly expressed in a cell, recombinantly expressed in cells transplanted into an animal, or recombinantly expressed in a transgenic animal, as well as administered as a protein to an animal or cell using delivery vehicles described below. The cells can be immobilized, be in solution, be injected into an animal, or be naturally occurring in a transgenic or non-transgenic animal.
Modulation of gene expression is tested using one of the in vitro or in vivo assays described herein. Samples or assays are treated with a dominant negative protein and compared to control samples without the test compound, to examine the extent of modulation.
The effects of the dominant negative proteins can be measured by examining any of the parameters described above. Any suitable gene expression, phenotypic, or physiological change can be used to assess the influence of a dominant negative protein. When the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as tumor growth, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots or oligonucleotide array studies), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGMP.
Assays for dominant negative protein regulation of endogenous gene expression can be performed in vitro. In one preferred in vitro assay format, dominant negative protein regulation of endogenous gene expression in cultured cells is measured by examining protein production using an ELISA assay (see Examples VI and VII). The test sample is compared to control cells treated with an empty vector or an unrelated dominant negative protein that is targeted to another gene.
In another embodiment, dominant negative protein regulation of endogenous gene expression is determined in vitro by measuring the level of target gene mRNA expression. The level of gene expression is measured using amplification, e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNase protection, dot blotting. RNase protection is used in one embodiment (see Example VIII and
Alternatively, a reporter gene system can be devised using the target gene promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or β-gal. The reporter construct is typically co-transfected into a cultured cell. After treatment with the dominant negative protein of choice, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
Another example of an assay format useful for monitoring dominant negative protein regulation of endogenous gene expression is performed in vivo. This assay is particularly useful for examining dominant negative proteins that inhibit expression of tumor promoting genes, genes involved in tumor support, such as neovascularization (e.g., VEGF), or that activate tumor suppressor genes such as p53. In this assay, cultured tumor cells expressing the dominant negative protein of choice are injected subcutaneously into an immune compromised mouse such as an athymic mouse, an irradiated mouse, or a SCID mouse. After a suitable length of time, preferably 4-8 weeks, tumor growth is measured, e.g., by volume or by its two largest dimensions, and compared to the control. Tumors that have statistically significant reduction (using, e.g., Student's T test) are said to have inhibited growth. Alternatively, the extent of tumor neovascularization can also be measured. Immunoassays using endothelial cell specific antibodies are used to stain for vascularization of the tumor and the number of vessels in the tumor. Tumors that have a statistically significant reduction in the number of vessels (using, e.g., Student's T test) are said to have inhibited neovascularization.
Transgenic and non-transgenic animals are also used as a preferred embodiment for examining regulation of endogenous gene expression in vivo. Transgenic animals typically express the dominant negative protein of choice. Alternatively, animals that transiently express the dominant negative protein of choice, onto which the dominant negative protein has been administered in a delivery vehicle, can be used. Regulation of endogenous gene expression is tested using any one of the assays described herein.
Nucleic Acids Encoding Dominant Negative Proteins and Gene Therapy
Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids encoding engineered dominant negative proteins in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding dominant negative proteins to cells in vitro. Preferably, the nucleic acids encoding dominant negative proteins are administered for in vivo or ex vivo gene therapy uses. Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808-813 (1992); Nabel & Feigner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Böhm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
Methods of non-viral delivery of nucleic acids encoding engineered dominant negative proteins include lipofection, microinjection, ballistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. No. 5,049,386, U.S. Pat. No. 4,946,787; and U.S. Pat. No. 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Feigner, WO 91/17424, WO 91/16024. Delivery can be to cells (ex vivo administration) or target tissues (in vivo administration).
The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
The use of RNA or DNA viral based systems for the delivery of nucleic acids encoding engineered dominant negative protein take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells are administered to patients (ex vivo). Conventional viral based systems for the delivery of dominant negative proteins could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vector that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT/US94/05700).
In applications where transient expression of the dominant negative protein is preferred, adenoviral based systems are typically used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system. Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
In particular, at least six viral vector approaches are currently available for gene transfer in clinical trials, with retroviral vectors by far the most frequently used system. All of these viral vectors utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn et al., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138 (1997)). PA317/pLASN was the first therapeutic vector used in a gene therapy trial. (Blaese et al., Science 270:475-480 (1995)). Transduction efficiencies of 50% or greater have been observed for MFG-S packaged vectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoff et al., Hum. Gene Ther. 1:111-2 (1997).
Recombinant adeno-associated virus vectors (rAAV) are a promising alternative gene delivery systems based on the defective and nonpathogenic parvovirus adeno-associated type 2 virus. All vectors are derived from a plasmid that retains only the AAV 145 by inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system. (Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther. 9:748-55 (1996)).
Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used for colon cancer gene therapy, because they can be produced at high titer and they readily infect a number of different cell types: Most adenovirus vectors are engineered such that a transgene replaces the Ad E1a, E1b, and E3 genes; subsequently the replication defector vector is propagated in human 293 cells that supply deleted gene function in trans. Ad vectors can transduce multiply types of tissues in vivo, including nondividing, differentiated cells such as those found in the liver, kidney and muscle system tissues. Conventional Ad vectors have a large carrying capacity. An example of the use of an Ad vector in a clinical trial involved polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)). Additional examples of the use of adenovirus vectors for gene transfer in clinical trials include Rosenecker et al., Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:7 1083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarez et al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther. 5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).
Packaging cells are used to form virus particles that are capable of infecting a host cell: Such cells include 293 cells, which package adenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the protein to be expressed. The missing viral functions are supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.
In many gene therapy applications, it is desirable that the gene therapy vector be delivered with a high degree of specificity to a particular tissue type. A viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface. The ligand is chosen to have affinity for a receptor known to be present on the cell type of interest. For example, Han et al., PNAS 92:9747-9751 (1995), reported that Moloney murine leukemia virus can be modified to express human heregulin fused to gp70, and the recombinant virus infects certain human breast cancer cells expressing human epidermal growth factor receptor. This principle can be extended to other pairs of virus expressing a ligand fusion protein and target cell expressing a receptor. For example, filamentous phage can be engineered to display antibody fragments (e.g., FAB or Fv) having specific binding affinity for virtually any chosen cellular receptor. Although the above description applies primarily to viral vectors, the same principles can be applied to nonviral vectors. Such vectors can be engineered to contain specific uptake sequences thought to favor uptake by specific target cells.
Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.
Ex vivo cell transfection for diagnostics, research, or for gene therapy (e.g., via re-infusion of the transfected cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject organism, transfected with a dominant negative protein nucleic acid (gene or cDNA), and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transfection are well known to those of skill in the art (see, e.g., Freshney et al., Culture of Animal Cells, A Manual of Basic Technique (3rd ed. 1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).
In one embodiment, stem cells are used in ex vivo procedures for cell transfection and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34+ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
Stem cells are isolated for transduction and differentiation using known methods. For example, stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4+ and CD8+ (T cells), CD45+(panB cells), GR-1 (granulocytes), and Tad (differentiated antigen presenting cells) (see Inaba et al., J. Exp. Med. 176:1693-1702 (1992)).
Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic dominant negative protein nucleic acids can be also administered directly to the organism for transduction of cells in vivo. Alternatively, naked DNA can be administered. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. Suitable methods of administering such nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention, as described below (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).
Delivery Vehicles for Dominant Negative Proteins
An important factor in the administration of polypeptide compounds, such as the dominant negative proteins of the present invention, is ensuring that the polypeptide has the ability to traverse the plasma membrane of a cell, or the membrane of an intra-cellular compartment such as the nucleus. Cellular membranes are composed of lipid-protein bilayers that are freely permeable to small, nonionic lipophilic compounds and are inherently impermeable to polar compounds, macromolecules, and therapeutic or diagnostic agents. However, proteins and other compounds such as liposomes have been described, which have the ability to translocate polypeptides such as dominant negative proteins across a cell membrane.
For example, “membrane translocation polypeptides” have amphiphilic or hydrophobic amino acid subsequences that have the ability to act as membrane-translocating carriers. In one embodiment, homeodomain proteins have the ability to translocate across cell membranes. The shortest internalizable peptide of a homeodomain protein, Antennapedia, was found to be the third helix of the protein, from amino acid position 43 to 58 (see, e.g., Prochiantz, Current Opinion in Neurobiology 6:629-634 (1996)). Another subsequence, the h (hydrophobic) domain of signal peptides, was found to have similar cell membrane translocation characteristics (see, e.g., Lin et al., J. Biol. Chem. 270:1 4255-14258 (1995)).
Examples of peptide sequences which can be linked to a dominant negative protein of the invention, for facilitating uptake of dominant negative protein into cells, include, but are not limited to: an 11 animo acid peptide of the tat protein of HIV; a 20 residue peptide sequence which corresponds to amino acids 84-103 of the p16 protein (see Fahraeus et al., Current Biology 6:84 (1996)); the third helix of the 60-amino acid long homeodomain of Antennapedia (Derossi et al., J. Biol. Chem. 269:10444 (1994)); the h region of a signal peptide such as the Kaposi fibroblast growth factor (K-FGF) h region (Lin et al., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare, Cell 88:223-233 (1997)). Other suitable chemical moieties that provide enhanced cellular uptake may also be chemically linked to dominant negative proteins. For example, nuclear localization signals may be appended to enhance uptake into the nuclear compartment of cells.
Toxin molecules also have the ability to transport polypeptides across cell membranes. Often, such molecules are composed of at least two parts (called “binary toxins”): a translocation or binding domain or polypeptide and a separate toxin domain or polypeptide. Typically, the translocation domain or polypeptide binds to a cellular receptor, and then the toxin is transported into the cell. Several bacterial toxins, including Clostridium perfringens iota toxin, diphtheria toxin (DT), Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracis toxin, and pertussis adenylate cyclase (CYA), have been used in attempts to deliver peptides to the cell cytosol as internal or amino-terminal fusions (Arora et al., J. Biol. Chem., 268:3334-3341 (1993); Perelle et al., Infect. Immun., 61:5147-5156 (1993); Stenmark et al., J. Cell Biol. 113:1025-1032 (1991); Donnelly et al., PNAS 90:3530-3534 (1993); Carbonetti et al., Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295 (1995); Sebo et al., Infect. Immun. 63:3851-3857 (1995); Klimpel et al., PNAS U.S.A. 89:10277-10281 (1992); and Novak et al., J. Biol. Chem. 267:17186-17193 1992)).
Such subsequences can be used to translocate dominant negative proteins across a cell membrane. Dominant negative proteins can be conveniently fused to or derivatized with such sequences. Typically, the translocation sequence is provided as part of a fusion protein. Optionally, a linker can be used to link the dominant negative protein and the translocation sequence. Any suitable linker can be used, e.g., a peptide linker.
The dominant negative protein can also be introduced into an animal cell, preferably a mammalian cell, via a liposomes and liposome derivatives such as immunoliposomes. The term “liposome” refers to vesicles comprised of one or more concentrically ordered lipid bilayers, which encapsulate an aqueous phase. The aqueous phase typically contains the compound to be delivered to the cell, i.e., a dominant negative protein.
The liposome fuses with the plasma membrane, thereby releasing the drug into the cytosol. Alternatively, the liposome is phagocytosed or taken up by the cell in a transport vesicle. Once in the endosome or phagosome, the liposome either degrades or fuses with the membrane of the transport vesicle and releases its contents.
In current methods of drug delivery via liposomes, the liposome ultimately becomes permeable and releases the encapsulated compound (in this case, a dominant negative protein) at the target tissue or cell. For systemic or tissue specific delivery, this can be accomplished, for example, in a passive manner wherein the liposome bilayer degrades over time through the action of various agents in the body. Alternatively, active drug release involves using an agent to induce a permeability change in the liposome vesicle. Liposome membranes can be constructed so that they become destabilized when the environment becomes acidic near the liposome membrane (see, e.g., PNAS 84:7851 (1987); Biochemistry 28:908 (1989)). When liposomes are endocytosed by a target cell, for example, they become destabilized and release their contents. This destabilization is termed fusogenesis. Dioleoylphosphatidylethanolamine (DOPE) is the basis of many “fusogenic” systems.
Such liposomes typically comprise a dominant negative protein and a lipid component, e.g., a neutral and/or cationic lipid, optionally including a receptor-recognition molecule such as an antibody that binds to a predetermined cell surface receptor or ligand (e.g., an antigen). A variety of methods are available for preparing liposomes as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, 4,946,787, PCT Publication No. WO 91\17424, Deamer & Bangham, Biochim. Biophys. Acta 443:629-634 (1976); Fraley, et al., PNAS 76:3348-3352 (1979); Hope et al., Biochim. Biophys. Acta 812:55-65 (1985); Mayer et al., Biochim. Biophys. Acta 858:161-168 (1986); Williams et al., PNAS 85:242-246 (1988); Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al., Chem. Phys. Lip. 40:89 (1986); Gregoriadis, Liposome Technology (1984) and Lasic, Liposomes: from Physics to Applications (1993)). Suitable methods include, for example, sonication, extrusion, high pressure/homogenization, microfluidization, detergent dialysis, calcium-induced fusion of small liposome vesicles and ether-fusion methods, all of which are well known in the art.
In certain embodiments of the present invention, it is desirable to target the liposomes of the invention using targeting moieties that are specific to a particular cell type, tissue, and the like. Targeting of liposomes using a variety of targeting moieties (e.g., ligands, receptors, and monoclonal antibodies) has been previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044).
Examples of targeting moieties include monoclonal antibodies specific to antigens associated with neoplasms, such as prostate cancer specific antigen and MAGE. Tumors can also be diagnosed by detecting gene products resulting from the activation or over-expression of oncogenes, such as ras or c-erbB2. In addition, many tumors express antigens normally expressed by fetal tissue, such as the alphafetoprotein (AFP) and carcinoembryonic antigen (CEA). Sites of viral infection can be diagnosed using various viral antigens such as hepatitis B core and surface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virus antigens, human immunodeficiency type-1 virus (HIV1) and papilloma virus antigens. Inflammation can be detected using molecules specifically recognized by surface molecules which are expressed at sites of inflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g., ELAM-1) and the like.
Standard methods for coupling targeting agents to liposomes can be used. These methods generally involve incorporation into liposomes lipid components, e.g., phosphatidylethanolamine, which can be activated for attachment of targeting agents, or derivatized lipophilic compounds, such as lipid derivatized bleomycin. Antibody targeted liposomes can be constructed using, for instance, liposomes which incorporate protein A (see Renneisen et al., J. Biol. Chem., 265:16337-16342 (1990) and Leonetti et al., PNAS 87:2448-2451 (1990).
Doses of Dominant Negative Proteins
For therapeutic applications of dominant negative proteins, the dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. In addition, particular dosage regimens can be useful for determining phenotypic changes in an experimental setting, e.g., in functional genomics studies, and in cell or animal models. The dose will be determined by the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular compound or vector in a particular patient.
The appropriate dose of an expression vector encoding a dominant negative protein can also be calculated by taking into account the average rate of dominant negative protein expression from the promoter and the average rate of dominant negative protein degradation in the cell. Preferably, a weak promoter such as a wild-type or mutant HSV TK is used.
In determining the effective amount of a dominant negative protein to be administered in the treatment or prophylaxis of disease, the physician evaluates circulating plasma levels of the dominant negative protein or nucleic acid encoding the dominant negative protein, potential dominant negative protein toxicities, progression of the disease, and the production of anti-dominant negative protein antibodies. Administration can be accomplished via single or divided doses.
Pharmaceutical Compositions and Administration
Dominant negative proteins and expression vectors encoding dominant negative proteins can be administered directly to the patient for modulation of gene expression and for therapeutic or prophylactic applications, for example, cancer, ischemia, diabetic retinopathy, macular degeneration, rheumatoid arthritis, psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease, muscular dystrophy, neurodegenerative diseases, vascular disease, cystic fibrosis, stroke, and the like. Examples of microorganisms that can be inhibited by dominant negative protein gene therapy include pathogenic bacteria, e.g., chlamydia, rickettsial bacteria, mycobacteria, staphylococci, streptococci, pneumococci, meningococci and conococci, klebsiella, proteus, serratia, pseudomonas, legionella, diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, and Lyme disease bacteria; infectious fungus, e.g., Aspergillus, Candida species; protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g., Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas, Giardia, etc.); viral diseases, e.g., hepatitis (A, B, or C), herpes virus (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), HIV, Ebola, adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus, rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus, HTLV virus, dengue virus, papillomavirus, poliovirus, rabies virus, and arboviral encephalitis virus, etc.
Administration of therapeutically effective amounts is by any of the routes normally used for introducing dominant negative protein into ultimate contact with the tissue to be treated. The dominant negative proteins are administered in any suitable manner, preferably with pharmaceutically acceptable carriers. Suitable methods of administering such modulators are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.
Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed. 1985)).
The dominant negative proteins, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be “nebulized”) to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration, such as, for example, by intravenous, intramuscular, intradermal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. In the practice of this invention, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. The formulations of compounds can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
Functional Genomics Assays
Dominant negative proteins also have use for assays to determine the phenotypic consequences and function of gene expression. The recent advances in analytical techniques, coupled with focussed mass sequencing efforts have created the opportunity to identify and characterize many more molecular targets than were previously available. This new information about genes and their functions will speed along basic biological understanding and present many new targets for therapeutic intervention. In some cases analytical tools have not kept pace with the generation of new data. An example is provided by recent advances in the measurement of global differential gene expression. These methods, typified by gene expression microarrays, differential cDNA cloning frequencies, subtractive hybridization and differential display methods, can very rapidly identify genes that are up or down-regulated in different tissues or in response to specific stimuli. Increasingly, such methods are being used to explore biological processes such as, transformation, tumor progression, the inflammatory response, neurological disorders etc. One can now very easily generate long lists of differentially expressed genes that correlate with a given physiological phenomenon, but demonstrating a causative relationship between an individual differentially expressed gene and the phenomenon is difficult. Until now, simple methods for assigning function to differentially expressed genes have not kept pace with the ability to monitor differential gene expression.
Using conventional molecular approaches, over expression of a candidate gene can be accomplished by cloning a full-length cDNA, subcloning it into a mammalian expression vector and transfecting the recombinant vector into an appropriate host cell. This approach is straightforward but labor intensive, particularly when the initial candidate gene is represented by a simple expressed sequence tag (EST). Under expression of a candidate gene by “conventional” methods is yet more problematic. Antisense methods and methods that rely on targeted ribozymes are unreliable, succeeding for only a small fraction of the targets selected. Gene knockout by homologous recombination works fairly well in recombinogenic stem cells but very inefficiently in somatically derived cell lines. In either case large clones of syngeneic genomic DNA (on the order of 10 kb) should be isolated for recombination to work efficiently.
The dominant negative protein technology can be used to rapidly analyze differential gene expression studies. Engineered dominant negative proteins can be readily used to up or down-regulate any endogenous target gene. This makes the dominant negative protein technology ideal for analysis of long lists of poorly characterized differentially expressed genes.
This specific example of using engineered dominant negative proteins to add functional information to genomic data is merely illustrative. Any experimental situation that could benefit from the specific up or down-regulation of a gene or genes could benefit from the reliability and ease of use of engineered dominant negative proteins.
Additionally, greater experimental control can be imparted by dominant negative proteins than can be achieved by more conventional methods. This is because the production and/or function of an engineered dominant negative protein can be placed under small molecule control. Examples of this approach are provided by the Tet-On system, the ecdysone-regulated system and a system incorporating a chimeric factor including a mutant progesterone receptor. These systems are all capable of indirectly imparting small molecule control on any endogenous gene of interest or any transgene by placing the function and/or expression of a dominant negative protein under small molecule control.
Transgenic Mice
A further application of the dominant negative protein technology is manipulating gene expression in transgenic animals. Conventional down-regulation of gene expression in transgenic animals is plagued by technical difficulties. Gene knockout by homologous recombination is the method most commonly applied currently. This method requires a relatively long genomic clone of the gene to be knocked out (ca. 10 kb). Typically, a selectable marker is inserted into an exon of the gene of interest to effect the gene disruption, and a second counter-selectable marker provided outside of the region of homology to select homologous versus non-homologous recombinants. This construct is transfected into embryonic stem cells and recombinants selected in culture. Recombinant stem cells are combined with very early stage embryos generating chimeric animals. If the chimerism extends to the germline homozygous knockout animals can be isolated by back-crossing. When the technology is successfully applied, knockout animals can be generated in approximately one year. Unfortunately two common issues often prevent the successful application of the knockout technology; embryonic lethality and developmental compensation. Embryonic lethality results when the gene to be knocked out plays an essential role in development. This can manifest itself as a lack of chimerism, lack of germline transmission or the inability to generate homozygous back crosses. Genes can play significantly different physiological roles during development versus in adult animals. Therefore, embryonic lethality is not considered a rationale for dismissing a gene target as a useful target for therapeutic intervention in adults. Embryonic lethality most often simply means that the gene of interest can not be easily studied in mouse models, using conventional methods.
Developmental compensation is the substitution of a related gene product for the gene product being knocked out. Genes often exist in extensive families. Selection or induction during the course of development can in some cases trigger the substitution of one family member for another mutant member. This type of functional substitution may not be possible in the adult animal. A typical result of developmental compensation would be the lack of a phenotype in a knockout mouse when the ablation of that gene's function in an adult would otherwise cause a physiological change. This is a kind of false negative result that often confounds the interpretation of conventional knockout mouse models.
A few new methods have been developed to avoid embryonic lethality. These methods are typified by an approach using the cre recombinase and lox DNA recognition elements. The recognition elements are inserted into a gene of interest using homologous recombination (as described above) and the expression of the recombinase induced in adult mice post-development. This causes the deletion of a portion of the target gene and avoids developmental complications. The method is labor intensive and suffers form chimerism due to non-uniform induction of the recombinase.
The use of engineered dominant negative proteins to manipulate gene expression can be restricted to adult animals using the small molecule regulated systems described in the previous section. Expression and/or function of a dominant negative protein can be switched off during development and switched on at will in the adult animals. This approach relies on the expression of the dominant negative protein only; homologous recombination is not required. Because the dominant negative proteins are trans dominant, there is no concern about germline transmission or homozygosity. These issues dramatically affect the time and labor required to go from a poorly characterized gene candidate (a cDNA or EST clone) to a mouse model. This ability can be used to rapidly identify and/or validate gene targets for therapeutic intervention, generate novel model systems and permit the analysis of complex physiological phenomena (development, hematopoiesis, transformation, neural function etc.). Chimeric targeted mice can be derived according to Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, (1988); Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, Robertson, ed., (1987); and Capecchi et al., Science 244:1288 (1989).
All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
The following examples are provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.
The Tat-hybrid assay, in which Tat fused to a heterologous RNA-binding domain (RBD) elicits activation of an HIV-1 LTR reporter plasmid containing a cognate RNA-binding site, has been useful for studying RNA-protein interactions in living cells6. However, as with other types of fusion protein assays, dominant negative proteins can be generated unintentionally that score as false negatives. We discovered a novel class of highly potent dominant negatives, exemplified by Tat fusions to splicing factors, whose potency appears to be dictated by cotranscriptional recruitment to the HIV promoter.
We devised a dual-fluorescence Tat-hybrid assay to monitor RNA-binding specificity using two pairs of orthogonal reporters and Tat fusions, herein referred to as T-fusions. To calibrate the assay, T-BIVRBD, a fusion between the HIV Tat activation domain (AD) and the RBD of bovine immunodeficiency virus (BIV) Tat, was used to activate a BIV TAR (BTAR)-DsRed reporter, while T-SF1, a Tat fusion to human splicing factor SF1, was used to activate a branch point sequence (BPS)-GFP reporter (
Using a more quantitative luciferase reporter, we found that inhibition was remarkably potent, with a stoichiometric amount of T-SF1 plasmid DNA (5 ng) sufficient to almost completely block activation mediated by the BIV Tat-BTAR interaction (
If the targeting hypothesis is correct, then T-fusions to other RNAP II-localized splicing factors might show a similar phenotype. Indeed, T-U2AF65 is an even more potent inhibitor (
The specificity of inhibition for the HIV promoter was assessed by measuring effects of T-U2AF65 on other reporter-activator combinations. No inhibition was observed in any case, including activation by the P-TEFb-dependent MHC class II transactivator (CIITA) and heat-shock factor 1 (HSF1), as well as p53 and GAL4-VP16, and constitutive expression from the cytomegalovirus (CMV) promoter (
To begin examining the effect of localization on inhibitor activity, we first asked whether nuclear localization alone might account for some of its potency, particularly because a variety of T-fusions showed activity, albeit not as strong as T-U2AF65 (
The RS domains of U2AF65 and other splicing factors help recruit these proteins to regions of active splicing within the nucleus8,9 and also are believed to interact with RNAP II during transcription complex asembly9. The presence of RNAP II and splicing and mRNA-export factors suggests an active role for the “speckle-associated patches” in mRNA processing, although they are otherwise considered mainly as storage sites for factors involved in mRNA metabolism10,11. To test the possible involvement of RS domains in dominant negative inhibition, we generated a T-fusion lacking the RS domain (T-U2AF65ΔRS, which contains U2AF65 residues 91-475) and a second with the RS domain alone (T-RS, which contains U2AF65 residues 2-73). Of these, only T-RS remained a potent inhibitor (
To examine the recruitment of T-U2AF65 to the transcriptional machinery, we first analyzed possible interactions with RNAP II by co-immunoprecipitation using antibodies against the Ser5-phosphorylated CTD (Ser5P-CTD), known as RNAP IIa. T-U2AF65-GFP, as well as the K41A Tat AD mutant, are complexed with RNAP IIa in a RNA-independent manner (
A primary function of Tat is to enhance transcription elongation3 but it also participates in pre-initiation complex assembly3,16. RNase protection experiments using promoter proximal (Pp) and distal (Pd) probes indicate that the T-U2AF65 dominant negative primarily inhibits elongation (
The specificity of dominant negative inhibition for the HIV promoter is clear (
The high potency of the Tat dominant negatives and the requirement of Tat for viral replication suggested that they might be effective HIV inhibitors. To analyze this we generated SupT1 lymphocyte cell lines stably expressing T-U2AF65, T-HIVRBD-U2AF65, or T-BIVRBD-U2AF65 dominant negatives or the non-fusion controls, TatAD, Tat, T-BIVRBD, or U2AF65, and monitored HIV replication rates using viruses dependent on either the HIV or BIV Tat-TAR interactions18. We observed striking specificity of the dominant negatives in which replication was inhibited only in viruses driven by a non-cognate RNA-protein interaction. Expression of T-U2AF65, which contains no TAR RNA-binding domain, markedly suppressed replication of both viruses compared to the TatAD or U2AF65 controls, with no p24 antigen detectable until 18-20 days after infection (
To assess whether the RBD of Tat contributes to the dominant negative activity, we generated U2AF65 fusions to full-length Tat or TatAD and measured their effects using an LTR-BTAR-RL reporter and Tat-BIVRBD activator. Indeed, both T-HIVRBD-U2AF65 and T-U2AF65 inhibited activation more than 10-fold at sub-stoichiometric plasmid DNA levels relative to the activator (
Immunofluorescence experiments showed that the T-Rev activator and T-U2AF65 dominant negative were expressed similarly and localized to the nucleus (
The potent inhibition observed with T-SF1 and T-U2AF65 prompted us to evaluate whether fusions to other transcription or RNA processing factors might also act as dominant negatives. While T-SF1 was slightly less potent than T-U2AF65, a fusion to the SR-protein 9G8 (T-9G8) was nearly as potent as T-U2AF65 (
Deleting the RS domain of T-U2AF65 eliminates dominant negative activity (see T-U2AF65ΔRS in
An even more striking subnuclear localization pattern is seen for T-RS-GFP bearing only the U2AF65 RS-domain in which only a few (10-30) bright clusters are observed (
To assess expression levels of the Tat activators (Tat, TatAD and T-BIVRBD) and dominant negative inhibitors (T-U2AF65, T-HIVRBD-U2AF65, and T-BIVRBD-U2AF65) in the stable SupT1 populations used for the viral replication assays, we first determined mRNA steady-state levels for each protein by quantitative real-time RT-PCR, using two sets of primers that amplify Tat or U2AF65 portions of the mRNAs. While the RNA expression levels varied widely between samples, all were clearly detectable, with the SupT1-Tat population expressing the highest levels (normalized expression level of 370 units), followed by T-BIVRBD (120 units), TatAD (100 units), TatAD-U2AF65 and T-BIVRBD-U2AF65 (35 units), and Tat-U2AF65 (7 units). We next characterized expression in a more functional assay in vivo by transfecting each stable cell population with an activatable GFP reporter, depending on the Tat protein expressed, and monitored activity by flow cytometry (data not shown). All stable SupT1 populations expressing the full-length Tat moiety activated an LTR-HTAR-GFP reporter, varying from 9-20 fold, while cell lines expressing TatAD did not activate. Stable cell populations expressing T-BIVRBD activated an LTR-BTAR-GFP reporter about 7-9 fold but not an LTR-HTAR-GFP reporter. The TatAD-U2AF65-expressing population weakly activated an LTR-BPS-GFP reporter, through its polypyrimidine tract (PPT) binding site [9]. This weak activity likely reflected the generally lower activation observed with the U2AF65-PPT interaction [9] and, probably, the low transfection efficiency of the SupT1 cells. Thus, expression of each Tat or Tat-fusion protein could be confirmed by RT-PCR and functional assays, but expression levels generally appeared low, as expected for a stable cell population transduced by a retrovirus but not clonally selected [10]. Weak expression was further confirmed by Western blot and immunofluorescence analysis using an anti-Tat antibody where expression was virtually undetectable (data not shown).
We also estimated the activities of the integrated dominant negatives in the SupT1 cell lines using functional assays. Cells were co-transfected with a fixed amount of the LTR-HTAR-FFL or LTR-BTAR-FFL reporter and varying concentrations of the corresponding Tat activator and levels of inhibition were measured. For example, SupT1 cells expressing T-U2AF65 and T-BIVRBD-U2AF65 were co-transfected with the LTR-HTAR-FFL reporter and HIV Tat, and no significant activation was observed at low levels (0.1-1 ng) of transfected activator (
Transcriptional squelching has been described for many dominant negative transcription factors, such as yeast Gal4, and herpes simplex virus VP16, where common components of the transcriptional apparatus become “titrated of” of promoters[11,12]. Typically, these dominant negatives are rather promiscuous because the target co-activators do not need to be bound to the specific promoter. For HIV Tat, for example, it has been shown that Tat over-expression leads to decreased transcription from an MHC class II promoter, because both Tat and the class II transactivator (CIITA) require P-TEFb to function [13]. Because the Tat dominant negatives described here apparently operate via co-transcriptional recruitment to the HIV promoter, we suspected that they might display promoter specificity, unlike the more traditional dominant negatives. Reporter experiments show that T-U2AF65 has specificity for the HIV promoter versus other P-TEFb-regulated promoters (
We observed that virus eventually emerged after 18-20 days in the inhibitor-containing cell lines but with low replication kinetics and reaching a low steady-state plateau of p24 expression (
Conclusions
The potent Tat dominant negative inhibitors described in this work represent a new mechanistic class in which we hypothesize that a transcription factor AD is efficiently recruited to its promoter via a tethering signal, in this case an RS domain, among other specific contacts with the transcriptional apparatus. Unlike other dominant negatives, these Tat inhibitors function at stoichiometric or even sub-stoichiometric levels and do not require the considerable over-expression typically required for squelching or other simple competition mechanisms1,19. We speculate that their specificity and potency is imposed by localization, first at the sub-cellular and sub-nuclear levels and second by efficient recruitment to the promoter. Ptashne and Gann proposed the concept of “regulated localization”, where specificity typically is imposed by simple binding interactions between a locator, the transcriptional machinery, and the DNA20. We propose that combining localization functions within a single polypeptide can substantially enhance activity. In the case of the T-U2AF65 inhibitor, it appears that the Tat AD provides the dominant negative function, in part through interactions with P-TEFb at the HIV promoter, while the RS domain, provides additional localization and timing functions utilizing co-transcriptional mechanisms that RNA-processing factors, including SR proteins, use to load into transcription complexes10,11. This hypothesis is supported by the observations that RS-domain-containing proteins localize to sub-nuclear speckles, which are thought to anchor splicing factors to the nuclear matrix and facilitate assembly with RNAP II,21 and that Tat and P-TEFb co-localize to nuclear speckles22. It remains to be determined if other transcription factors, including those that do not function at the elongation step, can be efficiently localized and assembled into transcription complexes in a similar manner, and if other types of targeting domains may be used.
HIV replication is substantially inhibited by low-level expression of the dominant negative in stable cell lines (
Methods and Materials
Transcriptional Activation and Inhibition Reporter Assays
HeLa cells were transfected with GFP or firefly luciferase (FFL) reporter plasmids (typically 25 ng), appropriate amounts of Tat activator and inhibitor plasmids, and 5 ng of a CMV-Renilla luciferase (RL) plasmid using the Polyfect lipid transfection reagent (Qiagen) in a 48-well format. Reporter activity was measured 48 hr post-transfection using a Becton-Dickinson FACS Calibur (
Microsopy
HeLa or stably-integrated HeLa LTR-RREIIB-FFL cells were grown to 50% confluence on glass cover slips, transfected with 100 ng of plasmid DNAs, fixed in 4% paraformaldehyde in 1× PBS buffer (pH 7.6) 24 hr post-transfection, rinsed twice with PBS, and permeabilized with PBS-Triton 0.5% for 10 min at 4° C. Nonspecific antibody sites were blocked in 1× PBS, 3% goat serum, and 4% BSA for 1 hr at room temperature, cells were incubated with primary antibodies for 1 hr at room temperature, washed three times with PBS, incubated with appropriate Alexa 488- or Alexa 546-coupled secondary antibodies (Molecular Probes) for 1 hr at room temperature, and washed three times with PBS. Cells were mounted on DAPI-containing Vecta-shield slides (Vector Labs). Light microscopy was done using an LSM510 confocal microscope (Zeiss) and images were processed using LSM (Zeiss) software.
Co-Immunoprecipitation
To examine association of dominant negative inhibitors with RNAP II, HeLa cells were transiently transfected with T-U2AF65-GFP, T(K41A)-U2AF65-GFP, or T-NLS-GFP, and nuclear extracts were prepared with RIPA buffer. Half of the extract was used directly for the immunoprecipitation and the remaining half was treated with 1 μg of RNAse A, which was sufficient to quantitatively digest the RNA from 106 HeLa cells. RNAP II was immunoprecipitated using agarose-conjugated to 8WG16 and H14 antibodies overnight at 4° C. with mild shaking. Similarly, GFP-tagged proteins were immunoprecipitated using agarose-conjugated GFP-antibodies. After centrifuging and washing the beads immunocomplexes were dissociated by boiling for 10 min in 2× gel loading buffer, samples were separated by 10% SDSPAGE, transferred to PVDF, and analyzed by Western blot.
RNase Protection Assay
HeLa cells were transfected with the pLTR-HTAR-FFL reporter alone or with activator and inhibitor-expressing plasmids, total RNA was extracted using TRIzol (Invitrogen), and 15 μg of each sample was hybridized with proximal and distal probes corresponding to HIV promoter and luciferase ORF regions, respectively. The antisense probes were synthesized using a T3/T7 MaxiScript kit (Ambion) from plasmid templates linearized at a KpnI site, hybridization was performed with approximately 10,000 cpm of 32P-CTP-labeled probe (in 80% formamide, 40 mM PIPES, 400 mM NaCl, 1 mM EDTA) incubated at 42° C. overnight, RNase digestion was performed for 1.5 hr at 30° C. (in 10 mM Tris pH 8.0, 300 mM NaCl, 5 mM EDTA, 11 units/ml of RNase A, 11 units/ml RNase T1), samples were treated with proteinase K, extracted with phenol/chloroform, and RNA duplexes were precipitated with ethanol and glycogen carrier. RNAs were separated on a 6% polyacrylamide/8 M urea gel and visualized and quantified using a Typhoon phosphorimager (Molecular Dynamics). Experiments were performed in duplicate, with errors bars representing the SD of the mean.
Selection of a HeLA LTR-RREIIB-FFL Reporter Cell Line and ChIP Assays
HeLa cells were transfected in 6-well plates with a pcDNA3.1-derived plasmid (Invitrogen) bearing the LTR-RREIIB-FFL using Polyfect reagent (Qiagen). Clones were selected over more than four weeks in D-MEM-10% FBS supplemented with 750 μg/ml of G418 (Gibco). Twenty clones were analyzed for activation by pSV-T-Rev-HA by luciferase assays and a single highly active clone was chosen for ChIP analyses. ChIP assays were performed as described29 with minor modifications. HeLa LTR-RREIIB-FFL reporter cells were transfected with various expressor plasmids (5 μg each) using 30 μl of Lipofectamine 2000 (Invitrogen) per 25 cm culture dish, incubated for 36 hr, and washed in PBS. Chromatin was cross-linked with 1% formaldehyde for 15 min at RT and the reaction stopped by adding glycine to 125 mM. Cells were washed with PBS and harvested in RIPA buffer, and samples were sonicated to generate DNA fragments <500 bp. For immuno-precipitations, 1 mg of protein extract was pre-cleared for 2 hr with 40 μl of a 50% slurry of 50:50 protein A/G-agarose and -then incubated with protein A/G-agarose and the appropriate antibodies overnight at 4° C. preblocked with 1 mg/ml and 0.3 mg/ml of salmon sperm DNA. Immunocomplexes were recovered using anti-rabbit IgG/protein A/G-agarose beads (Santa Cruz), beads were washed twice with RIPA buffer, four times with ChIP wash buffer (100 mM Tris-HCl, pH 8.5, 500 mM LiCL, 1% v/v Nonidet P-40, 1% w/v deoxycholic acid), twice with RIPA buffer, and twice with 1× TE buffer. Immunocomplexes were eluted in 1% SDS for 10 min at 65° C. and cross-linking was reversed by adjusting to 200 mM NaCl and incubating for 5 hr at 65° C. A fraction of purified DNA was used for PCR amplification, with 25-32 cycles performed in the exponential range depending on the particular primers and antibodies. To ensure linearity, control PCR reactions were performed for one cycle using twice and half the amount of sample. PCR products (100-250 bp) were quantified by incorporation of SyBr Green and fluorescence detection (MJ Research) and by visualization on 2% agarose gels stained with ethidium bromide, using PCR products from known input DNAs as standards and IQMac 1.2 for analysis. Primer sequences are provided in Supplementary Information.
Dominant Negative-Expressing SupT1 Cells and Viral Replication Kinetics.
Plasmids expressing TatAD, Tat, T-BIVRBD, T-U2AF65, T-HIVRBD-U2AF65, T-BIVRBD-U2AF65, and U2AF65 were constructed in a pBMN retroviral vector (kindly provided by G. Nolan), using an SV40 promoter to express the Tat or Tat-fusion proteins. Plasmids were transfected into ONX packaging cells using the Polyfect reagent, and the retrovirus-containing supernatant recovered after 48 hr was used to transduce human CD4+ SupT1 cells. Populations of stable integrants were selected by growing cells in 2 mg/ml G418 (Invitrogen) for at least 4 weeks. Relative expression levels for each protein were assessed by real-time RT-PCR, transcriptional activation of transfected reporter plasmids and Western bloting (Supplementary Information). Each stable SupT1 population was infected with an HIV Tat-TAR-dependent (R7HTat/HTAR) or BIV Tat-TAR-dependent (R7 HBTat/BTAR) virus18 at an m.o.i of 1. Supernatant samples were harvested at different intervals following infection and the amount of viral replication was monitored by p24 antigen expression using ELISA (Immuno Diagnostics, Inc.) over a period of 110 days. Each experiment was performed in duplicate and mean values of p24 were calculated.
RNA Isolation and Expression Levels by Quantitative Real-Time RT-PCR
Total RNA was isolated from cells using the Trizol reagent according to manufacturer instructions (Invitrogen). Randomly primed cDNA was prepared from 1 μg of total RNA using MMULV reverse transcriptase (New England Biolabs). One twentieth of the resultant cDNA was amplified in 35 μl reactions containing 1.25 units of Taq DNA polymerase (ABI), 1.5 mM MgCl2, 300 nM of each primer, 0.5 mM dNTP mix, and 0.2× SYBR green I dye (Molecular Probes) in 1× Taq polymerase buffer. Real-time PCR was performed in an Opticon 2 DNA Engine (MJ Research) and analyzed using the Ct method (Applied Biosystems Prism 7700).
Expression Analysis by Western Blot
To more quantitatively assess relative inhibitor and activator expression levels, HeLa cells were co-transfected with 300 ng of pEGFPN3 (Clontech) and either 1.35 μg of pSV2-T-Rev-HA, 1.35 μg pSV2-T-U2AF65-HA, or both plasmids in 6-well plates. Nuclear extracts were prepared using NE-PER reagents (Pierce), samples were separated on a 12.5% SDS-PAGE gel, transferred to nitrocellulose, and probed with anti-HA, anti-GFP, or anti-nucleolin antibodies.
Functional Analysis of Protein Expression and Activity in SupT1 Cell Lines
Stable SupT1 G418-resistant cell populations (3×106 cells) were transfected by electroporation (Bio-Rad, 250V, 0.975 μF) with LTR-HTAR-GFP or LTR-BPS-BTAR-GFP reporters to assess the activities of integrated plasmids expressing Tat or T-fusion proteins. After 48 hours, cells were analyzed by flow cytometry and GFP activity was quantitated using Celquest software (Becton Dickinson). Populations expressing Tat and derivatives were transfected with LTR-HTAR-GFP, populations expressing T-BIVRBD and derivatives were transfected with LTR-BPS-BTAR-GFP, and populations expressing U2AF65 fusions were transfected with LTR-BPS-BTAR-GFP, which contains a BPS and PPT that binds U2AF65 cooperatively with SF1 [9]. For luciferase assays, we used the LTR-HTAR-FFL or LTR-BTAR-FFL reporters and CMV-RL as an internal control for data normalization.
Genomic DNA Extraction from SupT1-Infected Cells and Viral Genome Sequencing
SupT1-T-U2AF65, SupT1-T-BIVRBD-U2AF65, and SupT1-T-HIVRBD-U2AF65 infected populations (about 1×106 cells) were harvested 25 days post-infection and genomic DNA was extracted using Flexigene according to manufacturer instructions (Qiagen). DNA was amplified by PCR using Turbo Pfu (Stratagene), with primer pair specific to regions of the HIV LTR promoter and surrounding Tat coding sequence. PCR-amplified DNA was gel purified (Qiagen) and cloned into a TOPO vector (Invitrogen). Eight clones from each cell population were sequenced, and sequences were compared to the original viral isolate, HXB2, using the NCBI BLAST algorithm.
B. anthracis str. Am B. coronavirus B. indicusB. mori B. taurus
C. aethiops C. albicans C. briggsae C. burnetii RSA 493 C. carpio
C. elegans C. familiaris C. griseus
C. jacchus C. jejuni RM1221
C. porcellus C. torquatus atys D. discoideum D. erecta D. ethenogenes 195
D. mauritiana D. melanogaster D. pseudoobscura D. rerio D. sechellia
D. simulans D. sp. D. virilis D. yakubaE. caballus F. catus G. gallus
G. gorilla G. gorilla gorilla G. sulfurreducens PCH. lar H. sapiens
L. major
L. monocytogenes str M. auratus M. capsulatus str. B
M. fascicularis M. fuscata fuscata M. monax M. mulatta
M. musculus M. musculus castaneu M. musculus domestic M. musculus
molossin M. musculus musculus
M. natalensis M. nemestrina M. parviflora
M. unguiculatus O. aries O. cuniculus O. kitabensis O. longistaminata
O. mykiss O. nivara O. officinalis O. sativa O. sativa (indica cu O. sativa
P. sativum P. syringae pv. toma P. syringae pv. toma P. troglodytes
Panicum
R. norvegicus R. sp. S. cerevisiae S. coronavirus S. oedipus
S. oneidensis S. oneidensis MR-1 S. pombe S. pomeroyi DSS-3
S. sciureus S. scrofa S. sp. PCC 6803 T. brucei T. brucei TREU927
T. cambridgei T. vulpecula V. arvensis
V. cholerae O1 biova V. odorata
X. laevis X. tropicalis
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The present application claims the benefit of U.S. Ser. No. 60/817,927, filed Jun. 30, 2006, herein incorporated by reference in its entirety.
This invention was made with government support under Grant Nos. R01 AI29135 and R41CA 103407, awarded by the National Institutes of Health. The government has certain rights in this invention.
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| Number | Date | Country | |
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| 60817927 | Jun 2006 | US |
