Patent Application
20060069023
The present invention relates generally to a method for the treatment and/or prophylaxis of conditions arising from or otherwise associated with aberrations in hormone signalling. The present invention further provides an animal model useful for screening for agents capable of agonizing or antagonizing hormone signalling. More particularly, the present invention contemplates a method for the treatment and/or prophylaxis of conditions arising from or otherwise associated with aberrations in growth hormone signalling. The present invention further comprises a genetically modified animal. More particularly, the animals are genetically modified such that they have altered growth hormone signalling. The genetically modified animals of the present invention range from laboratory animals useful inter alia for animal models for studying hormone regulation and for the development of therapeutic protocols predicated, in part, on modulating hormone signalling to livestock animals. The latter may be manipulated for improved food production.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia or in any other country.
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Cells continually monitor their environment in order to modulate physiological and biochemical processes which in turn effects future behaviour. Frequently, a cell's initial interaction with its surroundings occurs via receptors expressed on the plasma membrane. Activation of these receptors, whether through binding endogenous ligands (such as cytokines) or exogenous ligands (such as antigens), triggers a biochemical cascade from the membrane through the cytoplasm to the nucleus.
Of the endogenous ligands, cytokines represent a particularly important and versatile group. Cytokines are proteins which regulate the survival, proliferation, differentiation and function of a variety of cells within the body (1). The haemopoietic cytokines have in common a four-alpha helical bundle structure and the vast majority interact with a structurally related family of cell surface receptors, the type I and type II cytokine receptors (2, 3). In all cases, ligand-induced receptor aggregation appears to be a critical even in initiating intracellar signal transduction cascades. Some cytokines, for example, growth hormone, erythropoietin (Epo) and granulocyte-colony-stimulating factor (G-CSF), trigger receptor homodimerization, while for other cytokines, receptor heterodimerization or heterotrimerization is crucial. In the latter cases, several cytokines share common receptor subunits and on this basis can be grouped into three subfamilies with similar patterns of intracellular activation and similar biological effects (4). Interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) use the common β-receptor subunit (βc) and each cytokine stimulates the production and functional activity of granulocytes and macrophages. IL-2, IL-4, IL-7, IL-9, and IL-15 each use the common γ-chain (γc), while IL-4 and IL-13 share an alternative γ-chain (γ′c or IL-13 receptor α-chain). Each of these cytokines plays an important role in regulating acquired immunity in the lymphoid system. Finally, IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin (CT) share the receptor subunit gp130. Each of these cytokines appears to be highly pleiotropic, having effects both within and outside the haemopoietic system (1).
In all of the above cases, at least one subunit of each receptor complex contains the conserved sequence elements, termed box 1 and box 2, in their cytoplasmic tails (5). Box 1 is a proline-rich motif which is located more proximal to the transmembrane domain than the acidic box 2 element. The box-1 region serves as the binding site for a class of cytoplasmic tyrosine kinases termed JAKs (Janus kinases). Ligand-induced receptor dimerization serves to increase the catalytic activity of the associated JAKs through cross-phosphorylation. Activated JAKs then tryosine phosphorylate several substrates, including the receptors themselves. Specific phosphotyrosine residues on the receptor then serve as docking sites for SH2-containing proteins, the best characterized of which are the signal transducers and activators of transcription (STATs) and the adaptor protein, shc. The STATs are then phosphorylated on tyrosines, probably on JAKs, dissociate from the receptor and form either homodimers or heterodimers through the interaction of the SH2 domain of one STAT with the phosphotyrosine residue of the other. STAT dimers then translocate to the nucleus where they bind to specific cytokine-responsive promoters and activate transcription (6, 7, 8). In a separate pathway, tyrosine phosphorylated shc interacts with another SH2 domain-containing protein, Grb-2 leading ultimately to activation of members of the MAP kinase family and in turn transcription factors such as fos and jun (9, 10). These pathways are not unique to members of the cytokine receptor family since cytokines that bind receptor tyrosine kinases also being able to activate STATs and members of the MAP kinase family (9, 10, 11, 12).
Four members of the JAK family of cytoplasmic tyrosine kinases have been described, JAK1, JAK2, JAK3 and TYK2, each of which binds to a specific subset of cytokine receptor subunits. Six STATs have been described (STAT1 through STAT6), and these too are activated by distinct cytokine/receptor complexes. For example, STAT1 appears to be functionally specific to the interferon system, STAT4 appears to be specific to IL-12, while STAT6 appears to be specific for IL-4 and IL-13. Thus, despite common activation mechanism some degrees of cytokine specificity may be achieved through the use of specific JAKs and STATs.
In addition to those described above, there are clearly other mechanisms of activation of these pathways. For example, the JAK/STAT pathway appears to be able to activate MAP kinases independent of the shc-induced pathway (13) and the STATs themselves can be activated without binding to the receptor, possibly by direct interaction with JAKs (14). Conversely, full activation of STATs may require the action of MAP kinase in addition to that of JAKs (13, 15).
While activation of these signalling pathways is becoming better understood, little is known of the regulation of these pathways, including employment of negative or positive feedback loops. This is important since once a cell has begun to respond to a stimulus, it is critical that the intensity and duration of the response is regulated and that signal transduction is switched off. It is likewise desirable to increase the intensity of a response systemically or even locally as the situation requires.
In International Patent Application No. PCT/AU97/00729 [WO 98/20023], a new family of negative regulators of signal transduction were identified. The new family is the “suppressor of cytokine signalling” or “SOCS” family. It has now been surprisingly determined that modulating the levels of expression of SOCS genes in animal models has an effect on hormone and in particular growth hormone signalling.
Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. SEQ ID NO: 1, SEQ ID NO: 2, etc. A sequence listing is provided after the claims.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
One aspect of the present invention contemplates a method for modulating hormone signalling in an animal, said method comprising up-regulating or down-regulating expression of a genetic sequence encoding a SOCS protein or its derivative or homologue or increasing or decreasing the activity of a SOCS protein or its derivative or homologue in said animal.
Another aspect of the present invention provides a method of modulating hormone signalling in an animal and in particular a human, said method comprising up-regulating or down-regulating expression of a genetic sequence encoding a SOCS protein or increasing or decreasing the activity of a SOCS protein in said animal and wherein said SOCS protein comprises a protein:molecule interacting region such as but not limited to an SH2 domain, WD40 repeats and/or ankyrin repeats, N terminal of a SOCS box, wherein said SOCS box comprises the amino acid sequence:
wherein:
Still another aspect of the present invention contemplates a method for controlling hormone signalling such as growth hormone signalling in an animal such as a human or livestock animal, said method comprising modulating expression of a genetic sequence encoding a SOCS protein comprising a SOCS box and a protein:molecule interacting region N-terminal of said SOCS box wherein said SOCS box comprises the amino acid sequence:
wherein:
Yet another aspect of the present invention contemplates a method for controlling growth hormone signalling in an animal such as human or livestock animal, said method comprising administering to said animal a control-effective amount of a SOCS protein or functional part or homologue or analogue thereof or an antagonist or agonist of a SOCS protein for a time and under conditions sufficient to modulate growth hormone signalling.
Even yet another aspect contemplates genetically modified animals such as livestock animals.
Another aspect of the present invention provides a genetically modified animal exhibiting altered hormone and in particular growth hormone signalling.
Still another aspect of the present invention provides a genetically modified animal wherein the animal, before genetic modification, comprises a genetic sequence which comprises a sequence of nucleotides substantially corresponding to the nucleotide sequence set forth in SEQ ID NO: 1 or its complementary form or is able to hybridize under low stringency conditions at 42° C. to SEQ ID NO: 1 or its complementary form and wherein said genetic modification either results in a deletion of one or more nucleotides from said genetic sequence from said animal or substantially prevents, reduces or down-regulates expression of said genetic sequence.
Yet another aspect of the present invention provides a genetically modified animal comprising a substantial deletion in a genetic sequence comprising a nucleotide sequence substantially corresponding to SEQ ID NO: 1 or capable of hybridizing to SEQ ID NO: 1 under low stringency conditions at 42° C. and wherein said animal exhibits altered hormone regulation such as growth hormone regulation.
One aspect of the present invention contemplates a method for modulating hormone signalling in an animal, said method comprising up-regulating or down-regulating expression of a genetic sequence encoding a SOCS protein or its derivative or homologue or increasing or decreasing the activity of a SOCS protein or its derivative or homologue in said animal.
Reference herein to “SOCS” encompasses any or all members of the SOCS family. Specific SOCS molecules may be defined numerically such as, for example, SOCS-1, SOCS-2 and SOCS-3. The species from which the SOCS has been obtained may be indicated by a preface of single letter abbreviation where “h” is human, “m” is murine and “r” is rat. Accordingly, “mSOCS-2”, for example, is a specific SOCS from a murine animal. Reference herein to “SOCS” is not to imply that the protein solely suppresses cytokine-mediated signal transduction, as the molecule may modulate other effector-mediated signal transductions such as by hormones or other endogenous or exogenous molecules, antigen, microbes and microbial products, viruses or components thereof, ions, hormones and parasites. The term “modulates” encompass up-regulation, down-regulation as well as maintenance of particular levels.
Reference herein to a “hormone” includes protein hormones and cytokines as well as non-proteinaceous hormones. One particularly useful hormone is growth hormone. Another useful hormones are insulin-like growth factor I (IGF-I) and prolactin.
An “animal” is preferably a mammal such as but not limited to a human, primate, livestock animal (e.g. sheep, cow, pig, horse, donkey), laboratory test animal (e.g. rabbit, mouse, rat, guinea pig), companion animal (e.g. cat, dog) or captive wild animal. The animal may be in the form of an animal model. Useful animals for this purpose are laboratory test animals. Genetically modifying livestock animals is useful in assisting in food production. The preferred animal is a human, primate animal or laboratory test animal. The most preferred animal is a human.
Reference herein to “SOCS” includes a protein comprising a SOCS box in its C-terminal region comprising the amino acid sequence:
wherein:
The SOCS protein also comprises a protein:molecule interacting region such as but not limited to one or more of an SH2 domain, WD40 repeats and/or ankyrin repeats, N-terminal of the SOCS box.
Modulating hormone signalling includes modulating growth control mechanisms. In addition, modulating growth hormone signalling has applications in increasing strength in elderly people, obesity control treatment of catabolic diseases, treatment of chronic inflammatory disease, treatment and/or prophylaxis of osteoporosis, treatment of cardiomyopathy and in the treatment of complicated fracture.
Another aspect of the present invention provides a method of modulating hormone signalling in an animal and in particular a human, said method comprising up-regulating or down-regulating expression of a genetic sequence encoding a SOCS protein or increasing or decreasing the activity of a SOCS protein in said animal and wherein said SOCS protein comprises a protein:molecule interacting region such as but not limited to an SH2 domain, WD40 repeats and/or ankyrin repeats, N terminal of a SOCS box, wherein said SOCS box comprises the amino acid sequence:
wherein:
The present invention extends to any SOCS molecule such as those disclosed in International Patent Application No. PCT/AU99/00729 [WO 98/20023] which is herein incorporated by reference. However, in a particularly preferred embodiment, the present invention is directed to manipulating levels of SOCS-2, which murine form comprises the nucleotide and corresponding amino acid sequence as set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The present invention is hereinafter described with reference to murine SOCS-2 (mSOCS-2), however, this is done with the understanding that the present invention encompasses the manipulation of levels of any SOCS molecule, such as but not limited to human SOCS-2 (hSOCS-2). The nucleotide sequence of hSOCS-2 and its corresponding amino acid sequence can be found at Gen Bank Accession Number AF037989 (see also reference 19). Reference herein to a “SOCS” molecule such as SOCS-2 includes any mutants thereof such as functional mutants. An example of a mutant is a single or multiple amino acid substitution, addition and/or deletion or truncation to the SOCS molecule or its corresponding DNA or RNA. Another useful SOCS molecule is SOCS-3 and manipulating levels of SOCS-3 is encompassed by the present invention.
The present invention is predicated in part on the determination that knock out mice for a SOCS gene exhibit altered hormone signalling. In particular, mice which substantially do not produce SOCS-2 exhibit disrupted growth hormone signalling and, as a result, have a growth advantage over their littermates.
This provides the basis for developing therapeutic protocols for subjects which either require more or require less growth hormone signalling. It also has important implications in the veterinary industry for manipulating animals to provide same with a growth advantage. This may be important for food production. Alternatively, where a reduction in the size of an animal is desired, facilitating expression of a SOCS gene or facilitating SOCS protein activity will result in greater growth hormone signalling.
Although the present invention is specifically exemplified in relation to growth hormone, the instant invention extends to any hormone signalling such as facilitated by protein hormones (i.e. cytokines) and non-protenaceous hormones.
Accordingly, another aspect of the present invention contemplates a method for controlling hormone signalling such as growth hormone signalling in an animal such as a human or livestock animal, said method comprising modulating expression of a genetic sequence encoding a SOCS protein comprising a SOCS box and a protein:molecule interacting region N-terminal of said SOCS box wherein said SOCS box comprises the amino acid sequence:
wherein:
Preferably, the SOCS protein-encoding genetic sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 1 or a nucleotide sequence having at least 60% similarity thereto or a nucleotide sequence capable of hybridizing to SEQ ID NO: 1 or its complementary form under low stringency conditions at 42° C. Even more preferably, the SOCS protein in a human homologue of the nucleotide sequence set forth in SEQ ID NO: 1.
Alternatively, the SOCS protein is SOCS-3.
The term “similarity” as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et al. (16). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (17).
The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm=69.3+0.41 (G+C)% (18). However, the Tm of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (19). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.
In an alternative embodiment, the modulation of growth hormone signalling is accomplished at the protein level using either a SOCS antagonist or agonist or using a composition comprising SOCS such as SOCS-2 or its derivative, homologue or analogue.
Accordingly, another aspect of the present invention contemplates a method for controlling growth hormone signalling in an animal such as human or livestock animal, said method comprising administering to said animal a control-effective amount of a SOCS protein or functional part or homologue or analogue thereof or an antagonist or agonist of a SOCS protein for a time and under conditions sufficient to modulate growth hormone signalling.
Preferably, the SOCS protein is SOCS-2. Where a SOCS protein is administered, it may be an isolated form of the naturally occurring SOCS protein or it may be a derivative, homologue or analogue thereof.
A further aspect contemplates genetically modified animals such as livestock animals. Such animals, having altered hormone signalling, are useful inter alia for food production.
A “derivative” includes a part, portion or fragment thereof such as a molecule comprising a single or multiple amino acid substitution, deletion and/or addition. A “homologue” includes a functionally similar molecule from either the same species or another species.
Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Cα and Cβ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The present invention further contemplates chemical analogues of SOCS molecules capable of acting as antagonists or agonists of a SOCS or which can act as functional analogues of SOCS. Chemical analogues may not necessarily be derived from SOCS but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of SOCS. Chemical analogues may be chemically synthesized or may be detected following, for example, natural product screening.
Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.
The present invention extends to compositions such as pharmaceutical compositions comprising one or more of a SOCS protein, a derivative, homologue or analogue thereof and/or an antagonist or agonist of a SOCS protein and one or more pharmaceutically acceptable carriers and/or diluents.
Such compositions are useful for modulating growth hormone signalling in an animal such as a human or livestock animal. Preferably, the composition comprises SOCS-2 or a derivative, homologue or analogue thereof.
In order to facilitate passage of the SOCS protein through various membranes, the molecule may be fused to or co-expressed with TAT, (from HIV) or penetratin. Alternatively, the SOCS protein may be administered following laser treatment.
The preparation of pharmaceutical compositions is well known in the art and is described, for example, in Remington's Pharmaceutical Sciences, Eaton Publishing, Pennsylvania USA or in International Patent Publication No. PCT/AU99/00729 [WO 98/20023].
The present invention also provides for the genetic control of SOCS levels in animals.
For example, compositions comprising antisense RNA or DNA, ribozymes or sense molecules (for co-suppression) may be administered either locally or systemically to manipulate expression of SOCS genes or translation of SOCS mRNA.
Another aspect of the present invention provides a genetically modified animal exhibiting altered hormone and in particular growth hormone signalling.
More particularly, the present invention is directed to a genetically modified animal exhibiting increased or decreased levels of a SOCS protein or a derivative or homologue thereof wherein the SOCS protein, in a generally unaltered animal, comprises a protein:molecule interacting portion, N-terminal of a SOCS box, which SOCS box comprises the amino acid sequence:
wherein:
Preferably, the SOCS protein is SOCS-2 or its derivative or homologue and comprises the amino acid sequence substantially as set forth in SEQ ID NO: 2 or an amino acid sequence having at least about 60% similarity thereto.
In a particularly preferred embodiment, the present invention provides a genetically modified animal wherein the animal, before genetic modification, comprises a genetic sequence which comprises a sequence of nucleotides substantially corresponding to the nucleotide sequence set forth in SEQ ID NO: 1 or its complementary form or is able to hybridize under low stringency conditions at 42° C. to SEQ ID NO: 1 or its complementary form and wherein said genetic modification either results in a deletion of one or more nucleotides from said genetic sequence from said animal or substantially prevents, reduces or down-regulates expression of said genetic sequence.
The term “expression” includes transcription and/or translation of a genetic sequence.
In a particularly preferred embodiment, the present invention provides a genetically modified animal comprising a substantial deletion in a genetic sequence comprising a nucleotide sequence substantially corresponding to SEQ ID NO: 1 or capable of hybridizing to SEQ ID NO: 1 under low stringency conditions at 42° C. and wherein said animal exhibits altered hormone regulation such as growth hormone regulation.
Genetically modified animals contemplated by the present invention include mice, rats, guinea pigs, rabbits and livestock animals (e.g. pigs, cows, sheep, donkeys, horses). Genetically modified livestock animals are particularly useful in food production.
The genetically modified animals of the present invention are also useful animal models for screening for therapeutic agents capable of modulating growth hormone signalling.
The present invention further extends to agents identified using the animal model of this aspect of the present invention.
The present invention is further described by the following non-limiting Examples.
A genomic SOCS-2 fragment extending approximately 2.0 kb from the protein initiation ATG was generated by PCR. This fragment was fused to the ATG of β-galactosidase via the BamHI site in the plasmid vector pβgalpAloxneo (24). The 3′ arm, an EcoRI fragment extending 3.7 kb downstream from the termination codon was blunted and ligated into the XhoI (blunted) site of pβgalpAloxneo that already contained the 5′ Arm. This targetting vector was linearized with NotI and electroporated into C57BL/6 embryonic stem cells. Transfected cells were selected in G418 and resistant clones picked and expanded. Clones in which the targeting vector had recombined with the endogenous SOCS-2 gene were identified by hybridizing EcoRV-digested genomic DNA with a 0.8 kb BamHI-NheI fragment situated in 5′ SOCS-2 genomic sequence just outside the targeting vector (
Peripheral blood white cell and platelet counts were determined manually using haemocytometers. Single cell suspensions from femoral bone marrow, spleen and liver were prepared and differential counts of peripheral blood, bone marrow and spleen were performed from stained smears and cytocentrifuge preparations. Clonal cultures of 2.5×104 adult bone marrow cells were performed in 0.3% agar as previously described (26). Cultures were stimulated with recombinant purified GM-CSF, G-CSF, M-CSF, IL-3 (each at 10 ng/ml), SCF (100 ng/ml), IL-6 (100 ng/ml) or Flk-ligand (500 ng/ml) plus LIF (103 U/ml). Agar cultures were fixed, sequentially stained for acetylcholinesterase, Luxol fast Blue and hematoxylin, and the cellular composition of each colony determined microscopically. Tissue sections were prepared by standard techniques, stained with haematoxylin and eosin and examined by light microscopy.
MUP Analysis
To analyze MUP levels, 6-7 week old mice were made to urinate before samples were collected 3 and 5.5 hours later. Samples were pooled and centrifuged (13,000 rpm×3 min) before 0.5 μl of supernatant was electorphoresed in 12% SDS-polyacrylamide gels and stained with Coomassie blue.
Cohorts of mice were weighed at weekly intervals for 12 weeks from birth. After sacrifice, animals were pinned down through the oral cavity and lightly stretched by the tail for nose-anus (body length) and anus-tail (tail length) measurements. To measure skeletal dimensions, limbs were subsequently removed and oriented in a consistent manner for X-ray photography and bone length measurement.
Serum IGF-I levels in serum from orbital bleeds were determined using an EIA kit (rat IGF DSL-10-2900 Diagnostic Systems Laboratories, Webster, Tx) according to the manufacturer's instructions. IGF-I RNA expression was determined in RNase protections assays as previously described (23) using β-actin as an internal standard.
To construct the SOCS-2 targeting vector, a 5′ arm extending approximately 2.0 kb from the protein initiation ATG was generated by PCR using specific SOCS-2 oligonucleotides and genomic clone pgmSOCS-2 57-60-1-45 as template. This fragment was fused to the ATG of β-galactosidase via the BamHI site in the plasmid vector pβgalpAloxneo. The 3′ arm, a 3.7 kb EcoRI fragment from pgmSOCS-2 57-60-145 was blunted and ligated into the XhoI (blunted) site of pβgalphAloxneo that already contained the 5′ arm. This targeting vector was linearized with NotI and electroporated into C57BL/6 embryonic stem cells. Transfected cells were selected in G418 and resistant clones picked and expanded. Clones in which the targeting vector has recombined with the endogenous SOCS-2 gene were identified by hybridising EcoRV-digested genomic DNA with a 1.8 kb EcoRI-EcoRV fragment from pgmSOCS-2 57-60-1-45. This probe (probe A,
SOCS-2−/− mice appeared outwardly normal, however, male mice and perhaps to a lesser extend female mice developed to a significantly larger body weight than their normal littermates (
A thorough hematological survey has not identified any hematological abnormalities in SOCS-2−/− mice (Table 3).
*p < 0.0O5 in Student's t-test for comparison of sex-matched SOCS-24−/− and SOCS-2+/− data with SOCS-2+/+ mice (n = 6-12).
The increased growth of mice is considered to be due to disruption in the pulsatile nature of growth hormone signalling in animals and in particular male animals.
To further analyze this, the phenomenon is studied and compared in animals homozygous and heterozygous for animals homozygous and heterozygous for the SOCS-2 gene. In these animals, levels of growth hormone and growth hormone controlled factors such as IGF-1 is determined.
The activation status of STAT5a and in particular STAT5b, which are major mediators of growth hormone and prolactin signalling, is determined in the heterozygous and homozygous mice. In addition, growth hormone, pulse-regulated, sexually dimorphic gene expression of genes such as MUP and CYP is also determined. Furthermore, the sensitivity of knock out mice to growth hormone signalling is further analyzed.
To more directly examine the effects of SOCS-2 on the growth hormone system, the inventors initiated crosses of the SOCS-2−/− mice with STAT5b−/− and little mice. STAT5b is required for the sexually dimorphic effects of growth hormone. Consequently, male mice lacking SOCS-5b grow no larger than female mice, which are themselves normal size. A range of outcomes to this experiment is envisaged. If SOCS-2 is required to regulate growth hormone (GH) signalling, mice lacking both SOCS-2 and STAT5b are expected to reproduce a STAT-5b-deficient phenotype as GH signalling would not properly operate in the absence of this key signalling molecule making the presence or absence of SOCS-2 irrelevant. If SOCS-2 acts independently of GH signalling, an intermediate phenotype might ensue. Conversely, given that little mice are not entirely
GH-deficient, if SOCS-2 acts to regulate GH signalling, a little mouse that is also deficient in SOCS-2 might exhibit amelioration of dwarfism.
If SOCS-2 acts on the GH or IGF-I signalling pathways, a strong prediction would be that the SOCS-2-deficient cells or the SOCS-2−/− mice themselves would be significantly more sensitive than their wild-type counterparts to the effects of these cytokines. The deregulating of GH signalling might result in heightened activation of STAT5b, the key STAT involved in sexually dimorphic growth in mice. This is investigated in the livers of unmanipulated male SOCS-2−/− mice, together with those from unmanipulated wild-type mice and wild-type mice after injection with a maximally stimulating dose of GH. The levels of expression of downstream markers of pulse-regulated GH signalling, including MUP, and cytochrome P450-catalysed testosterone hydroxylase, are determined in Northern blot analysis of liver RNA and/or Western blot analysis of protein extracts. Differences in GH sensitivity is also investigated by comparing the cytokine concentration required to stimulate phosphorylation of STAT5b in SOCS-2−/− and normal mice in dose response studies using preparations of primary hepatocytes and/or fibroblasts.
The size parameters of SOCS-2/STAT5b are shown in Table 4. The data in the table show that male SOCS-2−/− STAT5b−/− mice have a growth phenotype intermediate to the large SOCS-2−/− STAT5b+/+ and small SOCS-2+/+ STAT5b−/− mice. In female mice, SOCS-2−/− STAT5b−/− also appear to be intermediate in this regard compared with mice lacking SOCS-2 or STAT5b singly. This result implies that the excessive growth in SOCS-2−/− mice is dependent, at least in part, on STAT5b. Also, although not proof, this is further evidence consistent with abnormal growth hormone signalling in SOCS-2−/− mice.
mean ± std dev of data from 2-8 mice per data point
The possibility that SOCS-2 acts directly in the IGF-I signalling cascade is also being investigated. To achieve this, the phosphorylation status of the IGF-I receptor and key downstream signalling molecules in cells or tissues of SOCS-2−/− mice is investigated. Primary embryo fibroblast cultures are established from SOCS-2−/− and normal control mice. Initially, the extent and duration of phosphorylation of the IGF-I receptor, as well as the downstream effectors IRS and shc is measured following stimulation of these cells, or in primary hepatocyte preparations, with a maximally-stimulating dose of IGF-I. Differences in IGF-I sensitivity are also investigated by comparing the minimal cytokine concentration required to stimulate receptor and substrate phosphorylation in SOCS-2−/− and normal fibroblasts in dose response studies.
Transgenic mice are generated which constitutively express SOCS-2 ubiquitously. The murine cDNA encoding SOCS-2 is linked to human ubiquitin C promoter, which drives high-level transgene expression in most mouse tissues. If the transgenic mice are small, and show evidence of resistance to GH or IGF-I, this provides strong evidence consistent with the hypothesis that SOCS-2 acts within the GH/IGF-I axis.
The inventors suspected that asspects of GH and/or IGF-I signalling might be deregulated in SOCS-2−/− mice. To directly examine the GH/IGF-I pathway, the inventors first investigated levels of major urinary protein (MUP), a GH pulse-dependent product that is down-regulated in GH-overexpressing transgenic mice, where the usual pulsatile pattern of GH signalling is disrupted (21). To analyze MUP levels, 6-7 week old mice were made to urinate before samples were collected 3 and 5.5 hours later. Samples were pooled and centrifuged (13,000 rpm×3 min) before 0.5 μl of supernatant was electorphoresed in 12% w/v SDS-polyacrylamide gels and stained with Coomassie blue. Consistent with deregulated GH signalling, less MUP was observed in samples of urine from each of 6 male and 5 female SOCS-2−/− mice compared with a similar number of sex-matched wild-type samples (
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
| Number | Date | Country | Kind |
|---|---|---|---|
| PQ 4082 | Nov 1999 | AU | national |
| PQ 7812 | May 2000 | AU | national |
The present application is a continuation of U.S. patent Ser. No. 10/130,039, filed Oct. 3, 2002, which is a 371 of PCT/AU00/01398 having an international filing date of Nov. 16, 2000.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10130039 | Oct 2002 | US |
| Child | 11231101 | Sep 2005 | US |
