Abstract:
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. More particularly, the present invention contemplates a method for the treatment and/or prophylaxis of conditions, the amelioration of symptoms of which, are facilitated by an over-expression of a gene encoding a suppressor of cytokine signalling molecule. The present invention further contemplates agents useful for the prophylaxis and/or treatment of such renditions in mammals including humans.

Description:
FIELD OF THE INVENTION  
         [0001]    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.  
           [0002]    More particularly, the present invention contemplates a method for the treatment and/or prophylaxis of conditions, the amelioration of symptoms of which are facilitated by an over-expression of a gene encoding a suppressor of cytokine signalling molecule. The present invention further contemplates agents useful for the prophylaxis and/or treatment of such conditions in mammals including humans.  
         BACKGROUND OF THE INVENTION  
         [0003]    Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.  
           [0004]    Reference to any prior art in this specification is not, and should not be taken as, au acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.  
           [0005]    The gene encoding Suppressor of Cytokine Signaling-1 (SOCS-1), the SOCS protein family prototype, was discovered in a functional genetic screen designed to identify inhibitors of cytokine signalling. Comparison to existing sequences on genetic databases identified a number of additional proteins that could be grouped into a “SOCS protein family” on the basis of homology within a novel COOH-terminal ‘SOCS-box’ sequence motif. Proteins containing the SOCS-box could be further divided into sub-families on the basis of additional protein sequence motifs including, for example, SH2 domains (SOCS1-7), WD40 repeats (WSB1, 2), ankyrin repeats (ASB1-3) and a SPRY domain (SSB1-3). Subsequent analysis has revealed tat SOCS-1 and other SOCS family members, most notably those which incorporate an SH2 domain, represent the key components of a classic negative feedback loop that regulates cytokine signalling. SOCS protein expression is induced by cytokine signaling and SOCS proteins interact with components of that process to tub signaling off.  
           [0006]    SOCS-1, which inhibits the in vitro activity of a variety of cytokines including IL-6, LIF, and type 19 interferons, binds directly to, and inhibits the action of, Janus kinses (JAKs). Published analysis indicates that this activity against JAKs may be mediated by three distinct functional domains within SOCS-1: the SH2 domain and preceding 12 ammo acids (extended SH2 subdomain) of SOCS-1 are required for binding to the phosphorylated (Y1007) activation loop of JAK2; an additional 12 N-terminal amino acids (kinase inhibitory region) of SOCS-1 contribute to high affinity binding to the JAK2 tyrosine e dowry and are required for the inhibition of JAK2 activity; and the SOCS-box has been found to mediate the inter on of SOCS proteins with elongin B and elongin C, intracellular proteins responsible for targeting proteins for degradation within the cell.  
           [0007]    In addition to inhibiting the activity of cytokines that signal through the JAK/STAT pathway, SOCS-1 has also been reported to inhibit TNFα activities such as induction of cell death (1). Although the mechanism for this activity remains unclear, there is some evidence to suggest that SOCS-1 regulates the activity of p38 MAP kinase which in turn may act as a survival factor in TNF treated cells.  
           [0008]    SOCS-3 has also been demonstrated to inhibit the in vitro activity of LIF and IL-6, however, in contrast to SOCS-1, it does not appear to bind directly to JAKs. Structure-function studies have identified an interaction between SOCS-3 and he cytoplasmic domain of shared receptor component gp130. In particular a single peptide representing the amino acid stretch 750764 of gp130 and centred around the phosphorylated tyrosine residue 757 (pY757) is able to bind to the SOCS-3 protein with high affinity (dd-42 nm). Thus, SOCS proteins appear to inhibit cytokine signalling by at least two mechanisms: they are able to bind to, and inhibit the activity of signalling intermediates activated following receptor oligermerization (e.g. JAKs) or they interact with receptor components (e.g. gp130) to inhibit the phosphorlyation and activation of downstream substrates.  
           [0009]    Cytokines are key mediators of a number of severe and debilitating diseases. For example, a number of cytokines including IL-1, IL-6, TNFα, GM-CSF and type I/II interferons are central to the pathophysiology of both acute and chronic inflammatory disease. This is reflected in the development and marketing of new therapeutic strategies which focus on inhibition of cytokine action. For example, specific antagonists of TNFα (monoclonal antibodies, soluble receptors) are now used successfully in the treatment of rheumatoid arthritis and Chrones disease.  
           [0010]    As potent negative regulators of cytokine signalling SOCS proteins provide for a new approach to the treatment of cytokine mediated disease such as rheumatoid arthritis. Targeted over-expression of SOCS proteins (i.e. SOCS proteins as gene therapeutics) should turn off cytokine signalling and ameliorate cytokine-mediated disease. Rheumatoid arthritis represents a useful example. When over-expressed, SOCS-1 has been demonstrated to interact with and inhibit the activity of JAKs. JAK activation and subsequent action represents an important downstream event in signalling through both IL-6 and GM-CSF receptors. Furthermore SOCS-1 has also been demonstrated to be a potent antagonist of TNFα mediated activities. In work leading up to the present invention, the inventors reasoned that over-expression of SOCS-1 could be expected to interfere in IL-6, GM-CSF and TNF signalling, all key mediators of rheurmatoid arthritis.  
           [0011]    For SOCS therapeutics to be effective, it is likely that they will need to be expressed at a high level such as being over-expressed in the majority of target cells within a pathological lesion Gene based therapies clearly represent the best way to achieve this, with viral vectors such as adenovirus, adeno-associated virus (AAV) and retrovirus likely to represent the delivery mechanism of choice.  
         SUMMARY OF THE INVENTION  
         [0012]    Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ED NOs: correspond numerically to the sequence identifiers &lt;400&gt;1, &lt;400&gt;2, etc. A sequence listing is provided after the claims.  
           [0013]    Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comiprising”, 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.  
           [0014]    The present invention is predicated in part on the use of genetic therapeutic protocols to increase, enhance or otherwise facilitate expression of nucleotide sequences encoding a SOCS molecule in a cell. Overexpression of such nucleotide sequences thereby elevates levels of the SOCS protein or other expression products (e.g. mRNA or spliced out introns from mRNA encoded by genomic DNA). The “over-expression” in this context means, in one particular embodiment, a level of expression statistically greater than a standardized normal control. However, the present invention also contemplates maintenance of normal expression levels. The “level” of expression may readily be determined by, for example, nuclear run-on analysis or determination of SOCS protein levels amongst other methods.  
           [0015]    Accordingly, one aspect of the present invention contemplates a method for modulating cytokine or hormone signalling in an animal, said method comprising up-regulating expression of a genetic sequence encoding a SOCS protein or its derivative or homolog in said animal.  
           [0016]    Another aspect of the present invention provides a method of modulating cytokine or hormone signalling in an animal and in particular a human, said method comprising up-regulating expression of a genetic sequence encoding 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, WD-40 repeats and/or ankyrin repeats, N terminal of a SOCS box, wherein said SOCS box comprises the amino acid sequence:  
           X 1 X 2 X 3 X 4 X 5 X 7 X 8 X 9 X 10 X 11 X 12  X 13 X 14 X 15 X 16 [X i ] n X 17 X 18 X 19 X 20 X 21 X 22 X 23 [X j ] X 24 X 25 X 26 X 27 X 28    
           [0017]    wherein:  
           [0018]    X 1  is L, I, V, M, A or P;  
           [0019]    X 2  is any amino acid residue;  
           [0020]    X 3  is P, T or S;  
           [0021]    X 4  is L, I, V, M, A or P;  
           [0022]    X 5  is any amino acid;  
           [0023]    X 6  is any amino acid;  
           [0024]    X 7  is L, I, V, M, A, F, Y or W;  
           [0025]    X 8  is C, T or S;  
           [0026]    X 9  is R, K or H;  
           [0027]    X 10  is any amino acid;  
           [0028]    X 11  is any amino acid;  
           [0029]    X 12  is L, I, V, M, A or P;  
           [0030]    X 13  is any amino acid;  
           [0031]    X 14  is any amino acid;  
           [0032]    X 15  is any amino acid;  
           [0033]    X 16  is L, I, V, M, A, P, G, C, T or S;  
           [0034]    [X i ] n  is a sequence of n amino acids wherein u is from 1 to 50 amino acids and wherein the sequence X i  may comprise the same or different amino acids selected from any amino acid residue;  
           [0035]    X 17  is L, I, V, M, A or P;  
           [0036]    X 18  is any amino acid;  
           [0037]    X 19  is any amino acid;  
           [0038]    X 20  is L, I, V, M, A or P;  
           [0039]    X 21  is P;  
           [0040]    X 22  is 1L, I, V, M, A, P or G;  
           [0041]    [X j ] n  is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the X i  may comprise the same or different amino acids selected from any amino acid residue,  
           [0042]    X 24  is L, V, A or P;  
           [0043]    X 25  is any amino acid;  
           [0044]    X 26  is any amino acid,  
           [0045]    X 27  is Y or F;  
           [0046]    X 28  is L, I, V, M, A or P.  
           [0047]    Still another aspect of the present invention contemplates a method for controlling cytokine or hormone signalling, such as pro-inflammatory cytokine signalling (i.e. 16, GM-CSF, TNFα), 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;  
           X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 [X i ] n X 17 X 18 X 19 X 20 X 21 X 22 X 23 [X j ] n X 24 X 25 X 26 X 27 X 28    
           [0048]    wherein:  
           [0049]    X 1  is L, I, V, M, A or P;  
           [0050]    X 2  is any amino acid residue;  
           [0051]    X 3  is P, T or S;  
           [0052]    X 4  is L, I, V, M, A or P;  
           [0053]    X 5  is any amino acid;  
           [0054]    X 6  is any amino acid;  
           [0055]    X 7  is L, I, V, M, A, F, Y or W;  
           [0056]    X 8  is C, T or S;  
           [0057]    X 9  is R, K or H;  
           [0058]    X 10  is amino acid;  
           [0059]    X 11  is any amino acid;  
           [0060]    X 12  is L, V, M, A or P;  
           [0061]    X 13  is any amino acid;  
           [0062]    X 14  is any amino acid;  
           [0063]    X 15  is any amino acid;  
           [0064]    X 16  is L, I, V, M, A, P, G, C, T or S;  
           [0065]    [X i ] n  is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X i  may comprise the same or different amino acids selected from any amino acid residue;  
           [0066]    X 17  is L, I, V, M, A or P;  
           [0067]    X 18  is any amino acid;  
           [0068]    X 19  is any amino acid;  
           [0069]    X 20  is L, I, V, A or P;  
           [0070]    X 21  is P;  
           [0071]    X 22  is L, I, V, M, A, P or G;  
           [0072]    X 23  is P or N;  
           [0073]    [X 24 ] n  is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the X j  may comprise the same or different amino acids selected from any amino acid residue;  
           [0074]    X 24  is L, I, V, M, A or P;  
           [0075]    X 25  is any amino acid;  
           [0076]    X 26  is amino acid;  
           [0077]    X 27  is Y or F;  
           [0078]    X 28  is L, I, V, M, A or P.  
           [0079]    Yet another aspect of the present invention contemplates a method for controlling cytokine or hormone signaling in an animal such as human or livestock animal, said method comprising administering to said animal a genetic molecule encoding a SOCS protein for a time and under conditions sufficient to modulate growth hormone signaling.  
           [0080]    Another aspect of the present invention contemplates a method for the treatment of cytokine-mediated disease in an animal, said method comprising modulating cytokine or hormone signalling in an animal by up-regulating the expression of a genetic sequence encoding a SOCS protein or its derivative or homologue in said animal.  
           [0081]    In a preferred embodiment, the SOCS gene is expressed at a high level such as being overexpressed.  
           [0082]    A summary of sequence identifiers used throughout the subject specification is provided below.  
         SUMMARY OF SEQUENCE IDENTIFIERS  
         [0083]    [0083]                                                                 SEQUENCE ID NO:   DESCRIPTION                                        1   Mouse SOCS-1 (nucleotide)           2   Mouse SOCS-1 (amino acid)           3   Mouse SOCS-3 (nucleotide)           4   Mouse SOCS-3 (amino acid)           5   Human SOCS-1 (nucleotide)           6   Human SOCS-1 (amino acid)           7   Rat SOCS-1 (nucleotide)           S   Rat SOCS-1 (amino acid)           9   Primer           10   Primer           11   Primer           12   Primer           13   Primer           14   Primer                        
       
    
    
     BRIEF DESCRIPTION OF THE FIGURES  
       [0084]    [0084]FIG. 1 is a graphical representation of SOCS-1 +/+  IFN-γ −/− mice (▪) compared to SOCS-1 +/+  IFN-γ −/−  (□) mice following injection of BSA and L-1 subcutaneously to knee joints in three daily injections. A histological score was measured in oxodate, synovitis, pannus, cartilage and bone.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0085]    One aspect of the present invention contemplates a method for modulating cytokine or hormone signalling in an animal, said method comprising up-regulating expression of a genetic sequence encoding a SOCS protein or its derivative or homolog in said animal.  
         [0086]    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 mouse and “r” is rat. Accordingly, “mSOCS-2”, for ale, 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 to “modulates” encompasses up-regulation as well as at least maintenance of particular levels. Preferably, the expression is up-regulated. Reference herein to “murine” includes both mouse and rat.  
         [0087]    Reference herein to a “hormone” includes protein hormones 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. A cytokine refers to any cytokine or cytokine-like molecule such as interleukin (e.g. IL-1, IL-6), tumour necrosis factor (e.g. TNFα), a colony stimulating factor (e.g. GM-CSF) or an interferon.  
         [0088]    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 annual 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.  
         [0089]    Reference herein to “SOCS” includes a protein comprising a SOCS box in its C-terminal region comprising the amino acid sequence:  
         X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 [X i ] n X 17 X 18 X 19 X 20 X 21 X 22 X 23 [X j ] n X 24 X 25 X 26 X 27 X 28    
         [0090]    wherein:  
         [0091]    X 1  is L, I, V, M, A or P;  
         [0092]    X 2  is any amino acid residue;  
         [0093]    X 3  is P, T or S;  
         [0094]    X 4  is L, I, V, M, A or P;  
         [0095]    X 5  is any amino acid;  
         [0096]    X 6  is any amino acid;  
         [0097]    X 7  is L, I, V, M, A, F, Y or W;  
         [0098]    X 8  is C, T or S;  
         [0099]    X 9  is R, K or I;  
         [0100]    X 10  is any amino acid;  
         [0101]    X 11  is any amino acid;  
         [0102]    X 12  is L, I, V, M, A or P;  
         [0103]    X 13  is any amino acid;  
         [0104]    X 14  is any amino acid;  
         [0105]    X 15  is any amino acid;  
         [0106]    X 16  is L, I, V, M, A, P, G, C, T or S;  
         [0107]    [X i ] n  is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X i  may comprise the same or different amino acids selected from any in acid residue;  
         [0108]    X 17  is L, I, V, M, A or P;  
         [0109]    X 18  is amino acid;  
         [0110]    X 19  is any amino acid;  
         [0111]    X 20  is L, V, M, A or P;  
         [0112]    X 21  is P;  
         [0113]    X 22  is L, I, V, W, A, P or G;  
         [0114]    X 23  is P or N,  
         [0115]    [X j ] n  is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the X j  may comprise the same or different amino acids selected from any amino acid residue;  
         [0116]    X 24  is L, I, V, M, A or P;  
         [0117]    X 25  is any amino acid;  
         [0118]    X 26  is any amino acid;  
         [0119]    X 27  is Y or F;  
         [0120]    X 28  is L, I, V, M, A or P.  
         [0121]    The SOCS protein also comprises a protein:molecule interacting region such as but not limited to one or more of an SH2 domain, WD-40 repeats and/or ankyrin repeats, N-terminal of the SOCS box.  
         [0122]    In an important aspect, the present invention contemplates un-regulating expression of a nucleotide sequence encoding a SOCS protein in the treatment of inflammatory diseases such as rheumatic arthritis.  
         [0123]    Another aspect of the present invention provides a method of modulating cytokine or hormone signalling in an animal and in particular a human, said method comprising up-regulating expression of a genetic sequence encoding a SOCS protein in said anima and wherein said SOCS protein comprises a protein:molecule interacting region such as but not limited to an SE domain, WD-40 repeats and/or ankyrin repeats, N terminal of a SOCS box, wherein said SOCS box comprises the amino acid sequence:  
         X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 [X i ] n X 17 X 18 X 19 X 20 X 21 X 22 X 23 [X j ] n X 24 X 26 X 27 X 28    
         [0124]    wherein:  
         [0125]    X 1  is L, I, V, M, A or P;  
         [0126]    X 2  is any amino acid residue;  
         [0127]    X 3  is P, T or S;  
         [0128]    X 4  is L, I, V, M, A or P;  
         [0129]    X 5  is any amino acid;  
         [0130]    X 6  is any amino acid;  
         [0131]    X 7  is L, I, M, A, F, Y or W;  
         [0132]    X 8  is C, T or S;  
         [0133]    X 9  is R, K or H;  
         [0134]    X 10  is any amino acid;  
         [0135]    X 11  is any amino acid;  
         [0136]    X 12  is L, I, V, M, A or P;  
         [0137]    X 13  is any amino acid;  
         [0138]    X 14  is any amino acid;  
         [0139]    X 15  is any amino acid;  
         [0140]    X 16  is L, I, V, M, A, P, G, T or S;  
         [0141]    [X i ] n  is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X i  may comprise the same or different amino acids selected from any amino acid residue;  
         [0142]    X 17  is L, V, M, A or P;  
         [0143]    X 18  is any amino acid;  
         [0144]    X 19  is any amino acid;  
         [0145]    X 20  is L, I, V, M, A or P;  
         [0146]    X 21  is P;  
         [0147]    X 22  is L, I, V, M A, P or G;  
         [0148]    X 23  is P or N;  
         [0149]    [X j ] n  is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the X j ; may comprise the same or different amino acids selected from any amino acid residue;  
         [0150]    X 24  is L, I, V, M, A or P;  
         [0151]    X 25  is any amino acid;  
         [0152]    X 26  is any amino acid;  
         [0153]    X 27  is Y or F;  
         [0154]    X 28  is L, I, V, M, A or P.  
         [0155]    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 incorporated herein by reference. However, in a particularly preferred embodiment, the present invention is directed to manipulating levels of SOCS-1, which murine form (mSOCS-1) 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-1 (mSOCS-1), 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 films SOCS-2 (hSOCS-2). Reference herein to a “SOCS” molecule such as SOCS-1 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 Accordingly, another aspect of the present invention contemplates a method for controlling cytokine or hormone signaling such as pro-inflammatory cytokine signalling (i.e. IL-6, GM-CSF, TNFα), 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:  
         X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 X 15 X 16 [X i ] n X 17 X 18 X 19 X 20 X 21 X 22 X 23 [X j ] n X 24 X 26 X 27 X 28    
         [0156]    wherein:  
         [0157]    X 1  is L, I, V, M, A or P;  
         [0158]    X 2  is any amino acid residue;  
         [0159]    X 3  is P, T or S;  
         [0160]    X 4  is L, I, V, M, A or P;  
         [0161]    X 5  is any amino acid;  
         [0162]    X 6  is any amino acid;  
         [0163]    X 7  is L, I, M, A, F, Y or W;  
         [0164]    X 8  is C, T or S;  
         [0165]    X 9  is R, K or H;  
         [0166]    X 10  is any amino acid;  
         [0167]    X 11  is any amino acid;  
         [0168]    X 12  is L, I, V, M, A or P;  
         [0169]    X 13  is any amino acid;  
         [0170]    X 14  is any amino acid;  
         [0171]    X 15  is any amino acid;  
         [0172]    X 16  is L, I, V, M, A, P, G, T or S;  
         [0173]    [X i ] n  is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X i  may comprise the same or different amino acids selected from any amino acid residue;  
         [0174]    X 17  is L, V, M, A or P;  
         [0175]    X 18  is any amino acid;  
         [0176]    X 19  is any amino acid;  
         [0177]    X 20  is L, I, V, M, A or P;  
         [0178]    X 21  is P;  
         [0179]    X 22  is L, I, V, M A, P or G;  
         [0180]    X 23  is P or N;  
         [0181]    [X j ] n  is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the X j ; may comprise the same or different amino acids selected from any amino acid residue;  
         [0182]    X 24  is L, I, V, M, A or P;  
         [0183]    X 25  is any amino acid;  
         [0184]    X 26  is any amino acid;  
         [0185]    X 27  is Y or P;  
         [0186]    X 28  is L, I, M, A or P.  
         [0187]    Preferably, the SOCS protein-encoding genetic sequence comprises a nucleotide sequence substantially as set forth in SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 or a nucleotide sequence having at least 60% similarity hereto or a nucleotide sequence capable of hybridizing to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 or its complementary form under low stringency conditions at 42° C. Even more preferably, the SOCS protein in a human homolog of the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.  
         [0188]    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 tat 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.  
         [0189]    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 similarity” 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 computerized 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. (2). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (3).  
         [0190]    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.  
         [0191]    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.  
         [0192]    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 T m  69.3+0.41 (G+C)% (4). However, the T m  of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (5). 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 2542° 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.  
         [0193]    Most preferably, an expression vector is administered capable of expressing high levels of a SOCS gene.  
         [0194]    Another aspect of the present invention contemplates a method for the treatment of cytokine-mediated disease in an animal, said method comprising modulating cytokine or hormone signalling in an animal by up-regulating the expression of a genetic sequence encoding a SOCS protein or its derivative or homolog in said animal.  
         [0195]    In accordance with the this and other aspects of the present invention, the expression of a genetic sequence encoding a SOCS protein is preferably unregulated by the administration to the animal of an expression vector comprising a SOCS gene.  
         [0196]    The present invention contemplates a range of derivatives of the SOCS molecule.  
         [0197]    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 “homolog” includes a functionally similar molecule from either the same species or another species.  
         [0198]    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.  
         [0199]    The present invention provides, therefore, the genetic control of SOCS levels in animals in the treatment of a range of physiological conditions. Preferably, the level of SOCS protein is increased by the administration of an expression vector comprising the SOCS gene.  
         [0200]    Preferably, the expression vector is a viral vector, such as an adenovirus, adeno-associated virus (AAV) or retrovimus, although other vectors, including plasmid-based vectors, are contemplated.  
         [0201]    Preferably, the genetic sequence encoding a SOCS protein is the SOCS-i genetic sequence encoding the SOCS-1 protein.  
         [0202]    For example, compositions comprising antisense RNA or sense or antisense 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.  
         [0203]    The present invention is further described by the following non-limiting Examples.  
       EXAMPLE 1  
     Construction of Recombinant Adenovirus for Expression of Selected SOCS Proteins  
       [0204]    Recombinant human adenovirus type 5 expressing selected SOCS proteins (for analysis in mouse models of disease mouse SOCS proteins are preferable) are generated following recombination between an adenovirus shuttle vector, into which a SOCS encoding cDNA has been cloned, and a mutant adenovirus. The E1 region has been deleted in the mutant adenovirus rendering it incapable of replication except in a packaging cell line that complements the defect (for example, human 293 cells expressing viral E1A and E1B proteins). Recombination, and subsequent selection of recombinants, can be carried out in the packaging cell line but a bacterial system, referred to as the pAdEasy system is preferred (6)  
         [0205]    The pAdEasy system is used to generate recombinant adenovirus expressing murine SOCS proteins by the following means.  
         [0206]    Murine SOCS-1 cDNA is amplified by the polymerase chain reaction (PCR), using the following primer set: 5′primer-ATATCTCGAGGCCACCATGGTAGCACGCAACCAGG [SEQ ID NO: 9]; 3′primer-ATATAAGCTTTCAGATCTGGAAGGGGAAGG [SEQ ID NO:10]. The 5′ primer contains a Kozak sequence and XhoI restriction site, while the 3′ primer contains a HindIII restriction site.  
         [0207]    Murine SOCS-2 cDNA is amplified by PCR, using the following primer set: 5′ primer- 
                                                                 [SEQ ID NO:11]                    3′ primer - ATATGCGGCCGCGCCACCATGACCCTGCGGTGCCT;                        [SEQ ID NO:12]                    5′ primer - ATATTCTAGATTATACCTGGAATTTATATTCTTCC.              
 
         [0208]    contains a Kozak sequence and a NotI restriction site, and the 3′ primer contains a XbaI restriction site.  
         [0209]    Murine SOCS-3 cDNA was amplified by PCR, using the following primer set: 5′ primer-TATAGCGGCCGCGCCACCATGGTCACCCACAGCAA [SEQ ID NO:13]; 3′ primer-ATATAAGCTTTTAAAGTGGAGCATCATACTA [SEQ ID NO:14]. The 5′ primer contains a Kozak sequence and a NotI restriction site, and the 3′ primer contains a HindIII restriction site.  
         [0210]    All three SOCS genes are amplified under the same PCR conditions: one cycle at 96° C. for 2 mins then 35 cycles of 96° C. for 10 seconds, 55° C. for 10 seconds and 72° C. for 1 minute.  
         [0211]    PCR products are cloned into the adenovirus shuttle vector, pShuttle-CMV, (6) by standard ligation reactions. Generation of recombinant adenovirus plasmids by homologous recombination is then carried out in the  E. coli  strain BJ5183 (6). 1 μg of pShuttle-CMV (containing selected SOCS gene) was linearized with PmeI restriction enzyme and purified with a DNA purification kit (Qiagen), then mixed with 100 ng of the adenovirus backbone plasmid; pAdEasy-1. The DNA was then electroporated into  E.coli  BJ5183, which was then plated out onto LB-agar plates containing 30/g/ml of kanamycin and left at 37° C. for 18 hrs. The smallest colonies were picked and grown in 2 ml LB broth containing 30 μg/ml of kanamycin and placed at 37° C. for 8 hrs. Adenovirus plasmid DNA was extracted from each culture and was screened for the presence of recombinant adenoviral DNA by restriction enzyme digestion in comparison with pAdEasy-1. Direct sequencing of the recombinant adenovirus DNA clones confirmed the presence of SOCS encoding sequence.  
         [0212]    Production of recombinant adenovirus for in vivo studies is carried out in 293 cells (viral E1 transformed). 93 cells are cultured in 25 cm 2  flasks, in OptiMEM media (Gibco BRL), at 37° C. and 10% CO 2  until they are 70% confluent 4 μg of recombinant adenovirus, digested with the Pac1 restriction enzyme, is transfected into 293 cells with, Lipofectamine (Gibco-BRL), according to the manufacturer&#39;s instructions. Cells are left for 7-10 days and then harvested by scrapping cells off the bottom of the flask into PBS. Cells are subjected to 5 cycles of a freeze/thawing, and the supernatant can then be used to infect more 293 cells to build up viral stocks. Cell lysis should be evident in the majority of cells approximately 3 days post infection, and should be harvested as described above.  
         [0213]    To purify the recombinant adenovirus, the infected 293 cells are harvested and spun at 7000 g 4° C. for 10 minutes. The supernatant is discarded and the cells are resuspended in 10 ml of PBS and subject to 5 cycles of a freeze/thawing. The recombinant adenovirus is then purified through a CsCl gradient, comprising two layers of 1.5 ml and 2.5 ml at densities of 1.45 g/ml and 1.25 g/ml respectively. The CsCl is made-up in 5 mM Tris Cl, 1 mM EDTA pH 7.8. The CsCl gradient containing the recombinant adenovirus spun at 90,000 g for 2 hrs and the virus fraction collected with a 19-gauge needle.  
         [0214]    The adenovirus is subject to a second round of CsCl purification. The adenovirus is diluted in CsCl solution at a density of 1.33 g/ml and centrifuged at 105 g for 18 hrs. The adenovirus is recovered with a 19-gauge needle and then placed through a G-25 Sephadex column (Amersham) and the virus fractions collected in PBS containing 10% glycerol. The recombinant adenovirus can then be stored at −70° C. until ready for use.  
       EXAMPLE 2  
     Adenovirus Expressing SOCS-1 have a Beneficial Therapeutic Effect in a Mouse Model of Rheumatoid Arthritis  
       [0215]    Collagen-induced arthritis (CIA) is a model of chronic arthritis that is induced following intradermal immunization of mice with collagen in Complete Freund&#39;s Adjuvant. It affects articular joints and is characterized by synovial hyperplasia and inflammation, pannus formation and progressive cartilage and bone degradation. The importance of individual cytokines such as GM-CSF and TNFα in CIA has been extensively studied by antibody neutralisation in vivo over the course of disease or by initiating disease in cytokine gene knockout mice.  
         [0216]    For induction of CIA, type II collagen (of bovine or chick origin for example) is dissolved to a concentration of 2 mg/ml in 10 mM acetic acid (overnight at 4° C.) then emulsified in an equal volume of Complete Freunds Adjuvant. Male DBA/1 mice are injected intradermally at several sights into the base of the tail with a total of 100 microliters of the emulsion containing 100 micrograms of collagen. On day 21 mice are given an intraperitoneal booster injection of 100 microgram of type 11 collagen dissolved in phosphate buffered saline with onset of arthritis occurring at around day 25-28.  
         [0217]    Just prior to expected onset of CIA, mice are scored visually for appearance of arthritis. Mice without macroscopic signs of arthritis in their paws are selected for treatment groups. Alternatively, to study the impact of treatment on existing disease, mice can be left for longer and those that develop overt arthritis selected for treatment groups.  
         [0218]    For treatment selected mice are anaesthetized and a small incision in the skin of the knee joint is performed for the intra-articular injection procedure. Intra-articular injection is performed with 10 7 /6 microlitre of either a SOCS-1 (or other SOCS protein) expressing or an empty or β-galactosidase expressing control recombinant adenovirus. At days 1, 5, 10 and 20 after treatment mice are sacrificed and the skin of the knee joint removed. The appearance of arthritis was assessed and severity score was recorded as per routine methods described elsewhere (7). For histological assessment whole knee joints are removed, fixed, decalcified and paraffin embedded Tissue sections are stained with hematoxylin and eosin and evaluated without knowledge of the treatment groups. Histological changes can be scored according to standard methods. For example, infiltration of cells is scored on a scale of 0-3, depending on the amount of inflammatory cells in the synovial cavity (exudate) and synovial tissue (infiltrate). A characteristic parameter in CIA is the progressive loss of bone. This destruction can be graded on a scale of 0-3, ranging from no damage to complete loss of bone structure. Additional analysis may encompass, for example, immunohistological determination of other cell surface/tissue specific markers of disease progression and severity.  
         [0219]    Over-expression of SOCS-1 (or other selected SOCS proteins) within the joint may decrease both incidence and severity of CIA and his may be reflected in histological analysis where cellular-accumulation within the joint and/or the level of bone and cartilage destruction is significantly ameliorated  
       EXAMPLE 3  
     Analysis of Arthritis in an Animal Model Demonstrates a Regulatory Role for SOCS-1 and Supports the Use of SOCS-Based Gene Therapy for the Treatment of Human Inflammatory Disease  
       [0220]    Genetically modified mice with a targeted deletion of the SOCS-1 gene (SOCS-1 −/− ) die within 3 weeks of birth. The primary mediator of this lethal phenotype is interferon-γ. SOCS-1 −/−  animals crossed onto an IFN-γ −/−  background survive as do SOCS-1 −/−  treated with an antibody that inhibits IFN-γ activity. SOCS-1- −/− IFN-γ −/−  mice are ideal for studying the role of SOCS-1 in the development of various disease pathologies. In the present example, the role of SOCS-1 in regulating the activity of the pr-inflammatory cytokines responsible for the development of arthritis was assessed,  
         [0221]    SOCS-1 +/+  IFN-γ −/−  and SOCS-1 −/−  IFN-γ −/−  mice were anaesthetiZed and injected intra-articularly into the knee joint with 10 id of a 20 mg/ml solution of methylated bovine serum albumin (mBSA). At the same time, mice were also injected with 250 ng recombinant human IL-1β subcutaneously into the rear footpad The IL-1 injection was repeated on the next 2 days. The mice were sacrificed on day 7 and the knee joints fixed in 10% v/v neutral buffered formalin for at least 2 days, decalcified and embedded in pal Frontal sections of the knee joints were cut at 4 depths, approximately 100 μm apart and stained with haemotoxylin and eosin.  
         [0222]    Assessment of Arthritis:  
         [0223]    Joint pathology was assessed in a blinded manner and 5 parameters of arthritis were graded for severity from 0 (normal) to 5 (severe). Exudate was scored according to the presence and relative numbers of inflammatory cells and fibrin-like debris in the joint space. Synovitis was defined as thickening of the synovial lining layer and soft tissue inflammation in the infrapatellar fat pad, joint capsule and the area adjacent to the periosteal sheath Pannus was defined as the encroachment of hyperplastic synovium over the articular surface or at the cartilage-bone junction. Cartilage degradation was evaluated on patellofemoral and tibiofemoral articular surfaces. Done degradation was evaluated as the extent and depth of subchondral and periosteal bone erosion. The Mann-Whitney 2-sample rank test was used to compare mean histologic scores of test and control groups.  
         [0224]    The results demonstrate a role for SOCS-1 in down-regulating/controlling the development of arthritis, in this model of the disease. SOCS-1 −/− IFN-γ −/−  animals develop more severe arthritis gm control SOCS-1 +/+ IFN-γ −/−  animals (FIG. 1). The severity of the disease in the SOCS-1 +/+ IFN-γ −/−  animals was identical to that routinely observed in wildtype controls (not shown) indicating that the lack of functional SOCS-1 and not INF-γ was responsible for the exacerbation in disease phenotype. Given the clearly demonstrated role for SOCS-1 in the negative regulation of cytokine signalling it is assumed that the exacerbation of disease is the result of the increased activity of proinflammatory cytokines. Over-expression of SOCS-1, following SOCS-1 based gone therapy would inhibit pro-inflammatory cytokine activity and thus ameliorate disease pathology.  
         [0225]    The results are shown in tabular form in Table 1 and graphically in FIG. 1.  
         [0226]    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.  
                                                             TABLE 1                       Exudate   Synovitis   Pannus   Cartilage loss Bone loss                                2980   3   3.75   2.5   2.5   2.5   14.25       2981   3.33   4.67   2.33   2   1.67   14       2982   3   4   3.25   2.5   3.25   16       2983   3   3.75   3   2.5   2.75   15       2984   3   3   2.5   2.75   2   13.25       2985   3.25   3.5   2   1.25   1   11       Average   3.096666667   3.77833333   2.59666667   2.25   2.195   13.9166667       Std.   0.062003584   0.22576413   0.18577166   0.2236068   0.32943133   0.69721669       Dev.       2986   2   2.25   1.25   1   1.25   7.75       2987   2   3   3   2.25   2.75   13       2988   1   2   1.75   2   1.25   8       2989   2   4   2.75   2   2   12.75       2990   2   3.75   1.75   15   2   11       2991   1.5   2.5   2.75   2.5   2   11.25       2992   2.5   3   2   1.25   1.75   10.5       2993   1   2   2   1   1.5   7.5       2994   2   2.75   2.75   2   1.5   11       2995   2   3   1.75   15   1   9.25       Average   1.8   2.825   2.175   1.7   1.7   10.2       Std. Dev   0.152752523   0.21424934   0.18652524   0.16583124   0.16158933   0.63113654       2996   4   4.75   3.5   2.75   2.5   17.5       2997   2.5   4   4   2.5   3.5   16.5       2998   4   5   4   3.5   3.5   20       2999   4   5   4   3.25   3.25   19.5       3000   3   4.5   3.5   3   3   17       3001   2   2.5   2.5   2   2   11       Average   3.25   4.29166667   3.58333333   2.83333333   2.95833333   16.9166667       Std. Dev   0.359397644   0.3895332   0.23863035   0.22047928   0.24509069   1.31286371       Ttest   0.000736214   0.0028622   0.00038333   0.00099983   0.0005242   0.00013435                  
 
       BIBLIOGRAPHY  
       [0227]    1. Moriata et al.,  PNAS  97: 5405-5410, 2000.  
         [0228]    2. Altschul et al.,  Nucl. Acids Res.  25:3389.1997.  
         [0229]    3. Ausubel et al., “Current Protocols in Molecular Biology” John Wiley &amp; Sons Inc, 1994-1998, Chapter 15.  
         [0230]    4. Bonner and Laskey,  Eur. J. Biochem.  46: 83, 1974.  
         [0231]    5. Marmur and Doty,  J. Mol. Biol.  5: 109, 1962.  
         [0232]    6. He et al.,  PNAS  95: 2509-2514, 1998.  
         [0233]    7. Campbell et al.,  Annals. Rheum. Dis.  56: 364-368, 1997.  
     
       
       
         1 
         
           
             14  
           
           
             1  
             1235  
             DNA  
             murine  
             
               CDS  
               (161)..(799)  
             
           
            1 

cgaggctcaa gctccgggcg gattctgcgt gccgctctcg ctccttgggg tctgttggcc     60 

ggcctgtgcc acccggacgc ccggctcact gcctctgtct cccccatcag cgcagccccg    120 

gacgctatgg cccacccctc cagctggccc ctcgagtagg atg gta gca cgc aac      175 
                                            Met Val Ala Arg Asn 
                                            1               5 

cag gtg gca gcc gac aat gcg atc tcc ccg gca gca gag ccc cga cgg      223 
Gln Val Ala Ala Asp Asn Ala Ile Ser Pro Ala Ala Glu Pro Arg Arg 
                10                  15                  20 

cgg tca gag ccc tcc tcg tcc tcg tct tcg tcc tcg cca gcg gcc ccc      271 
Arg Ser Glu Pro Ser Ser Ser Ser Ser Ser Ser Ser Pro Ala Ala Pro 
            25                  30                  35 

gtg cgt ccc cgg ccc tgc ccg gcg gtc cca gcc cca gcc cct ggc gac      319 
Val Arg Pro Arg Pro Cys Pro Ala Val Pro Ala Pro Ala Pro Gly Asp 
        40                  45                  50 

act cac ttc cgc acc ttc cgc tcc cac tcc gat tac cgg cgc atc acg      367 
Thr His Phe Arg Thr Phe Arg Ser His Ser Asp Tyr Arg Arg Ile Thr 
    55                  60                  65 

cgg acc agc gcg ctc ctg gac gcc tgc ggc ttc tat tgg gga ccc ctg      415 
Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe Tyr Trp Gly Pro Leu 
70                  75                  80                  85 

agc gtg cac ggg gcg cac gag cgg ctg cgt gcc gag ccc gtg ggc acc      463 
Ser Val His Gly Ala His Glu Arg Leu Arg Ala Glu Pro Val Gly Thr 
                90                  95                  100 

ttc ttg gtg cgc gac agt cgt caa cgg aac tgc ttc ttc gcg ctc agc      511 
Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys Phe Phe Ala Leu Ser 
            105                 110                 115 

gtg aag atg gct tcg ggc ccc acg agc atc cgc gtg cac ttc cag gcc      559 
Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg Val His Phe Gln Ala 
        120                 125                 130 

ggc cgc ttc cac ttg gac ggc agc cgc gag acc ttc gac tgc ctt ttc      607 
Gly Arg Phe His Leu Asp Gly Ser Arg Glu Thr Phe Asp Cys Leu Phe 
    135                 140                 145 

gag ctg ctg gag cac tac gtg gcg gcg ccg cgc cgc atg ttg ggg gcc      655 
Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg Arg Met Leu Gly Ala 
150                 155                 160                 165 

ccg ctg cgc cag cgc cgc gtg cgg ccg ctg cag gag ctg tgt cgc cag      703 
Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln Glu Leu Cys Arg Gln 
                170                 175                 180 

cgc atc gtg gcc gcc gtg ggt cgc gag aac ctg gcg cgc atc cct ctt      751 
Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu Ala Arg Ile Pro Leu 
            185                 190                 195 

aac ccg gta ctc cgt gac tac ctg agt tcc ttc ccc ttc cag atc tga      799 
Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe Pro Phe Gln Ile 
        200                 205                 210 

ccggctgccg ctgtgccgca gcattaagtg ggggcgcctt attatttctt attattaatt    859 

attattattt ttctggaacc acgtgggagc cctccccgcc tgggtcggag ggagtggttg    919 

tggagggtga gatgcctccc acttctggct ggagacctca tcccacctct caggggtggg    979 

ggtgctcccc tcctggtgct ccctccgggt cccccctggt tgtagcagct tgtgtctggg   1039 

gccaggacct gaattccact cctacctctc catgtttaca tattcccagt atctttgcac   1099 

aaaccagggg tcggggaggg tctctggctt catttttctg ctgtgcagaa tatcctattt   1159 

tatattttta cagccagttt aggtaataaa ctttattatg aaagtttttt tttaaaagaa   1219 

aaaaaaaaaa aaaaaa                                                   1235 

 
           
             2  
             212  
             PRT  
             murine  
           
            2 

Met Val Ala Arg Asn Gln Val Ala Ala Asp Asn Ala Ile Ser Pro Ala 
1               5                   10                  15 

Ala Glu Pro Arg Arg Arg Ser Glu Pro Ser Ser Ser Ser Ser Ser Ser 
            20                  25                  30 

Ser Pro Ala Ala Pro Val Arg Pro Arg Pro Cys Pro Ala Val Pro Ala 
        35                  40                  45 

Pro Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ser Asp 
    50                  55                  60 

Tyr Arg Arg Ile Thr Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe 
65                  70                  75                  80 

Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala 
                85                  90                  95 

Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys 
            100                 105                 110 

Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg 
        115                 120                 125 

Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Thr 
    130                 135                 140 

Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg 
145                 150                 155                 160 

Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln 
                165                 170                 175 

Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu 
            180                 185                 190 

Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe 
        195                 200                 205 

Pro Phe Gln Ile 
    210 

 
           
             3  
             2187  
             DNA  
             murine  
             
               CDS  
               (18)..(695)  
             
           
            3 

cgctggctcc gtgcgcc atg gtc acc cac agc aag ttt ccc gcc gcc ggg        50 
                   Met Val Thr His Ser Lys Phe Pro Ala Ala Gly 
                   1               5                   10 

atg agc cgc ccc ctg gac acc agc ctg cgc ctc aag acc ttc agc tcc       98 
Met Ser Arg Pro Leu Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser 
            15                  20                  25 

aaa agc gag tac cag ctg gtg gtg aac gcc gtg cgc aag ctg cag gag      146 
Lys Ser Glu Tyr Gln Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu 
        30                  35                  40 

agc gga ttc tac tgg agc gcc gtg acc ggc ggc gag gcg aac ctg ctg      194 
Ser Gly Phe Tyr Trp Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu 
    45                  50                  55 

ctc agc gcc gag ccc gcg ggc acc ttt ctt atc cgc gac agc tcg gac      242 
Leu Ser Ala Glu Pro Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp 
60                  65                  70                  75 

cag cgc cac ttc ttc acg ttg agc gtc aag acc cag tcg ggg acc aag      290 
Gln Arg His Phe Phe Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys 
                80                  85                  90 

aac cta cgc atc cag tgt gag ggg ggc agc ttt tcg ctg cag agt gac      338 
Asn Leu Arg Ile Gln Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp 
            95                  100                 105 

ccc cga agc acg cag cca gtt ccc cgc ttc gac tgt gta ctc aag ctg      386 
Pro Arg Ser Thr Gln Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu 
        110                 115                 120 

gtg cac cac tac atg ccg cct cca ggg acc ccc tcc ttt tct ttg cca      434 
Val His His Tyr Met Pro Pro Pro Gly Thr Pro Ser Phe Ser Leu Pro 
    125                 130                 135 

ccc acg gaa ccc tcg tcc gaa gtt ccg gag cag cca cct gcc cag gca      482 
Pro Thr Glu Pro Ser Ser Glu Val Pro Glu Gln Pro Pro Ala Gln Ala 
140                 145                 150                 155 

ctc ccc ggg agt acc ccc aag aga gct tac tac atc tat tct ggg ggc      530 
Leu Pro Gly Ser Thr Pro Lys Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly 
                160                 165                 170 

gag aag att ccg ctg gta ctg agc cga cct ctc tcc tcc aac gtg gcc      578 
Glu Lys Ile Pro Leu Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala 
            175                 180                 185 

acc ctc cag cat ctt tgt cgg aag act gtc aac ggc cac ctg gac tcc      626 
Thr Leu Gln His Leu Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser 
        190                 195                 200 

tat gag aaa gtg acc cag ctg cct gga ccc att cgg gag ttc ctg gat      674 
Tyr Glu Lys Val Thr Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp 
    205                 210                 215 

cag tat gat gct cca ctt taa ggagcaaaag ggtcagaggg gggcctgggt         725 
Gln Tyr Asp Ala Pro Leu 
220                 225 

cggtcggtcg cctctcctcc gaggcacatg gcacaagcac aaaaatccag ccccaacggt    785 

cggtagctcc cagtgagcca ggggcagatt ggcttcttcc tcaggccctc cactcccgca    845 

gagtagagct ggcaggacct ggaattcgtc tgaggggagg gggagctgcc acctgctttc    905 

ccccctcccc cagctccagc ttctttcaag tggagccagc cggcctggcc tggtgggaca    965 

atacctttga caagcggact ctcccctccc cttcctccac accccctctg cttcccaagg   1025 

gaggtgggga cacctccaag tgttgaactt agaactgcaa ggggaatctt caaactttcc   1085 

cgctggaact tgtttgcgct ttgatttggt ttgatcaaga gcaggcacct gggggaagga   1145 

tggaagagaa aagggtgtgt gaagggtttt tatgctggcc aaagaaataa ccactcccac   1205 

tgcccaacct aggtgaggag tggtggctcc tggctctggg gagagtggca aggggtgacc   1265 

tgaagagagc tatactggtg ccaggctcct ctccatgggg cagctaatga aacctcgcag   1325 

atcccttgca ccccagaacc ctccccgttg tgaagaggca gtagcattta gaagggagac   1385 

agatgaggct ggtgagctgg ccgccttttc caacaccgaa gggaggcaga tcaacagatg   1445 

agccatcttg gagcccaggt ttcccctgga gcagatggag ggttctgctt tgtctctcct   1505 

atgtggggct aggagactcg ccttaaatgc cctctgtccc agggatgggg attggcacac   1565 

aaggagccaa acacagccaa taggcagaga gttgagggat tcacccaggt ggctacaggc   1625 

caggggaagt ggctgcaggg gagagaccca gtcactccag gagactcctg agttaacact   1685 

gggaagacat tggccagtcc tagtcatctc tcggtcagta ggtccgagag cttccaggcc   1745 

ctgcacagcc ctcctttctc acctgggggg aggcaggagg tgatggagaa gccttcccat   1805 

gccgctcaca ggggcctcac gggaatgcag cagccatgca attacctgga actggtcctg   1865 

tgttggggag aaacaagttt tctgaagtca ggtatggggc tgggtggggc agctgtgtgt   1925 

tggggtggct tttttctctc tgttttgaat aatgtttaca atttgcctca atcactttta   1985 

taaaaatcca cctccagccc gcccctctcc ccactcaggc cttcgaggct gtctgaagat   2045 

gcttgaaaaa ctcaaccaaa tcccagttca actcagactt tgcacatata tttatattta   2105 

tactcagaaa agaaacattt cagtaattta taataaaaga gcactatttt ttaatgaaaa   2165 

aaaaaaaaaa aaaaaaaaaa aa                                            2187 

 
           
             4  
             225  
             PRT  
             murine  
           
            4 

Met Val Thr His Ser Lys Phe Pro Ala Ala Gly Met Ser Arg Pro Leu 
1               5                   10                  15 

Asp Thr Ser Leu Arg Leu Lys Thr Phe Ser Ser Lys Ser Glu Tyr Gln 
            20                  25                  30 

Leu Val Val Asn Ala Val Arg Lys Leu Gln Glu Ser Gly Phe Tyr Trp 
        35                  40                  45 

Ser Ala Val Thr Gly Gly Glu Ala Asn Leu Leu Leu Ser Ala Glu Pro 
    50                  55                  60 

Ala Gly Thr Phe Leu Ile Arg Asp Ser Ser Asp Gln Arg His Phe Phe 
65                  70                  75                  80 

Thr Leu Ser Val Lys Thr Gln Ser Gly Thr Lys Asn Leu Arg Ile Gln 
                85                  90                  95 

Cys Glu Gly Gly Ser Phe Ser Leu Gln Ser Asp Pro Arg Ser Thr Gln 
            100                 105                 110 

Pro Val Pro Arg Phe Asp Cys Val Leu Lys Leu Val His His Tyr Met 
        115                 120                 125 

Pro Pro Pro Gly Thr Pro Ser Phe Ser Leu Pro Pro Thr Glu Pro Ser 
    130                 135                 140 

Ser Glu Val Pro Glu Gln Pro Pro Ala Gln Ala Leu Pro Gly Ser Thr 
145                 150                 155                 160 

Pro Lys Arg Ala Tyr Tyr Ile Tyr Ser Gly Gly Glu Lys Ile Pro Leu 
                165                 170                 175 

Val Leu Ser Arg Pro Leu Ser Ser Asn Val Ala Thr Leu Gln His Leu 
            180                 185                 190 

Cys Arg Lys Thr Val Asn Gly His Leu Asp Ser Tyr Glu Lys Val Thr 
        195                 200                 205 

Gln Leu Pro Gly Pro Ile Arg Glu Phe Leu Asp Gln Tyr Asp Ala Pro 
    210                 215                 220 

Leu 
225 

 
           
             5  
             1094  
             DNA  
             human  
           
            5 

ctccggctgg ccccttctgt aggatggtag cacacaacca ggtggcagcc gacaatgcag     60 

tctccacagc agcagagccc cgacggcggc cagaaccttc ctcctcttcc tcctcctcgc    120 

ccgcggcccc cgcgcgcccg cggccgtgcc ccgcggtccc ggccccggcc cccggcgaca    180 

cgcacttccg cacattccgt tcgcacgccg attaccggcg catcacgcgc gccagcgcgc    240 

tcctggacgc ctgcggattc tactgggggc ccctgagcgt gcacggggcg cacgagcggc    300 

tgcgcgccga gcccgtgggc accttcctgg tgcgcgacag ccgccagcgg aactgctttt    360 

tcgcccttag cgtgaagatg gcctcgggac ccacgagcat ccgcgtgcac tttcaggccg    420 

gccgctttca cctggatggc agccgcgaga gcttcgactg cctcttcgag ctgctggagc    480 

actacgtggc ggcgccgcgc cgcatgctgg gggccccgct gcgccagcgc cgcgtgcggc    540 

cgctgcagga gctgtgccgc cagcgcatcg tggccaccgt gggccgcgag aacctggctc    600 

gcatccccct caaccccgtc ctccgcgact acctgagctc cttccccttc cagatttgac    660 

cggcagcgcc cgccgtgcac gcagcattaa ctgggatgcc gtgttatttt gttattactt    720 

gcctggaacc atgtgggtac cctccccggc ctgggttgga gggagcggat gggtgtaggg    780 

gcgaggcgcc tcccgccctc ggctggagac gaggccgcag accccttctc acctcttgag    840 

ggggtcctcc ccctcctggt gctccctctg ggtccccctg gttgttgtag cagcttaact    900 

gtatctggag ccaggacctg aactcgcacc tcctacctct tcatgtttac atatacccag    960 

tatctttgca caaaccaggg gttgggggag ggtctctggc tttatttttc tgctgtgcag   1020 

aatcctattt tatatttttt aaagtcagtt taggtaataa actttattat gaaagttttt   1080 

ttttttaaaa aaaa                                                     1094 

 
           
             6  
             211  
             PRT  
             human  
           
            6 

Met Val Ala His Asn Gln Val Ala Ala Asp Asn Ala Val Ser Thr Ala 
1               5                   10                  15 

Ala Glu Pro Arg Arg Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser 
            20                  25                  30 

Pro Ala Ala Pro Ala Arg Pro Arg Pro Cys Pro Ala Val Pro Ala Pro 
        35                  40                  45 

Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ala Asp Tyr 
    50                  55                  60 

Arg Arg Ile Thr Arg Ala Ser Ala Leu Leu Asp Ala Cys Gly Phe Tyr 
65                  70                  75                  80 

Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala Glu 
                85                  90                  95 

Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys Phe 
            100                 105                 110 

Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg Val 
        115                 120                 125 

His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Ser Phe 
    130                 135                 140 

Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg Arg 
145                 150                 155                 160 

Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln Glu 
                165                 170                 175 

Leu Cys Arg Gln Arg Ile Val Ala Thr Val Gly Arg Glu Asn Leu Ala 
            180                 185                 190 

Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe Pro 
        195                 200                 205 

Phe Gln Ile 
    210 

 
           
             7  
             2807  
             DNA  
             murine  
           
            7 

ggaaaccgag gcggggagac caggaggcct tggcctcaga gcttcagagt cgcgtggcag     60 

caaacagaga aacctgtaga gggcagtgtg cgtcacttag ctcagggaag ctgcacgcga    120 

aactcacccg ccttcattca taaacatcgt cagctaggca cctactcctg ggctttcagg    180 

acaaactgaa tcacgaaacc acagtgtcct taaaataggt ctgaccgcct gaatccctgg    240 

ccaaggtgtg tacggggcat gggagccctt gtgcagagat gcttgcagga gccttgaggg    300 

gctctgtaag acagaggcta ggaagacaaa gttgggggct acagcttctt gtcctgcccg    360 

gggcctcagt ttcttcggtt gcccacgtag gagtgcagag agtccagccc ctggggaccc    420 

aacccaaccc cgcccagttt ccgaggaact cgtccgggag cgggggcgcc cctcccgcac    480 

cgccttaggc ttcctttgaa gcctctgcgg tcaggccacc gcttcctggg aagcccaagc    540 

caaggccagg ccgagtggcc aacgggaggg gcccgcgcgc gattctggag gagggcggcg    600 

gccccacagg tctccagggc tggctagccg ggctcctaga gcggagactg ccaaggcctt    660 

cgggtcctgg gcaggaagga tcctggcagg gaggagttgc ttggggggtg ggggggaaag    720 

gctccaggcg cggtggagct ctgaccagga gaatgcacac actcggaggg gaggaggcgt    780 

gtcagcccca agctagcatc ccacccgggg agcagcgatg tggggcgaag gtagccagag    840 

caaaagagca ggcaccaggt gacacgaaac agaagattcc gggtagagcc agaaccccag    900 

aagtcccatt cagggaaggt gcgaggcgag aacgagttag gtggaccctc tccaggggca    960 

gccaaagaaa tctaaagaga acccgaagga cttgccggaa agagaaaccg aaagcggcgg   1020 

tgggcgggat cggtgggcgg ggcctccctg gtttaagagc ttgatgcagg ggcgggcagc   1080 

agcagagaga actgcggccg tggcagcggc acggctcccg gccccggagc atgcgcgaca   1140 

gcagccccgg aacccccagc cgcggcgccc cgcgtcccgc cgccaggtga gccgaggcag   1200 

ctgcgaagga gcaggcggga ggggatggga ggaaggggag cagagcctgg caggactatc   1260 

ctcgcagact gcatggcggg gtcgtggatg ctatgcctct ggcgcccgcc ccaccggctg   1320 

gcccaggcgg cccctcgcgc gcgcggggcg ccgtcagccc ctcctctccg gccctgagcc   1380 

cggatcgtcc gcccgggttc cagttcccgg cgtggccagt aggcggcaac cgcgaggcgg   1440 

caagccaccc agcggggacg gcctggagtc gggcccctct ccacgccccc ttctccacgc   1500 

gcgcggggag gcagggctcc accgccagtc tggaagggtt ccacatacag gaacggccta   1560 

cttcgcagat gagcccaccg aggctcaggc tccgggcgga ttctgcgtgt caccctcgct   1620 

ccttggggtc cgctggccgg cctgtgccac ccggacgccc ggttcactgc ctctgtctcc   1680 

cccatcagcg cagccccgga cgctatggcc cacccctcca gctggcccct cgagtaggat   1740 

ggtagcacgt aaccaggtgg aagccgacaa tgcgatctcc ccggcatcag agccccgacg   1800 

gcggccagag ccatcctcgt cctcgtcttc gtcctcgccg gcggccccgg cgcgtccccg   1860 

gccctgcccg gtggtcccgg ccccggctcc gggcgacact cacttccgca ccttccgctc   1920 

ccactctgat taccggcgca tcacgcggac cagcgctctc ctggacgcct gcggcttcta   1980 

ctggggaccc ctgagcgtgc atggggcgca cgaacggctg cgttccgaac ccgtgggcac   2040 

cttcttggtg cgcgacagtc gccagcggaa ctgcttcttc gcgctcagcg tgaagatggc   2100 

ttcgggcccc acgagcattc gtgtgcactt ccaggccggc cgcttccacc tggacggcaa   2160 

ccgcgagacc ttcgactgcc tcttcgagct gctggagcac tacgtggcgg cgccgcgccg   2220 

catgttgggg gccccactgc gccagcgccg cgtgcggccg ctgcaggagc tgtgtcgcca   2280 

gcgcatcgtg gccgccgtgg gtcgcgagaa cctggcacgc atccctctta acccggtact   2340 

ccgtgactac ctgagttcct tccccttcca gatctgaccg gctgccgccg tgcccgcaga   2400 

attaagtggg agcgccttat tatttcttat tattaattat tattattttt ctggaaccac   2460 

gtgggagccc tccccgccta ggtcggaggg agtgggtgtg gagggtgaga tccctcccac   2520 

ttctggctgg agaccttatc ccgcctctcg gggggcctcc cctcctggtg ctccctcccg   2580 

gtccccctgg ttgtagcagc ttgtgtctgg ggccaggacc tgaactccac gcctacctct   2640 

ccatgtttac atgttcccag tatctttgca caaaccaggg gtgggggagg gtctctggct   2700 

tcatttttct gctgtgcaga atattctatt ttatattttt acatccagtt tagataataa   2760 

actttattat gaaagttttt ttttttaaag aaacaaagat ttctaga                 2807 

 
           
             8  
             212  
             PRT  
             murine  
           
            8 

Met Val Ala Arg Asn Gln Val Glu Ala Asp Asn Ala Ile Ser Pro Ala 
1               5                   10                  15 

Ser Glu Pro Arg Arg Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser 
            20                  25                  30 

Ser Pro Ala Ala Pro Ala Arg Pro Arg Pro Cys Pro Val Val Pro Ala 
        35                  40                  45 

Pro Ala Pro Gly Asp Thr His Phe Arg Thr Phe Arg Ser His Ser Asp 
    50                  55                  60 

Tyr Arg Arg Ile Thr Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe 
65                  70                  75                  80 

Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ser 
                85                  90                  95 

Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys 
            100                 105                 110 

Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg 
        115                 120                 125 

Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly Asn Arg Glu Thr 
    130                 135                 140 

Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg 
145                 150                 155                 160 

Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln 
                165                 170                 175 

Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly Arg Glu Asn Leu 
            180                 185                 190 

Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu Ser Ser Phe 
        195                 200                 205 

Pro Phe Gln Ile 
    210 

 
           
             9  
             35  
             DNA  
             primer  
           
            9 

atatctcgag gccaccatgg tagcacgcaa ccagg                                35 

 
           
             10  
             30  
             DNA  
             primer  
           
            10 

atataagctt tcagatctgg aaggggaagg                                      30 

 
           
             11  
             35  
             DNA  
             primer  
           
            11 

atatgcggcc gcgccaccat gaccctgcgg tgcct                                35 

 
           
             12  
             35  
             DNA  
             primer  
           
            12 

atatgcggcc gcgccaccat gaccctgcgg tgcct                                35 

 
           
             13  
             35  
             DNA  
             primer  
           
            13 

atatgcggcc gcgccaccat gaccctgcgg tgcct                                35 

 
           
             14  
             35  
             DNA  
             primer  
           
            14 

atatgcggcc gcgccaccat gaccctgcgg tgcct                                35