AU784277B2 - An animal model for studying hormone signalling and method of modulating the signalling - Google Patents

Description

WO 01/35732 PCT/AU00/01398 An animal model for studying hormone signalling and method of modulating the signalling FIELD OF THE INVENTION 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.

BACKGROUND OF THE INVENTION 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 WO 01/35732 PCT/AU00101398 -2cytokines) 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 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 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 homodimcrization, 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 Interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) use the common 0receptor subunit (3c) 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 ychain while IL-4 and IL-13 share an alternative y-chain (Yc or IL-13 receptor achain). Each of these cytokines plays an important role in regulating acquired immunity in the lymphoid system. Finally, IL-6, IL-11, leukemia inhibitory factor (LLF), oncostatin (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin (CT) share the receptor subunit gpl30. Each of these cytokines appears to be highly pleiotropic, having effects both within and outside the haemopoietic system 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 Box 1 is a proline-rich motif which is located more proximal to the transmembrane domain than the acidic box 2 element. The box-I 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- WO 01/35732 PCT/AU00/01398 -3phosphorylation. 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, she. 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 7, In a separate pathway, tyrosine phosphorylated she 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, 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 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 (STATI 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 she-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, WO 01/35732 PCT/AU00/01398 -4- 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.

WO 01/35732 PCT/AU00/01398 SUMMARY OF THE INVENTION Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>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, repeats and/or ankyrin repeats, N terminal of a SOCS box, wherein said SOCS box comprises the amino acid sequence: Xi X 2

X

3 X X X X 8 X X 9

X

1 o X 1 I X 12

X

13

X

14

X

15

X

1 6 [Xi]n X17 X18 X 1 9

X

2 0

X

2 1

X

2 2

X

2 3 [XjJn X 2 4 X25 X 2 6

X

2 7

X

2 8 wherein: Xi is L, I, V, M, A or P;

X

2 is any amino acid residue; X3 is P, T or S;

X

4 is L, I, V, M, A or P; WO 01/35732 PCT/AU00/01398 -6is any amino acid;

X

6 is any amino acid;

X

7 is L, I, V, M, A, F, Y or W;

X

8 is C, T or S; X9 is R, K or H; Xlo is any amino acid; X, is any amino acid; Xl 2 is L, I, V, M, A or P;

X

i3 is any amino acid; X1 4 is any amino acid; Xi 5 is any amino acid;

X,

6 is L, I, V, M, A, P, G, C, T or S;

[X

i ]n is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X, may comprise the same or different amino acids selected from any amino acid residue;

X

1 7 is L, 1, V, M, A or P;

X

1 8 is any amino acid;

X

1 9 is any amino acid;

X

2 o is L, I, V, M, A or P;

X

2 1 is P;

X

22 is L, I, V, M, A, P or G;

X

23 is P or N; [Xj]n is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the Xj may comprise the same or different amino acids selected from any amino acid residue;

X

2 4 is L, I, V, M, A or P;

X

2 5 is any amino acid;

X

26 is any amino acid;

X

27 is Y or F;

X

28 is L, I, V, M, A or P.

WO 01/35732 PCT/AU00/01398 -7- 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:

X

1

X

2

X

3

X

4 Xs X6 X X 9 X9 1 X XX 1 3 4 X1 X 1 6 [Xj]n X 17

X

1 8

X

19

X

2 0

X

2 1

X

2 2

X

23 [Xj]n X 2 4

X

2 5

X

2 6

X

2 7

X

28 wherein: X 1 is L, I, V, M, A or P;

X

2 is any amino acid residue;

X

3 is P, T or S;

X

4 is L, I, V, M, A or P; X5 is any amino acid;

X

6 is any amino acid;

X

7 is L, 1, V, M, A, F, Y or W; X is C, T or S;

X

9 is R, K or H; XIo is any amino acid; X I is any amino acid;

X

12 is L, I, V, M, A or P;

X

1 3 is any amino acid;

X

14 is any amino acid;

X

1 5 is any amino acid;

X

1 6 is L, I, V, M, A, P, G, C, T or S; is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence Xi may comprise the same or different amino acids selected from any amino acid residue;

X

1 7 is L, 1, V, M, A or P;

X

18 is any amino acid; WO 01/35732 PCT/AU00/01398 -8-

X

1 9 is any amino acid;

X

2 0 is L, I, V, M, A or P;

X

2 i is P;

X

2 2 is L, I, V, M, A, P or G;

X

2 3 is P or N; [Xj]n is a sequence ofn amino acids wherein n is from 1 to 50 amino acids and wherein the Xj may comprise the same or different amino acids selected from any amino acid residue; X24 is L, I, V, M, A or P;

X

25 is any amino acid; X26 is any amino acid;

X

27 is Y or F;

X

28 is L, 1, V, M, A or P.

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 <400>1 or its complementary form or is able to hybridize under low stringency conditions at 42 0 C to <400>1 or its complementary form and wherein said WO 01/35732 PCT/AU0/01398 -9genetic 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 <400>1 or capable of hybridizing to <400>1 under low stringency conditions at 42 0 C and wherein said animal exhibits altered hormone regulation such as growth hormone regulation.

WO 01/35732 PCT/AU00/01398 BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a diagrammatic representation showing disruption of the SOCS-2 gene by homologous recombination. The functional murine SOCS-2 gene BamHI; Nh, Nhel; RV, EcoRV) with the exons containing the coding region as shaded boxes. In the targeted allele, the entire SOCS-2 coding region was replaced by a 3-gal-PGKneo cassette in which the 0-galactosidase coding region was fused to the SOCS-2 initiation codon. (b) Southern blot of EcoRV-digested genomic DNA from the tails of mice derived from a cross beween SOCS-2/" mice. The blot was hybridized with the 5' genomic SOCS-2 probe, which distinguishes between endogenous (16 kb) and mutant SOCS-2 (9 kb) alleles.

Northern blot showing lack of SOCS-2 expression in organs of SOCS-2-' mice. Top, the blot was hybridized with a coding region probe, which detects the 3.4-kb SOCS-2 transcript; bottom, the integrity of the RNA was confirmed by hybridization with GAPDH (1.4-kb transcript). Sal gl, salivary gland.

Figure 2 is a graphical representation showing excessive growth of SOCS-2- mice.

Growth curves for male and female SOCS-2 (squares), SOCS-2+" (triangles) and SOCS- 2" (circles) mice. Body weights from cohorts of mice weighed at weekly intervals are shown: each point represents mean s.d. for 5-20 mice.

Figure 3 is a graphical representation showing percentage increase in body, carcass and organ weights in SOCS-2 mice over that determined in age-matched wild-type mice (N= 7-8 three-month-old mice per measurement). measurements from SOCS-2-" mice that were significantly different from wild-type controls (P <0.05, Student's t-test). wt, weight; mes LN, mesenteric lymph node; sal gland, salivary gland; sem ves, seminal vesicles.

Figure 4 is a representation showing deregulated growth hormone signalling in SOCS-2"' mice. Decreased MUP in urine samples from male and female SOCS-2" mice. Each lane represents a sample from an individual SOCS-2 or age and sex-matched wild-type mouse. RNase protection assays of IGF-I expression in tissues of SOCS-2" mice. Each lane represents a sample from an individual SOCS-2-- or age and sex-matched wild-type WO 01/35732 PCT/AU00/01398 -11 mouse. Expression of IGF-I RNA is tissues of male SOCS-2' mice, expressed as a percentage of expression in age and sex-matched wild type mice. Data represents the mean and standard deviation for comparison of at least 4 pairs of mice, p< 0.05.

WO 01/35732 PCT/AUOO/01398 12- DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 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 is human, is murine and 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 effectormediated 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 nonproteinaceous 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 sheep, cow, pig, horse, donkey), laboratory test animal rabbit, mouse, rat, guinea pig), companion animal 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.

WO 01/35732 WO 0135732PCT/AUOO/01398 13 Reference herein to "SOCS" includes a protein comprising a SOCS box in its C-terminal region comprising the amino acid sequence: XI X 2

X

3 X4 5

X

5

X

6

X

7

X

8

X

9 XIO XI 1

X

12

X

13

X

14

X

15

X

16

X

1 7

X

18

X

19

X

2 0

X

21

X

22

X

23

X

24

X

25

X

26

X

27

X

2 9 wherein: X, is L, 1, V, M, A or P;

X

2 is any amino acid residue;

X

3 isP, Tor S;

X

4 is L, 1, V, M, A or P;

X

5 is any amino acid;

X

6 is any amino acid;

X

7 Is L, 1, V, M, A, F, Y or W; X is C, Tor S; Xgis R, K or H; Xio is any amino acid; X I is any amino acid; X1 2 is L, I, V, M, A or P; X 13 is any amino acid;

*X

14 is any amino acid;

*X

1 5 is any amino acid;

X

16 is L, 1, V, M, A, P, G, C, T or S; [XjI is a sequence of n amino acids wherein n is from I to 50 amino acids and wherein thc sequence X, may comprise the same or diffcrent amino acids selected from any amino acid residue;

X

17 is L, 1, V, M, A or P; X 18 is any amino acid;

X

19 is any amino acid;

X

2 is L,1, V,M, Aor P;

X

2 1 is P; WO 01/35732 PCT/AU00/01398 -14-

X

22 is L, I, V, M, A, P or G;

X

23 is P or N; [Xj]n is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the Xj may comprise the same or different amino acids selected from any amino acid residue;

X

24 is L, I, V, M, A or P;

X

25 is any amino acid;

X

26 is any amino acid;

X

2 7 is Y or F;

X

2 8 is L, I, V, M, A or P.

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, Nterminal 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, 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 Xs X 9

X

1 i XI X 2

X

1

X

3

X

4

X

1 5

X

1 6 X17 X18 X, 9

X

20

X

2 1

X

2 2

X

23 [Xj]n X 2 4

X

2 5

X

2 6

X

2 7

X

2 8 WO 01/35732 PCT/AU00/01398 wherein: Xi is L, I, V, M, A or P;

X

2 is any amino acid residue;

X

3 isP,TorS;

X

4 isL, I, V, M, A or P; Xs is any amino acid;

X

6 is any amino acid;

X

7 is L, I, V, M, A, F, Y or W; Xs is C, T or S;

X

9 is R, K or H; Xio is any amino acid; XII is any amino acid;

X

1 2 is L, I, V, M, A or P;

X

1 3 is any amino acid;

X

1 4 is any amino acid;

X

i5 is any amino acid;

X

16 is L, I, V, M, A, P, G, C, T or S; is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence X, may comprise the same or different amino acids selected from any amino acid residue;

X

1 7 is L, I, V, M, A or P;

X

1 8 is any amino acid; X19 is any amino acid;

X

20 is L, I, V, M, A or P;

X

21 is P;

X

22 is L, I, V, M, A, P or G;

X

23 is P or N; [Xj]n is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the X, may comprise the same or different amino acids selected from any amino acid residue;

X

24 is L, I, V, M, A or P; WO 01/35732 PCT/AU00/01398 -16-

X

25 is any amino acid;

X

26 is any amino acid;

X

27 is Y or F;

X

28 is L, I, V, M, A or P.

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 <400>1 and <400>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 WO 01/35732 PCT/AU00/01398 17the 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 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: X X2 X3 X XX X 5 X X X 7 X XI XI X X12 X 1 3 X 1 4 X 15 Xi6 [Xi]n X 1 7

X

18

X

1 9

X

2 1

X

2 2

X

2 3 [Xj]n X 24

X

2 5 X2 6

X

27

X

2 8 wherein: X 1 is L, I, V, M, A or P;

X

2 is any amino acid residue;

X

3 is P, T or S;

X

4 is L, I, V, M, A or P;

X

5 is any amino acid;

X

6 is any amino acid;

X

7 is L, I, V, M, A, F, Y or W; Xs is C, T or S;

X

9 is R, K or H; Xlo is any amino acid; Xii is any amino acid; Xi 2 is L, I, V, M, A or P;

X

13 is any amino acid;

X

1 4 is any amino acid; WO 01/35732 PCT/AU00/01398 -18-

X

5 s is any amino acid;

X

1 6 is L, I, V, M, A, P, G, C, T or S; [Xi]n is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence Xi may comprise the same or different amino acids selected from any amino acid residue;

X

1 7 is L, I, V, M, A or P;

X

1 8 is any amino acid;

X

1 9 is any amino acid;

X

2 o is L, I, V, M, A or P;

X

2 1 is P;

X

22 is L, I, V, M, A, P or G; X23 is P or N; is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the Xj may comprise the same or different amino acids selected from any amino acid residue;

X

24 is L, I, V, M, A or P;

X

25 is any amino acid;

X

26 is any amino acid; X27 is Y or F;

X

28 is L, 1, V, M, A or P.

Preferably, the SOCS protein-encoding genetic sequence comprises a nucleotide sequence substantially as set forth in <400>1 or a nucleotide sequence having at least 60% similarity thereto or a nucleotide sequence capable of hybridizing to <400>1 or its complementary form under low stringency conditions at 42 0 C. Even more preferably, the SOCS protein in a human homologue of the nucleotide sequence set forth in <400>l.

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, WO 01/35732 PCT/AU0/01398 -19- "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 a sequence only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and 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 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, WI, USA) or by inspection and the best alignment 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. A detailed discussion of sequence analysis can be found in Unit 19.3 ofAusubel et al. (17).

WO 01/35732 PCT/AU00/01398 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-bynucleotide 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 A, T, C, G, I) or the identical amino acid residue Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, 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 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, California, 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 0 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 (18).

WO 01/35732 PCT/AU00/01398 -21- Howevcr, the Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42 0 C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20 0 C to 65 0 C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65 0

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 WO 01/35732 PCT/AU00/01398 -22or 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 NaBH 4 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 NaBH 4 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 4chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with Nbromosuccinimide 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.

WO 01/35732 WO 0135732PCT/AUOO/OI 398 23 Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with jodoacetic 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-3acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenyiglycine, omnithine, sarcosine, 4-aniino-3-hydroxy-6-methylheptanoic acid, 2-thicnyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 1.

WO 01/35732PC/IO/39 PCT/AUOO/01398 -24- TABLE 1 Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid a-amino-oy-methylbutyrate aminocyclopropaflecarboxylate aminoisobutyric acid aminonorbomylcarboxylatc cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamine D-glutamic acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-ornithine D-phenylalanine D-proline D-senne D-threonine D-tryptophan Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Deys Dgln Dglu Dhis Dule Dleu Dlys Dmet Dom Dphe Dpro Dser Dthr Dtrp L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-Nmethylhstidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-mcthylnorleucinc L-N-methylnorvaline L-N-methylornithine L-N-methylphenylalanine L-N-methylproline L-N-methylseine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmhis Nmile Nmleu Nmlys Nmmet Nrnnle Nnmva Nmom Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva WO 01/35732 PTAO/19 PC'r/AUOO/01398 25 D-tyrosine D-valine D-a-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-ae-methylglutamnine D-a-methylhistidine D-a-methylisoleucine D-ca-methy1Ieucine D-a-methyllysine D-a-methylmethionine D-a-methylomithine D-a-methylphenylalanine D-a-methylproline D-a-methylserine D-a-methylthreonine D-c-methyltryptophan D-cr-methyltyrosine D-cr-methylvaline D-N-methylalanine D-N-methylarginine D-N-.methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-mcthylglutamine D-N-methylglutwmate D-N-methylh~istidine D-N-methylisoleucine Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dmmet Dmorn Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnala Dnmarg Dnmasn Drnasp Dnmcys Dnmgln Dnmglu Drimhis ce-methyl-aminoisobutyrate a-methyl--y-ami nobutyrate ce-methyleyclohexylaianine ce-methylcylcopentylalanine a-methyl-ct-napthylalanine a-methylpenicillamine N-(4-aininobutyl)glycine N-(2-aminoethyl)glycine N-(3-amninopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamylmethyl)glycine N-(2-carboxycthyl)glycine N-(carboxymethyl)glycine N.-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cyicododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl)glycinc N-(3,3 -diphenylpropyl)glycine -guanidinopropyl)glycine 1 -hydroxyethyl)glycine N-(hydroxyethyl))glycine Maib Mgabu Mcbexa Mcpen Mariap Mpen Nglu Naeg Nom Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Ntbr Nser Nhis Dnmile N-(imidazolylethyl))glycine PCT/AUOO/01398 WO 01/35732 C/10039 D-N-methylleucine D-N-methyllysine N-methylcyclohexylalanine D-N-methylomithine N-methylglycine N-mcthylaniinoisobutyrate I -methylpropyl)glycine N-(2-mcthylpropyl)glycine D-N-methyltryptophan D-N-methyltyrosine D-N-methylvaline yt-aminobutyric acid L-t-butylglycine L-ethylglycine L-homophenylalanine L-ci-methylarginine L-ci-niethylaspartate L-ce-methylcysteine L-ae-methylgiutainine L-ci-metliylhistidine L-cr-methylisoleucine L-cu-methyIleucine L-oa-methylniethionine L-ae-mcthylinorvaline L-ce-methylphenylalanine L-a -methylserine L-u-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl) carbamylmethyl)glycine Dnmleu Dnmlys Nmchexa Dnmom Nala Nmaib Nile Nleu Dnmtrp Drntyr Dnmval Gabu Tbug Etg Hphe Marg Masp Mcys Mgln mhlis Mile Mleu Mmet Mnva Mphe Mser Mtrp MvaI Nnbhmn N-(3-indolylyethyl)glycine N-methyl-y-aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methyltlireonine 1 -methylethyl)glycine N-methyla-napthylalanine N-methylpenicillamine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamine L-ce-methylalanine L-ce-methylasparagine L-a-nethyl-t-butylglycine L-methylethylglycine L-ci-methyiglutamate L-cr-methythomophenylalanine N-(2-methylthioethyl)glycine L-a-methyllysine L-a-methylnorleucine L-cy--methylornithine L-ci-methylproline L-ca-methylthreonine L-a-methyltyrosine L-N-methylhomophenylalanine N-(N-(3,3-diphenylpropyl) carbamylmethyl)glycine Nhtrp Nmgabu Dnmmet Nmcpen Dnphe Dnmpro Dnmnser Dnimthr NvaI Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys Mnle Momn Mpro Mthr Mtyr Nmhpbe Nnbhe WO 01/35732 PCT/AU00/01398 -27- 1-carboxy-l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilize 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH 2 )n spacer groups with n 1 to n 6, glutaraldehyde, N-hydroxysuccinimide esters and heterobifunctional reagents which usually contain an amino-reactive moiety such as Nhydroxysuccinimide 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 C, and Nmethylamino acids, introduction of double bonds between Co and Cg 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 WO 01/35732 PCT/AU00/01398 -28and/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: XX X; X 4 X4 X X X 8 X X9 Xi X 1

X

2 X1 3

X

1 4

XI

5 X16 [Xi]n X 1 7 Xs1 X9 X 20 WO 01/35732 PCT/AU00/01398 -29-

X

2 1

X

2 2

X

2 3 [Xj]n X 24

X

2 5

X

26

X

2 7

X

2 8 wherein: Xi is L, I, V, M, A or P;

X

2 is any amino acid residue;

X

3 is P, T or S;

X

4 is L, I, V, M, A or P; Xs is any amino acid;

X

6 is any amino acid;

X

7 is L, I, V, M, A, F, Y or W; XsisC,TorS; X9 is R, K or H; Xio is any amino acid;

X

1 I is any amino acid;

X

12 is L, I, V, M, A or P;

X

13 is any amino acid;

X

1 4 is any amino acid; is any amino acid;

X

1 6 is L, I, V, M, A, P, G, C, T orS; [Xi]n is a sequence of n amino acids wherein n is from 1 to 50 amino acids and wherein the sequence Xi may comprise the same or different amino acids selected from any amino acid residue;

X

17 is L, I, V, M, A or P; XIs is any amino acid;

X

19 is any amino acid; X20 is L, I, V, M, A or P;

X

21 is P;

X

22 is L, I, V, M, A, P or G;

X

23 is P or N; [Xj] is a sequence of n amino acids wherein n is from 0 to 50 amino acids and wherein the Xj may comprise the same or different amino acids selected from any amino acid residue; WO 01/35732 PCT/AU00/01398

X

24 is L, I, V, M, A or P;

X

25 is any amino acid;

X

2 6 is any amino acid;

X

2 7 is Y or F;

X

2 8 s is L, I, V, M, A or P.

Preferably, the SOCS protein is SOCS-2 or its derivative or homologue and comprises the amino acid sequence substantially as set forth in <400>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 <400>1 or its complementary form or is able to hybridize under low stringency conditions at 42 0 C to <400>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 downregulates 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 <400>1 or capable of hybridizing to <400>1 under low stringency conditions at 42 0 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 pigs, cows, sheep, donkeys, horses).

Genetically modified livestock animals are particularly useful in food production.

WO 01/35732 PCT/AUOO/01398 -31 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.

WO 01/35732 PCT/AU00/01398 -32- EXAMPLE 1 Generation ofES targeted cells and mutant mice 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 1-galactosidase via the BamHI site in the plasmid vector pgalpAloxneo 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 pBgalpAloxneo 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 (Figure This probe distinguished between the endogenous (16kb) and targeted (9 kb) SOCS-2 alleles. A targeted ES cell clone was injected into Balb/c blastocysts to generate chimaeric mice. Male chimaeras were mated with C57BL/6 females to yield SOCS-2 heterozygotes which were interbred to produce wild-type (SOCS-2! heterozygous (SOCS-2 and mutant (SOCS-2' mice on a pure C57BL/6 genetic background. The genotypes of offspring were determined by Southern Blot analysis of genomic DNA extracted from tail biopsies as described above. The deletion of SOCS-2 coding sequence and subsequent inability to produce SOCS-2 mRNA in mutant mice was confirmed in nucleic acid blots which were performed as previously described Northern blots were probed with a full-length SOCS-2 coding region probe and then with a 1.2kb PstI chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fragment.

EXAMPLE 2 Hematological and histological analysis 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 WO 01/35732 PCT/AU00/01398 -33performed from stained smears and cytocentrifuge preparations. Clonal cultures of 2.5 x 4 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.

EXAMPLE3 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 x 3 min) before 0.5 pl of supernatant was electorphoresed in 12% SDS-polyacrylamidc gels and stained with Coomassie blue.

EXAMPLE 4 Growth curves and linear measurements 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 noseanus (body length) and anus-tail (tail length) measurements. To measure skeletal dimensions, limbs were subsequently removed and oriented in a consistent manner for Xray photography and bone length measurement.

EXAMPLE IGF-I measurements 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 WO 01/35732 PCT/AU00/01398 -34manufacturer's instructions. IGF-I RNA expression was determined in RNase protections assays as previously described (23) using l-actin as an internal standard.

EXAMPLE 6 Generation and analysis ofSOCS-2 knockout mice 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 f-galactosidase via the BamHI site in the plasmid vector ppgalpAloxneo. The 3' arm, a 3.7 kb EcoRI fragment from pgmSOCS-2 57-60-1-45 was blunted and ligated into the XhoI (blunted) site of p/galphAloxneo 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, Figure which is located 3' to the SOCS-2 sequences in the targeting vector, distinguished between the endogenous (greater than 14kb) and targeted (7.5 kb) SOCS-2 loci (Figure Several targeted ES cells clones were identified, one of which was injected into Balb/c blastocysts to generate chimeric mice. Germline chimeras were mated with C57/BL/6 mice to produce SOCS-2+ mice which were interbred to yield SOCS-2-deficient animals. Southern blot analysis at weaning revealed that offspring of heterozygous parents included mice of each of the three expected genotypes in approximately Mendelian proportions (22:51:22 for SOCS- 2+ :S:SOCS-2 Northern blots of RNA extracted from a range of organs confirmed that SOCS-2 transcripts were absent in homozygous mutant mice. This result is consistent with the findings that the SOCS-2 gene had been functionally deleted.

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 (Figure 2; males at 3 months of age: SOCS-2 41.2 4.8, wild-type 29.3 WO 01/35732 PCT/AU00/01398 1.8; females: SOCS-2 25.3 2.2, wild-type 21.9 2.2 grams). In male mice the increased body weight was parallelled by increased size of several organs, in particular the pancreas, liver, lungs and heart (Figure Upon removal of all organs the resulting means carcass weight also highlighted the increased size of SOCS-2 mice (21.5 1.5 g), compared to wild-type controls (13.1 1.9 An extensive analysis has suggested that mice SOCS-2 organs were histologically normal, with the exception of a marked thickening of the skin which develops as a result of increase collagen deposition. Both male and female SOCS-2 have been found to be fertile. Adult SOCS-2"' males exhibited an intermediate body weight 36.6 1.0 g; 30.8 1.3 g; 1.6 g, at 12 weeks of age, n 5-20 mice per group). Increased growth was also significant but less dramatic in female SOCS-2' mice. Adult SOCS-2" females typically attained weights of wild-type male mice, but heterozygous SOCS-2 females were not significantly heavier that sex-matched wild-type littermates 26.2 1.5 g; 21.8 0.7 g; 20.5 1.4 g, at 12 weeks of age, n 7-20 mice per group). Consistent with this interpretation, the femur, tibia, radius and humerus in SOCS-2 mice were all significantly longer than in wild-type controls (Table Body length in male SOCS-2-deficient mice was also greater, although tail length was normal (Table The mean frequency of hepatic nuclei per high power fields in SOCS-2' liver sections was no greater than that in wild type mice (27.3 4.4, n 4 versus 27.7 2.6, n 4) and striated muscle cell width was normal in the thighs of SOCS-2-deficient animals. Thus, increased organ weights in these mice appear to have resulted from elevated cell numbers rather than increased cell size. A similar, but less pronounced trend was also observed when organ and carcass weights and body, tail and bone lengths were assessed in female SOCS-2 mice (Table 2).

A thorough hematological survey has not identified any hematological abnormalities in SOCS-2 mice (Table 3).

TABLE 2 Body, tail and bone lengths in SOCS-2 4 mice Length (mm) Male Female SOCS-2+'+ SOCS-2+- SOCS-2+'+ SOCS-2+'+ SOCS-2+- SOCS-2-- Body 95.0 1.9 98.8 107 90.1 2.1 91.3 1.2 100.3 ±2.1* Tail 88.3 1.5 87.7 ±2.1 89.2 ±1.5 84.8 1.4 88.1 1.6 88.9 ±1.9* Femur 18.0 0.2 18.7 19.5 17.3 0.2 17.7 0.2* 18.6 Q3* Tibia 20.0 0.2 20.6 20.9 19.5 0.2 19.9 0.2* 2 0.5 ±0.3* Radius 12.0: 0.2 12.4 12.7±0.2* 11.6 0.2 11.7 ±0.2 12.0 ±0.2* Humerus 13.8 ±0.2 14.4 Q3* 15.4 13.2.± 0.2 13.7 14.6 .1* p<0.005 in Student's t-test for comparison of sex-matched SOCS-2' and SOCS-2"- data with SOCS-2+/+ mice (n 6-12).

WO 01135732 WO 0135732PCT/AUOO/01398 37 TABLE 3 Hematological profile of SOCS-2 4 mice Genotype SOCS-2' SOCS-2 Peripheral Blood Platelets (x I Ob/ml) 855 220 899±183 Haematocrit 44±2 44±2 White cell count (x I0-6/ml) 7.3 ±3.7 4.4 ±1.2 Neutrophils 370±360 350±230 Lymphocytes (gl1 1 5070 ±1130 5980 2330 Monocytes 230± 70 310± 270 Eosinophils (plIF') 50 60 120± 140 Bone Marrow Blasts 3 ±3 3±1I Promyelocytes/Myelocytes 2 7 I Metamyelocytes/Neutrophils ()34 6 29 4 Lymphocytes ()25 ±7 27± 7 Monocytes 9 9±1 7 ±3 Eosinophils 3 3±2 4± 2 Nucleated erythroid cells 21 ±4 23 7 Spleen Weight 83±14 119 ±29 Blasts 2 22 2± 2 Promyelocytes/Myelocytes 0 0.5 1 Metamnyelocytes/Neutrophils 3 ±1 2 ±2 Lymphocytes 85±5 86±5 Monocytes (2 22 3±2 Eosinophils 1 1 2±1 Nucleated erythrold cells 7 ±6 5 ±2 Peritoneal cells Metamyelocytes/Neutrophils 1 2 0 ±0 Lymphocytes ()39 17 31 19 Monocytes ()66 20 58 17 Eosinophils I 1±1 0 ±0 Mast cells 2 ±2 2 ±0 WO 01/35732 PCT/AU00/01398 -38- EXAMPLE 7 Disruption of growth hormone signalling 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.

EXAMPLE 8 Effects of SOCS-2 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. 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 WO 01/35732 PCT/AU00/01398 -39- 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.

EXAMPLE 9 Effects of STA 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 4 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-24- STAT5b mice have a growth phenotype intermediate to the large SOCS-2 STAT5b and small SOCS-2+/* STAT5b-" mice. In female mice, SOCS-2"' 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.

TABLE 4 Size parameters for SOCS-2/STAT5b double knock-out mice Genotype SOCS-2+'+/STAT~b'+ SOCS-2--/STAT5b++ SOCS-2 1 +STAT5b+"- SOCS-2 Male mice Body weight 28.4 2.8 38.9 ±2.0 19.5±0.1 25.1 Carcass weight 7.8± 1.0 11.3 1.0 5.2±0.2 5.9 0.4 Body length (mm) 99.9 ±2.0 112.5 ±3.6 88.0 ±4.2 94.5 i-2.1 Female mice Body weight 22.2 ±2.6 28.3 ±3.3 21.8 ±2.2 24.4 Carcass weight 5.7 ±0.7 8.2 ±1.2 5.6 ±0.4 6.3 ±0.8 Body length (mam) 92.4 ±4.4 106 ±3.2 92.0 ±0.8 95.4 ±4.2 mean std dev of data fromn 2-8 mice per data point WO 01/35732 PCT/AUOO/01398 -41 EXAMPLE IGF signalling cascade 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 4 and normal control mice. Initially, the extent and duration of phosphorylation of the IGF-I receptor, as well as the downstream effectors IRS and she 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.

EXAMPLE 11 Constitutive SOCS-2 expression 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.

EXAMPLE 12 SOCS-2 regulation of major urinary protein (MUP) and insulin-like growth factor (IGF-1) 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 WO 01/35732 PCT/AU00/01398 -42- GH signalling is disrupted 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 x 3 min) before 0.5 il 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 wildtype samples (Figure 4a). Production of IGF-I is also stimulated by GH and, consistent with deregulated GH action, RNase protection assays revealed increased IGF-I production in several organs including the heart, lungs and spleen of SOCS-2 4 mice (Figures 4b,c).

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 3-actin as an internal standard. However, excess production was not evident in the liver, bone, fat or muscle. No increase in serum IGF-I concentration was observed in SOCS-2-/ mice (male 278 74 ng/ml; 282 female 310 62; 298 76; n 6 mice per group), consistent with normal production in the liver, which is the major source of circulating IGF-I (22, 23).

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.

WO 01/35732 WO 0135732PCT/AUOO/01 398 -43-

BIBLIOGRAPHY

1. Nicola, N.A. Guidebook to cytokine and their receptors. Oxford University Press.

Oxford, 1994.

2. Bazan, J.F. Immunology Today 11: 350-354, 1990.

3. Sprang et a, Curr. Opin. Structural Bio. 3: 8 15-827, 1993.

4. Hilton, D.J. Guidebook to cytokines and their receptors, 8-16: 1994.

Murakami et al, Proc. Natl. Acad Sci. USA 88: 11349-11353, 1991.

6. Darnell et a, Science 264: 1415-1421, 1994.

7. Ihle, J.N. Nature 377: 59 1-594, 1995.

8. Ihle et al, Annual Review of Immunology 13:~ 369-398, 1995.

9. Sato et al, Embo Journal 12: 4181-4189, 1993.

Culter et al, Journal of Biological Chemistry 268: 2 1463-21465, 1993.

11. David et at, Journal of Biological Chemistry 271: 9185-9188, 1996.

12. Shual et Nature 366: 580-583, 1993.

13. David et a, Science 269: 1721-1723, 1995.

14. Gupta et al, Embo Journal 15: 1075-1084, 1996.

Wen et al, Cell 82: 241-250, 1995.

16. Altschul et al, NucI. Acids Res. 25:3389. 1997.

17. Ausubel et al. "Current Protocols in Molecular Biology" John Wiley Sons Inc, 1994-1998, Chapter 18. Marmur and Doty, J Mat. Biol. 5: 109, 1962 19. Bonner and Laskey, Eur. I. Biochem. 46: 83, 1974 Dey et al, J Biol. Chem. 273:24095-24101, 1998 21. Norstedt et Cell 36:805-812, 1984 22. Yakar et a, Proc Natl Acad Sci USA 96:73 24-7329, 1999 23. Sjogren et al, Proc Natl Acad Sci USA 96:7088-7092, 1999 24. Stanr et at, Proc Nall Acad Sci USA 95:14395-14399, 1998 Alexander et al, EMBO J 14:5 569-5 578, 1995 26. Alexander et al, Blood 87:2162-2170, 1996 EDITORIAL NOTE APPLICATION NUMBER 13719/01 The following sequence listing pages 1 4 are part of the description. The claims pages follow on pages 44 46 WO 01/35732 PCT/AU00/01398 -1- SEQUENCE LISTING <110> THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RES <120> A METHOD <130> 2338102/EJH <140> International <141> 2000-11-16 <150> PQ4082 <151> 1999-11-16 <150> PQ7812 <151> 2000-05-29 <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 1121 <212> DNA <213> mouse <220> <221> CDS <222> (223) (816) <400> 1 gcgatctgtg ggtgacagtg tctgcgagag actttgccac accattctgc cggaatttgg agaaaaagaa ccagccgctt ccagtcccct ccccctccgc caccatttcg gacaccctgc 120 acactctcgt tttggggtac cctgtgactt ccaggcagca cgcgaggtcc actggcccca 180 WO 01/35732 WO 0135732PCT/AUOO/01398 -2gctcgggcga ccagctgtct gggacgtgtt gactcatctc cc atg acc ctg cgg Met Thr Leu Arg

I

tgc ctg gag ccc tcc Cys Leu Glu Pro Ser aat gga gcy gac Asn Gly Ala Asp acg cgg agc cay Thr Arg Ser Gin ggg acc gcg gg Gly Thr Ala Gly ccg gag gaa. cay tcc ccc gag gcg gcg Pro Giu Glu Gin Ser Pro Giu Ala Ala cgt ctg Arg Leu gga agt Gly Ser gcg aaa gcc Ala Lys Ala atg act gtt Met Thr Val cgc gag ctc agt Arg Glu Leu Ser aca gga tgg tac Thr Gly Trp Tyr aat gaa gcc aaa Asn Giu Ala Lys aaa. tta aaa gag gct cca gaa gga Lys Leu Lys Glu Ala Pro Glu Gly act ttc Thr Phe ttg att aga gat Leu Tie Arg Asp tcg cat tca gac Ser is Ser Asp cta cta act ata Leu Leu Thr Ile gtt aag acg tca.

Val Lys Thr Ser gga ccg act aac Gly Pro Thr Asn cgg att gag tac Arg Ilie Giu Tyr gat ggg aaa t-.c aga. ttg gat tct atc Asp Gly Lys Phe Arg Leu Asp Ser Ile 105 tgt gtc aag tcc Cys Val Lys Ser aag ctt Lys Leu 115 570 aaa cay ttt Lys Gin Phe agt gtg gtt cat Ser Val Val His att gac tac tat Ile Asp Tyr Tyr gtc cag atg Val Gin Met 130 tgc aag gat aaa cgg aca gyc cca gaa acc cca cgg aat ggg act gtt WO 01/35732 WO 0135732PCr/AUOO/01398 Cys Lys Asp 135 cac oty tao His Leu Tyr Lys Arg Thr Gly Glu Ala Pro Arq tat aca tca goa Tyr Tin Ser Ala 160 Gly Thr Val act otg cag 714 Thr Leu Gin oty aco aaa Leu Thr Lys 150 oat tto His Phe tyt oga oto Cys Ary Leu aao aaa tgt Asn Lys Cys gyt aog ato tgg Gly Thr Ile Trp cot tta oca aca aga ota aaa gat tao ttg gas gaa Pro Leu Pro Tin Arg Leu Lys Asp Tyr Leu Glu Glu 185 190 gta taagtatttc tctctctttt tcgttttttt ttaaaaaaaa tat ass Tyr Lys 195 aaaaacac cag Gin Val gcctoatata ygataaotgo aagatgtago ttoctatggo aaaaaaaaaa gga 762 Gly 180 tto 810 Phe at 866 octet 926 ytgtg 986 tttot 1046 aacaa 1106 1121 gactatotcc gaatgcagot atgtgaaaga gcagaattot otottasyga oagttgggot taggtatttt aaagttcccc ttaggtagtt tgctcaagat caaatggccc ttttaaatga aaaaa gasoocagag gcot oagtoctaaot tassy ttagotgaat gatgo aacaaaaoaa aacaa <210> 2 <211> 198 <212> PPT <213> mouse <400> 2 Met Tin Leu Arg Cys Leu Giu Pro Ser Gly Asn Gly Ala Asp Arg Tin 1 5 10 WO 01/35732 PCT/AU00/01398 Arg Ser Gin Trp Gly Thr Ala Gly Leu Pro Glu Glu Gin Ala Ala Arg Ala Lys Ala Glu Leu Ser Ser Pro Glu Thr Gly Trp Leu Lys Glu Tyr Trp Ala Pro Leu Leu Gly Ser Met Thr Glu Ala Lys Glu Glu Gly Thr Phe Iie Arg Asp Ser Ser His Ser Asp Thr Ile Val Lys Thr Ser Gly Pro Thr Asn Leu Arg Ile Glu Tyr Lys Ser Lys 115 Tyr Val Gin Gly Lys Phe Asp Ser Ile Lys Gin Phe Val Val His Ile Cys Val 110 Ile Asp Tyr Ala Pro Arg Met Cys Lys Arg Thr Gly 130 Asn Gly Thr Val His Leu Thr Lys Tyr Thr Ser Thr Leu Gin Cys Arg Leu Asn Lys Cys Thr Gly 175 Leu Glu Thr Ile Trp Pro Leu Pro Arg Leu Lys Asp Glu Tyr Lys Phe Gin Val

Claims (11)

10-01-'06 12:42 FROM- T-307 P005/010 F-025 44 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A method of enhancing growth hormone signaling in an animal, said method comprising introducing a modulator of expression to down-regulate expression of a genetic sequence encoding a SOCS-2 protein or a modulator of activity of SOCS-2 protein to decrease the activity of a SOCS-2 in said animal. 2. A method according to Claim I wherein the animal is a human, primate, livestock animal, laboratory test animal, companion animal or captive wild animal. S"3. A method according to Claim 2 wherein the animal is a human. 4. A method according to any one of Claims I to 3 for use in increasing strength in elderly people, obesity control treatment or catabolic diseases, treatment of chronic inflammatory disease, treatment and/or prophylaxis of osteoporosis, treatment of cardiomyopathy and in the treatment of complicated fracture. A method according to any one of Claims I to 4 wherein said genetic sequence encodes an amino acid sequence substantially as set forth in <400>2 or an amino acid sequence having at least 60% similarity thereto. 6. A method according to Claim 5 wherein the genetic sequence comprises the :nucleotide sequence set forth in <400>1 or a nucleotide sequence having at least similarity thereto or a nucleotide sequence capable of hybridizing to <400>1 or its complementary form under low stringency conditions at 42°C. 7. A genetically modified non-human animal exhibiting decreased expression of SOCS-2 protein, characterized in that a genomic sequence encoding said SOCS-2 protein in said animal has been manipulated to delete one or more nucleotides to thereby prevent, reduce or down regulate expression of said genornic sequence, and wherein the COMS ID No: SBMI-02365048 Received by IP Australia: Time 12:49 Date 2006-01-10 10-01-'06 12:42 FROM- T-YW7 PFbd/021 P .WitJtbis meg amdrssns ma u aoM=iWons decreased expression of SOCS-2 protein results in enhanced growth hormone signaling in said animal. 8. The genetically modified non-human animal according to Claim 7 wherein the SOCS-2 protein comprises the amino acid sequence set forth in <400>2 or an amino acid sequence having at least 60% similarity thereto. 9. The genetically modified non-human animal according to Claim 7 wherein the genomic sequence encoding the SOCS-2 protein comprises the nucleotide sequence set forth in <400>1 or a nucleotide sequence having at least 60% similarity thereto or a nucleotide sequence capable of hybridizing to the nucleotide sequence set forth in <400>1 or its complementary form under low stringency conditions at 420C. 10. The genetically modified non-human animal according to any one of Claims 7 to 9 wherein the animal is a mouse, rat, guinea-pig, rabbit or livestock animal.

11. The genetically modified non-human animal according to Claim 10 wherein the animal is a mouse.

12. A method of screening for modulators of growth hormone signaling said method comprising administering potential modulators to a genetically modified non- human animal according to Claim 7, and identifying modulators which alter growth o '...hormone signaling.

13. A method according to Claim 12 wherein the genetically modified non- human animal is a mouse.

14. Use of a genetically modified non-human animal according to Claim 7, in the screening for therapeutic molecules which modulate growth hormone signaling. COMS ID No: SBMI-02365048 Received by IP Australia: Time 12:49 Date 2006-01-10 10-01-'06 12:42 FROM- T-307 P007/010 F-025 -46- A modulator of expression of a genetic sequence encoding a SOCS-2 protein or a modulator of activity of SOCS-2 protein when used according to the method of anyone of Claims I to 6.

16. A method of inhibiting growth hormone signaling in an animal, said method comprising up-regulating expression of a genetic sequence encoding a SOCS-2 protein or a functional part thereof or increasing the activity of a SOCS-2 protein or a functional part thereof in said animal.

17. A method according to Claim 16 wherein the animal is a human, primate, livestock animal, laboratory test animal, companion animal or captive wild animal.

18. A method according to Claim 17 wherein the animal is a human.

19. A method according to any one of Claims 16 to 18 wherein said genetic sequence encodes an amino acid sequence substantially as set forth in <400>2 or an amino acid sequence having at least 60% similarity thereto.

20. A method according to Claim 19 wherein the genetic sequence comprises the nucleotide sequence set forth in <400>1 or a nucleotide sequence having at least similarity thereto or a nucleotide sequence capable of hybridizing to <400>1 or its complementary form under low stringency conditions at 42 0 C.

21. A method according to anyone of Claims 1 to 6 or 12 to13 or 20 or a genetically modified non-human animal of anyone of Claims 7 tol 1 or 14 substantially as hereinbefore described with reference to Figures and or Examples. DATED this 10th day of January, 2006 The Walter and Eliza Hall Institute of Medical Research by its Patent Attorneys DAVIES COLLISON CAVE COMS ID No: SBMI-02365048 Received by IP Australia: Time 12:49 Date 2006-01-10