WO2005020677A1

WO2005020677A1 - Method of selecting animal models from animals which have been subject to mutagenesis, and the use of myb transcription factors for screening

Info

Publication number: WO2005020677A1
Application number: PCT/AU2004/001167
Authority: WO
Inventors: Douglas J. Hilton; Warren S. Alexander; Marina Carpinelli
Original assignee: The Walter And Eliza Hall Institute Of Medical Research
Priority date: 2003-08-29
Filing date: 2004-08-27
Publication date: 2005-03-10
Also published as: EP1667516A1; EP1667516A4

Abstract

The present invention relates generally to target molecules for potential therapeutic and/or prophylactic intervention. More particularly, the present invention provides a physiological assessment system in the form of vertebrate animal models to identify genetic or proteinaceous drug targets therein associated with a disease or a particular condition or phenotype. In an illustrative embodiment, target molecules associated with modulating platelet levels and ameliorating the symptoms of thrombocytopenia are described. The target molecules are useful as therapeutic and/or prophylactic agents such as in antisense or iRNA form. The present invention also relates to the application of these drug targets in the identification or development of therapeutic and/or prophylactic agents which modulate the functional activity of the drug target or its interacting network of molecules.

Description

Method of Selecting Animal Models from Animals which have been subject to Mutagenesis, and the use of Myb Transcription Factors for Screening.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to target molecules for potential therapeutic and/or prophylactic intervention. More particularly, the present invention provides a 10 physiological assessment system in the form of vertebrate animal models to identify genetic or proteinaceous drug targets therein associated with a disease or a particular condition or phenotype. In an illustrative embodiment, target molecules associated with modulating platelet levels and ameliorating the symptoms of thrombocytopenia are described. The target molecules are useful as therapeutic and/or prophylactic agents such 15 as in antisense or iRNA form. The present invention also relates to the application of these drug targets in the identification or development of therapeutic and/or prophylactic agents which modulate the functional activity of the drug target or its interacting network of molecules. In particular, the invention relates to vertebrate and particularly rodent animal models or human diseases, exhibiting physiological characteristics such as one or more 20 symptoms of a disease or of a condition, which may be used to screen for randomly or non-randomly induced mutations one or more of which effectively ameliorate/s symptoms of the disease or effectively ameliorates the condition. Such mutations are proposed to be within potential targets for therapeutic intervention. As an alternative to mutagenesis with a mutagen, the vertebrate animal model may be used as a recipient for other potential 25 physiology modifying agents such as sense or antisense molecules and small chemical, nucleic acid or proteinaceous molecules.

DESCRIPTION OF THE PRIOR ART

30 Bibliographic details of references in the subject specification are also listed at the end of the specification. The priority documents associated herewith are incorporated herein in their entirety.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that this prior art forms part of the common general knowledge in any country.

The increasing sophistication of molecular biology is greatly facilitating research and development in many fields of science. Medical science is a particularly important beneficiary of this rapidly developing art. Notwithstanding the great strides that have been made, problems with drug target validation in the pharmaceutical sciences have been largely ignored by the research community.

Target validation is an essential and often technically difficult task which must be completed prior to committing huge resources to drug development. While experimental results may indicate that a molecule is involved with a disease or condition, it seldom follows that the molecule is a good drug target with potential to beneficially influence the interaction because, for example, other molecules in the subject may compensate by performing the same function.

Genetic screens in lower eukaryotic model organisms have been used very successfully to study gene function and understand biochemical pathways. Mutagenesis in mammalian models such as the mouse has been the basis for phenotype-driven forward genetic screens to identify mutants having an altered phenotype which might be indicative of which genes contributes to the onset or progression of disease. Databases and markers are now available, largely as a result of genome projects, which permit certain positional cloning to proceed with precision and with relative speed. However, although the genes identified through these processes may be shown to be interactive in a pathway or contribute to disease, significant further investigative work is required to discover whether or not these interactive molecules are in fact useful drug targets in subjects of interest to medical science. The problem remains how to establish which of the approximately 30,000 genes in the human genome are the ones to target for the treatment of a particular disease. One example of progress in this area has been the development of genetic network databases which attempt to trace the complex web of genes and their products which switch each other off and on. Genetic networks map interacting genes by placing characterized genes at nodes linked to other nodes based on interaction of the genes or their encoded products. Ultimately, it is proposed that such networks will facilitate modelling of processes such as disease processes.

There is a need for drug target evaluation systems to be available which identify validated drug targets. Thrombocytopenia is one disease for which validated drug targets are required. Thrombocytopenia may occur as an inherited illness or may occur as a result of autoimmune disease, viral infection or as a side effect of chemotherapy. The current treatment for thrombocytopenia is inadequate (Demetri G. D., Oncologist, 6 (Suppl.5):\5- 23, 2001).

Blood platelets are shed by megakaryocytes into the circulation where they are required for blood clotting and haemostasis. Thrombopoietin (TPO), acting through its specific cell surface receptor c-Mpl, is considered the principle cytokine controlling megakaryocyte and platelet numbers (Kaushanksy K. et al, Oncogene, 27:3359-3367, 2002; Murone M. et al, Stem Cells, 16: 1-6, 1998). Mice and humans lacking functional Tpo or Mpl genes are profoundly thrombocytopenic and have a corresponding reduction in the numbers of megakaryocytes, megakaryocyte progenitor cells and stem cells (Alexander, W. S. et al, Blood, 57:2162-2170, 1996; De Sauvage F. et al, J. Exp. Med., 183:651-656, 1996; de Sauvage, F. J. et al, Journal of Laboratory & Clinical Medicine, 757:496-501, 1998; Ghilardi N. et al, British Journal of Haematology, 707:310-316, 1999; Gurney A. L. et al, Science 265: 1445- 1447, 1994; Ihara K. et al, PNASU, 96: 132-3136, 1999; Kimura S. et al, PNASU, 95:1195-1200, 1998). Although several transcription factors, including FOG-1 (Tsang A. P. et al, Genes & Development, 72:1176-1188, 1998), GATA-1 (Shivdasani R. A. et al, EMBO, 16:3965-3973, 1997), NF-E2 (Shivdasani R. A. et al, Cell, 81:695-704, 1995), MYB (Emambokus N. et al, EMBO, 22:1-11, 2003) and p300 (Kasper L. H. et al, Nature, 419:738-743, 2002) have been shown to contribute to the specification and production of megakaryocytes and platelets, little is known of their relationship to the TPO signal transduction pathway.

SUMMARY OF THE INVENTION

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.

Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID NOs: correspond numerically to the sequence identifiers <400>1 (SEQ ID NO: 1), <400>2 (SEQ ID NO: 2), etc. A summary of sequence identifiers is provided in Table 1. A sequence listing is provided after the claims.

The present invention is predicated, in part, on the use of physiologically assessable animal models of a human disease or condition in a physiological assessment system to identify drug targets. It is proposed that the instant assessment system will be used as a method of doing business, for example, in contract testing to identify drug targets.

In one embodiment, mutations are introduced into an animal model of a disease or of a physiological state of interest (condition), and animals identified in which symptoms of the disease or condition are ameliorated (changed). The mutation may be a random or non- random change at a genetic locus or a change in its expression or activity. Alternatively, mutations may be introduced into a wild type precursor of the animal model which is subsequently further modified such as, for example, by the administration of an infectious or chemotherapeutic agent or cancerous cells in order to develop the physiological characteristics of the animal model. The present invention provides animal models comprising one or more mutations which further affect the assessable physiological characteristics of the animal model. The proposal is that a mutation has occurred in a drug target resulting in an expression pattern or activity profile which ameliorates the symptoms of the particular disease or changes the condition. The genetic molecules comprising the mutations are cloned positionally and they, or their encoded molecules including RNA or proteinaceous molecules, comprise validated drug candidates themselves or substrates for the development of agonists and antagonists useful in the treatment and/or prophylaxis of the disease or condition.

In one illustrative embodiment, mutations are introduced into an animal model of thrombocytopenia, and animals identified in which platelet levels are elevated and/or symptoms of thrombocytopenia ameliorated. Alternatively, mutations are introduced into wild type animals which are subsequently developed into the animal model. For example, thrombocytopenia may be induced by chemotherapy. Alternatively, the animal model may be induced by downregulation or inhibition of Mpl by various agents such as antibodies, receptor antagonists or antisense/iRNA molecules. A mutation occurring in a drug target for the treatment of thrombocytopenia or for modulating platelet levels results in an expression pattern or activity profile in the animal model which effectively modulates platelet levels and/or overcomes the reduced platelet levels and ameliorates the symptoms of thrombocytopenia. Identification of the drug target facilitates identification of further drug targets selected from the group of molecules with which the initial drug target is known to interact.

Animals with reduced Mpl activity exhibit inter alia low platelet counts and provide therefore a useful animal model for thrombocytopenia and other conditions associated with an over supply or under supply of platelets in a subject or tissue. Conveniently, this occurs by introducing a mutation into one or both Mpl alleles. In a preferred embodiment, a Mpl^" ^A mouse is used as a model of thrombocytopenia and used in a physiological assessment system to identify drug targets for the treatment of thrombocytopenia. The present invention provides the use of a rodent model of thrombocytopenia, preferably with reduced Mpl levels, to screen for drug targets for use in the treatment or development of treatment for thrombocytopenia. The description of the present invention is supported by the production as described herein of three mouse pedigrees comprising Plt3, Plt4 and Plt6 mutations developed in Mpl ^''^'mice and exhibiting inter alia elevated platelet levels and qualitative changes in megakarocytosis.

The Plt3 and Plt4 mutations have been mapped to the Myb transcription factor gene and it is demonstrated herein that mutation in this gene can ameliorate thrombocytopenia caused by lack of TPO signalling through its receptor Mpl. In addition and importantly, it is also shown that reduction in the function of Myb ameliorates thrombocytopenia that occurs following administration of chemotherapeutic agents of the type that are used in treating human cancer patients. As demonstrated herein, down regulation of Myb is important in hematopoiesis and platelet production and has been revealed to be a validated candidate for the development of small molecule pharmaceuticals with which to treat thrombocytopenia (see, for example Figure 13 A, B and C).

The present invention provides antagonists and agonists of the molecules identified in the subject physiological assessment system. In various embodiments of the present invention antagonists and agonists may comprise all or part of the target molecules themselves, or their complementary sequences, chemical analogues, mimetics, sense or antisense molecules including iRNA-type agents, antibodies, or other molecules in the genetic network to which the target molecules identified by the instant methods belong or their derivatives.

Alternatively, the antagonists or agonists may be synthetic chemicals or natural products identified by screens known in the art. Once the target molecules are identified, a wide range of screening strategies known in the art are available for the identification, production, design and development of antagonists or agonists. The rational design of molecules which interact with the active site of proteinaceous target molecules may be achieved using the solution structures of the target and/or target-ligand complexes. Spectroscopic and computer modelling techniques are generally used to determine a solution structure and subsequently the three dimensional structure can be displayed and manipulated using computer enhanced algorithms for the design of agonists or antagonists. The present invention presents pharmaceutical compositions comprising recombinant synthetic or isolated forms of the present drug targets and one or more pharmaceutically acceptable carriers, diluents or excipients and furthermore contemplates methods of their use in vitro or in vivo in methods for the treatment of subjects, and in particular humans.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1A, B, C, D and E are photographic representations depicting the results of Automated Hematological analysis to determine platelet counts in (A) Mpl+/+ mice, (B) Mpl'^' mice, (C) GI progeny, (D) progeny of G2 mouse No. 985.34 with about half the progeny exhibiting very low platelet counts characteristic of Mpl*^' mice (pedigree Plt3), and (E) progeny of G2 mouse No. 1118.11 with about half progeny exhibiting very low platelet counts characteristic of Mpl ^''^' mice (pedigree Ptl4).

Figure 2A, B, C, D and E are photographic representations depicting the results of Automated Hematological analysis to determine platelet counts in homozygous Plt3 and PU4 mice compared with (A) Mpl+/+ mice and (B) Mpl^1' mice. Figure 2 (C) represents the platelet counts of individual mice resulting from crosses between Plt3/+ Mpl^1' and +/+Mpl'^' or Plt3/+ Mpl'^' and PH3/+ Mpl'^'. Figure 2 (D) represents the platelet counts of individual mice resulting from crosses between Plt4/+ Mpl'^' and +/+Mpl'^~ or PU4/+ Mpl'^' and Plt4/+ Mpl'^'. The right hand column in D shows the platelet counts of progeny of a cross between putative Plt4 homozygotes and +/+ Mpl'^' mice showing that all the progeny had mildly elevated platelet numbers expected of PU4I+ Mpl^1' mice.

Figure 3A, B, C, D and E are graphical representations showing the increased production of platelets by precursors in mice harbouring the PU3 and Plt4 mutations. Figure 3 (A) depicts numbers of Bone Marrow Blast-CFC in homozygous Plt4/Plt4 Mpl'^' mice, Plt4/+ Mpl^''^' mice, +/+Mpl'^~ mice and +/+Mpl^{+ +} mice. Figure 3 (B) depicts numbers of Bone Marrow Megakaryocyte-CFC in PU4/PU4 Mpl'^' mice, Plt4/+ Mpl'^' mice, +/+Mpl'^' mice and +/+Mpl+/+ mice. Figure 3 (C) depicts numbers of Spleen Megakaryocyte-CFC in homozygous Plt4/Plt4 Mpl'^' mice, PU4/+ Mpl^1' mice, +/+Mpl'^~ mice and +/+Mpl+/+ mice. Figure 3 (D) depicts numbers of Bone Marrow Megakaryocyte in homozygous Plt4/Plt4 Mpl'^' mice, PU4/+ Mpl'^' mice, +/+Mpr'^' mice and +/+Mpl+/+ mice. Figure 3 (E) depicts numbers of Spleen Megakaryocyte in homozygous Plt4/Plt4 Mpl'^' mice, PU4/+ Mpl'^' mice, +/+Mpl'^~ mice and +/+Mpl+/+ mice. Figure 4A, B and C are photographic representations depicting Automated Hematological results providing platelet counts in the Mpl^+/+ mice (A), and Mpl^" mice (B). To distinguish compound heterozygotes from double heterozygotes, mice with the highest platelet levels (Figure 4C left hand column) were mated with +/+Mpl'^~. In this mating, compound heterozygotes yield progeny, all of which will be heterozygote (PU3/+ Mpl'^' or Plt4/+ Mpl'^'), where as double heterozygotes yield double heterozygotes (PU3/+ PU4/+ Mpl'^'), single heterozygotes (PU3/+ +/+ Mpl'^' or +/+ Plt4/+ Mpl'^') or wild type (+/+ +/+ Mpl'). In the case of Plt3 and Plt4, the mutations are clearly in the same gene or closely linked since the progeny of the above mating all have the phenotype of heterozygotes (Figure 4C right hand column).

Figure 5 is a representation showing the chromosomal location of Plt4. PU4/+ Mpl'^' mice on a C57BL/6 background were mated to +/+ Mpl'⁺ on a 129Sv background and the PU4/+ Mpl'^' (C57BL/6xl29Sv) Fi progeny were selected because of the elevated platelet numbers compared with +/+ Mpl ^' controls. These Fj mice were then intercrossed to generate an F2 generation, which were bled at 7 weeks of age and had platelet count that varied from very low levels expected of +/+ Mpl'^' mice, intermediate levels characteristic of Plt4/+ Mpl'^' mice and exceptionally high levels characteristic of Plt4/Plt4 Mpl'^' mice. These mice were sacrificed and their livers were removed and genomic DNA was produced. Using a set of simple sequence length polymorphisms (SSLPs) across the genome it was determined, for each F2 mouse, whether a marker (see Table 3) was homozygous C57BL/6, heterozygous C57BL/6-129Sv or homozygous 129Sv. For markers not linked to the Plt4 mutation one would expect no correlation between genotype and phenotype, whereas for markers very closely linked to the mutation, one would expect animals with the highest platelet counts (usually Plt4/Plt4 Mpl'^') to be homozygous C57BL/6, animals with an intermediate platelet count to be heterozygous (usually PU4/+ Mpl'^') and animals with the lowest platelet counts (usually +/+ Mpl'^') to be homozygous 129Sv. The only region of the genome to follow this pattern was at the centromeric end of chromosome 10 (Figure 5) between marker D10Mit80 and D10Mit38. Using additional markers, this interval was further narrowed and the only strong candidate remaining was the Myb gene. Figure 6 is a representation showing the chromosomal location of Plt3 on mouse chromosome 10. Pit 3/+ Mpl'^' mice on a C57BL/6 background were crossed with PU3/+ Mpf^' mice on a 129Sv background. Plt3/+ Mpl'^' FI animals were then identified at 7 weeks of age because of their elevated platelet counts and were backcrossed to +/+ Mpf^' mice on a 129Sv background to generate N2 mice. N2 mice that were Mpl'^' were identified and their platelet counts were determined at 7 weeks of age. Mice were then sacrificed and their livers were removed and genomic DNA prepared. SSLPs across chromosome 10 (Table 4) were then amplified and analysed as described for PU4 confirming the co-localisation of the two mutations in the Myb gene.

Figure 7 is a representation of results of sequencing of genomic DNA from Plt3 and Plt4 mice showing the nature of the mutation in these mice. Primers (see Table 5) were designed to amplify the exons and flanking intronic DNA from genomic DNA of P 4/PU4 Mpl'^', PU4/+ Mpl'^', P 3/PU3 Mpl'^', Plt3/+ Mpl'; +/+ Mpl'^' and +/+ Mpl+/+ mice all on a C57BL/6 background. Figure 7 (A) shows Plt3 mice in which nucleotide A455 is altered to T leading to a substitution of Val for Asp at amino acid 152. Figure 7 (B) shows the results for PU4 mice indicating a nucleotide alteration from A to T in the Plt4 mutation.

Figure 8 is a representation showing the nucleotide and amino acid sequence of mouse Myb. The mutation in PU3 mice is shown at nucleotide A455 leading to a substitution of Asp for Val at amino acid position 152. The mutation in Plt4 mice is shown at nucleotide position Al 151 where an A is replaced with a T, leading to a substitution of Val for Asp at amino acid 384.

Figure 9 is a representation showing the nucleotide and amino acid sequence of human Myb.

Figure 10A, B and C are graphical representations showing platelet counts in ENU- mutant mice, a, The platelet counts observed in Mpl^+/+ mice (1400 x 10⁶ ± 200 x 10⁶/ml, mean ± standard deviation, n=83) are shown in the left panel for comparison with the pedigree segregating the pltl mutation, which includes multiple thrombocytopenic individuals (platelet counts less than 800 xl0⁶/ml; shown in lilac) and the pedigree segregating the plt2 mutation includes multiple thrombocytotic individuals (platelet counts above 2,000 x 10⁶/ml; shown in plum), b, Mpl^1' mice are severely thrombocytopenic (platelet counts of 117 x 10⁶ ± 52 x 10⁶/ml, n=783, left panel), as were the vast majority of GI Mpl'^' mice generated (second panel from left). Heritable suppression of thrombocytopenia was observed for 3 of the 8 GI mice with platelet counts greater than 300 x 10⁶/ml. Breeding from Plt3 (centre panel), Plt4 (second panel from right) and Plt6 (right panel) mutants generated significant numbers of offspring with platelet counts greater than 300 x 10⁶/ml, which are more than 3 standard deviations above the mean observed for Mpl'^' mice), c, Plt3, Plt4 and PU6 result in semi-dominant suppression of thrombocytopenia, since inter-crossing mice heterozygous for these mutations yielded an additional phenotypic category of mice with supra-physiological platelet numbers. In the case of Plt4, these animals were shown to be homozygous since, when mated to Mpl'^' mice, all of the progeny have mild suppression of thrombocytopenia (455 x 10⁶ ± 124 xlO⁶ /ml, n=32), whereas when inter-crossed, all of the mice have massively elevated platelet numbers (4496 x 10⁶ ± 425 xlO⁶ /ml, n=28).

Figure 11 A, B, C and D are representations of results of an analysis to determine linkage between mutants, their map position and position within Myb, and the effect of the mutation on the ability of expressed protein to activate transcription. Plt3 and Plt4 are tightly linked on chromosome 10 and are alleles of c-Myb. a, Pit 3/+, P 4/+ and Plt6/+

Mpl'^' mice were inter-crossed to produce compound heterozygotes, single heterozygotes and mice wild type for both Pit mutations. The platelet counts of the progeny are shown and the genotype was inferred by analysis of the progeny derived from mating these animals to +/+ Mpl'^' mice. Note that the progeny derived from crossing PU3/PU4 Mpl'^' mice to +/+ Mpl'^' mice all have intermediate platelet counts typical of either PU3/+ or

Plt4/+ Mpl'^' animals, showing these mutations are tightly linked. In contrast the progeny derived from PU3/PU6 Mpl'^' or PU4/PU6 Mpl'^' to +/+ Mpl'^' exhibit all three phenotypic classes showing Plt3 and Plt4 are not linked to Plt6. b, To map Plt4, a (C57BL/6xl29Sv)

F2 generation was produced and mice were bled at 7 weeks of age and categorized as having low platelets (<150 x 10⁶/ml) characteristic of +/+ Mpl'^' mice, moderate numbers of platelets (150xl0⁵ - 2000 x 10⁶/ml) characteristic of Plt4/+ Mpl'^' mice or extremely high platelets (>2000 xl0⁶/ml) characteristic oϊ PU4/Plt4 Mpl'^' mice. Animals were then genotyped and markers found to be homozygous 129/Sv are shown in white, markers that were heterozygous are shown in grey and markers homozygous C57BL/6 are shown in black. The number of animals with each haplotype is shown below. The physical distance of each marker is shown on the left and is from the Feb 2003 assembly of the mouse genome (http://www.genome.ucsc.edu). The Plt4 mutation was localized to between D10Mitl24 and DlOMit 214, as indicated by the solid lines. To confirm Plt3 was located close to Plt4, a (C57BL/6xl29Sv)N₂ generation was produced. Platelet numbers were determined in these mice which were genotyped using the markers most closely linked to PU4. The right hand panels show the correlation between genotype and phenotype for markers at the centromeric region of chromosome 10. c, Sequence of PCR-amplified exons and intron boundaries of the c-Myb gene showing a single A to T transversion in Plt3 and Plt4, resulting in an Asp to Val substitution at positions 152 and 382 respectively. Representative traces are shown; a total of 3 PU3/P13 Mpl'^', 3 Plt3/+ Mpl'^' mice, 3 Plt4/Plt4 Mpl'^' mice, 3 PU4/+ Mpl'^' mice, 3 +/+ Mpl'^' mice and 3 +/+ Mpt'⁺ mice were analysed, d, The transactivation activity of wild type Myb, Plt3 and Plt4 mutant Myb and a constitutively activated truncation of Myb (CT3, Ramsay R. G. et al, Oncogene, 6: 1875- 1879, 1991) were compared by measuring production of CAT from a Myb responsive promoter (Chen R. H. et al, Mol. Cell Biol, 73:4423-4431, 1993). The activity of both Plt3 and Plt4 Myb were significantly lower than wild type; p=0017 and p=0.0003 respectively, n=9).

Figure 12A, B and C are representations of results of an analysis to determine the effect of PU3 and PU4 mutations in cells and tissue of the hematopoietic system in homozygous and heterozygous form. Mutation of c-Myb results in an elevation in progenitor cells, megakaryocytes and platelets independent of Mpl. a, The numbers of colony-forming units-spleen (CFU-s), a measure of multi-potential, relatively mature stem cells, in the bone marrow (first panel), clonogenic megakaryocyte progenitor cells (second panel), megakaryocytes (third panel) and platelets (fourth panel) in c-Myb^+/+ (+/+), c-Myb ^{P t4/+} (4/+), c-Myb ^PMIPM (4/4), c-Myb ^pι,3/+ (31+) and c-Myb ^p"^3/p"³ (3/3) mutants on a Mpl'^' or Mpl^+/+ background are shown. Assays were performed as described herein with the error bars representing the standard deviation from the mean of data from 3 to 7 (CFU-e), 2 to 6 (progenitor cell data), 2 to 7 (megakaryocyte data) and 4 to 50 (platelet data) mice, b, Flow cytometric analysis of B lymphoid cells in the spleen and erythroid cells in the bone marrow of Plt4 mutant mice showing marked reductions in pre-B (B220⁺IgM^") and B (B220⁺IgM⁺) lymphocytes and accumulation of more immature erythroid cells (CD71^hiTerl l9^med and CD71^hiTerl l9^hi; Ref 31) in Mpl'^' c-Myb^p"^{4 PM} mice, c, Histological sections of spleens from Mpl^+/+ c-Myb ^{+ +}, Mpl ^''^' c-Myb ^{+ +}, Mpl ^''^' c-Myb ^P"^4/PM and Mpl^{+ +} c-Myb ^PM/P"⁴ mice. Note the poor development of lymphoid follicles, and expanded red pulp displaying reduced cellularity and disrupted architecture.

Figure 13A, B and C are graphical representations showing that a reduction in the function of Myb ameliorates thrombocytopenia that occurs following administration of chemotherapeutic agents of the type used in treating cancer patients. c-Myb Plt4/Plt4 mice (▼) treated with carboplatin are resistant to thrombocytopenia compared with their control c-Myb+/+ littermates (A). Carboplatin was administered at the beginning of the experiment and the platelet counts of animals were measured at the indicated times afterwards. +/+ Mpl^'7', PU4/+ Mpl^"7" and Plt4/Plt4 Mpl^"7" mice were injected IP with 250 microlitres of saline (Figure 13 A) or 100 mg/kg carboplatin in approximately 250 microlitres saline (Figure 13B). Likewise, +/+ Mpl^{+ +}, Plt4/+ Mpl⁺⁷⁺ and Plt4/Plt4 Mpl^{+ +} were also injected with the same dose of carboplatin (Figure 13C). Note that the platelet levels of the Plt4/Plt4 mutants with reduced Myb function remain higher than the controls throughout the period. DETAILED DESCRIPTION OF THE INVENTION

For the purpose of describing the present invention, the following terms are defined below.

As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "an animal" includes a single animal, as well as two or more animals; reference to "a symptom" includes a single symptom, as well as two or more symptoms; and so forth.

The "animal models" of the present invention are selected from those vertebrate animals in which genetic studies are feasible. Murine animal model are preferred. Animal models express or are capable of expressing (i.e. they are used in precursor form) physiologically assessable symptoms of a disease or condition which occurs or which has substantial similarities to a disease or condition which occurs in man or other subjects of interest.

The terms "ameliorate" or "ameliorating" or "treating" or "therapy of or "treatment" are used in the broadest context and include any measurable or statistically significant change in one or more symptoms or frequency of symptoms of a disease or one or more assessable indications of a condition as well as complete recovery from the disease or elimination of an associated or other condition, its symptoms or its underlying cause. The present invention is applicable to a large range of diseases or conditions and the skilled addressee must determine the precise parameters of the assessment of phenotypes on a case by case basis. Conditions may be associated with one, or more than one, disease but there is no requirement for this. The amelioration of a condition encompasses any desirable physiological or behavioural change provided it is directly or indirectly assessable. In the example of thrombocytopenia, assessment is conveniently made of the level of platelets in the animal model. The parameters of this assessment include measuring the levels of haematopoietic cells and their precursors which are affected in thrombocytopenia by, for example, automated haemotological analysis. Platelet levels are a readily assessable physiological aspect of thrombocytopenia. Thrombocytopenia may also be associated with reduced levels of megakaryocytes and committed progenitor cells and levels of markers for these cell types provide an alternative means for physiological assessment. Other methods of assessment of ameliorating the symptoms of thrombocytopenia will be well known to those skilled in the art and reference is made to Alexander W. S., Int. J. of Biol. & Cell Biol, 37(10):1027-1035, 1999 and to citations therein.

By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject agent may be administered together with an agonistic agent in order to enhance its effects. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.

"Complementary" as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other.

The term "compound" refers to a chemical compound that induces a desired pharmacological and/or physiological effect. The term also encompass pharmaceutically acceptable and pharmacologically active ingredients of those compounds specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like. When the above term is used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, etc. The term "compound" is not to be construed narrowly but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof.

A "derivative" of a polypeptide of the present invention is a modified form of the polypeptide and also encompasses a portion or a part of a full-length parent polypeptide which may or may not retain the functional activity of the parent molecule. For example functional and non-functional derivatives may be used in determining the three dimensional structure of the target molecule. Where the derivative retains function activity, for example in the case of Myb, the derivative retains the transcription factor activity of the parent polypeptide which is important in enhancing megakaryocytopoiesis. Such "biologically-active fragments" include deletion mutants and small peptides, for example, of at least 10, preferably at least 20 and more preferably at least 30 contiguous amino acids, which exhibit the requisite activity. Functional or non-functional derivatives may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. For example, reference may be made to solution synthesis or solid phase synthesis as described, for example, in Chapter 9 entitled "Peptide Synthesis" by Atherton and Shephard which is included in a publication entitled "Synthetic Vaccines" edited by Nicholson and published by Blackwell Scientific Publications. Alternatively, peptides can be produced by digestion of an amino acid sequence of the invention with proteinases such as endoLys-C, endoArg- C, endoGlu-C and staphylococcus V8-protease. The digested fragments can be purified by, for example, high performance liquid chromatographic (HPLC) techniques. Any such fragment, irrespective of its means of generation, is to be understood as being encompassed by the term "derivative" as used herein.

"Genetic forms" of the subject target molecules may be DNA or RNA. When the genetic form is DNA it may be genomic DNA or cDNA. RNA forms of the genetic molecules of the present invention are generally mRNA. The genetic form may be in isolated form or integrated with other genetic molecules such as vector molecules and particularly expression vector molecules. In the context of this invention, "hybridization" means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

"Modulation" of a target molecule includes completely or partially inhibiting or reducing or down regulating all or part of its functional activity and enhancing or up regulating or potentiating all or part its functional activity. Where the target is a genetic sequence its functional activity may be modulated by, for example, modulating its binding capabilities or transcriptional or translational activity, or its half-life. Where the target is an encoded polypeptide, its functional activity may be modulated by, for example, modulating its binding capabilities, its half-life, location in a cell or membrane or its enzymatic capability. Modulators are agonists or antagonists which achieve modulation.

In the context of the subject invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. The term "oligonucleotide" refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non- naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

The terms "polypeptide" or "proteinaceous molecule" refer to a polymer of amino acids and its equivalent and does not refer to a specific length of the product, thus, peptides, oligopeptides and proteins are included within the definition of a polypeptide. This term also does not exclude modifications of the polypeptide, for example, glycosylations, aceylations, phosphorylations and the like. Soluble form of the subject proteinaceous molecules are particularly useful. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid including, for example, unnatural amino acids such as those given in Table 2b or polypeptides with substituted linkages. Such polypeptides may need to be able to enter the cell.

The phrase "a physiologically assessable symptom" includes a symptom (trait or phenotype) which is capable of being measured or detected using the level, activity or amount of any molecule or event which is associated with the symptom. Thus, the assessment system assesses the symptoms with respect to any reliable marker thereof. Examples of markers include, without limitation, genetic or proteinaceous molecules, cells, infectious agents, temperature, electrical conductance, levels of ions.

By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of one or more different molecule or events. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in detecting antigen bound antibody are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules. In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme- labeled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. The fluorescent labeled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The terms "sequence similarity" and "sequence identity" as used herein refer 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, He, 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 (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, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

Preferably, the percentage similarity between a particular sequence and a reference sequence (nucleotide or amino acid) is at least about 60% or at least about 70% or at least about 80% or at least about 90% or at least about 95% or above such as at least about 96%, 97%, 98%, 99% or greater. Percentage similarities or identities between 60% and 100% are also contemplated such as 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%.

Reference herein to a "low stringency" includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out T_m = 69.3 + 0.41 (G+C)% (Marmur et al, J. Mol. Biol. 5.T09, 1962). However, the T_m of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner et al, Eur. J. Biochem., 4(5:83, 1974). 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°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.

The terms "similarity" or "identity" as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, "similarity" includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, "similarity" includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and amino acid sequence comparisons are made at the level of identity rather than similarity.

Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25-or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, 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, (Nucl. Acids Res., 25:3389, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994- 1998, Chapter 15).

Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

"Subjects" contemplated in the present invention refer to the treatment or prophylaxis of a disease or condition in any animal of commercial or humanitarian interest including plants, primates, livestock animals including fish and birds, laboratory test animals, companion animals, or captive wild animals. Man is a preferred subject.

The phrases "target", "drug target" and "target molecule" are used interchangeably and refer herein to one or more genetic sequences or molecules within or derived from a subject vertebrate animal whose modulation in vivo or ex vivo effectively ameliorates a disease or condition. Thus, the drug targets are targets for prophylactic and/or therapeutic intervention. The term includes the same or homologous or variant genetic sequences or molecules within or derived from subjects to be treated, such as man.

Targets comprise the genetic sequences or their encoded products in the immediate vicinity of the mutation, or longer sequences encoding protein or nucleic acid molecules, complementary forms thereof and/or their regulatory/expression control regions. Targets also extend to functional homologs from other species or genera and functional derivatives having at least about 60% amino acid or nucleic acid similarity to all or an appropriately functional part or domain of the parent molecule after optimal alignment.

Where other interacting molecules in the genetic network comprising the target molecule are known, the invention extends to these interacting molecules. Accordingly, targets include without limitation, positive and negative regulators of transcription, precursors, upstream and downstream molecules, or co-factors.

The therapeutic compositions of the present invention interact with the target molecule. For the avoidance of doubt, the therapeutic compositions interact directly with a target molecule which may constitute an interacting molecule in the genetic or biochemical network comprising the target molecule, including molecules with which it interacts directly. In the example of the Myb transcription factor, Myb binds to a region of substrate nucleic acid in order to promote transcription. Molecules which interfere with this protein:DNA interaction will antagonise Myb factor functional activity. Thus, antagonists may comprise DNA binding molecule including nucleic acid or proteinaceous molecules.

The term "variant" refers to nucleotide sequences displaying substantial sequence identity with a reference nucleotide sequences or polynucleotides that hybridize with a reference sequence under stringency conditions that are defined hereinafter.

The terms "nucleotide sequence", "polynucleotide" and "nucleic acid molecule" may be used herein interchangeably and encompass polynucleotides in which one or more nucleotides have been added or deleted, or replaced with different nucleotides. In this regard, it is well understood in the art that certain alterations inclusive of mutations, additions, deletions and substitutions can be made to a reference nucleotide sequence whereby the altered polynucleotide retains the biological function or activity of the reference polynucleotide. The term "variant" also includes naturally-occurring allelic variants.

"Functional derivatives" of a target molecule include active portions of the target molecule whose modification in a subject ameliorates a disease or condition and which may be further modified to enhance this affect. A functional derivative of a target molecule in the form of a protein or peptide comprises a sequence of amino acids having at least 60% similarity to the target molecule or portion thereof. A "portion" in peptide form may be as small as an epitope comprising less than 5 amino acids or as large as several hundred kilodaltons. The length of the polypeptide sequences compared for homology will generally be at least about 16 amino acids, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues and preferably more than about 35 residues.

When in nucleic acid form, a functional derivative comprises a sequence of nucleotides having at least 60% similarity to the target molecule or portion thereof. A "portion" of a nucleic acid molecule is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides. This definition includes all sizes in the range of 10-35 nucleotides including 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides as well as greater than 35 nucleotides including 50, 100, 300, 500, 600 nucleotides or nucleic acid molecules having any number of nucleotides within these values.

The present invention provides, inter alia, the use of physiologically assessable vertebrate animal models of human diseases or conditions in a physiological assessment system to identify genetic or proteinaceous drug target molecules associated with the amelioration of symptoms of a disease or of a condition. Although humans are a preferred subject, other subjects are clearly contemplated and encompassed. Target molecules are particularly useful as therapeutic compounds themselves or in the development of agonists or antagonists as therapeutic compounds which ameliorate symptoms of a particular disease or condition.

Accordingly, the present invention provides both animal models and methods for their use in a physiological assessment system which will be particularly useful for pharmaceutical development, in business methods such as contract testing and for the sale of embryos and progeny of their animal models, including gametes and stem cells therefrom.

Accordingly, one aspect of the present invention provides a physiological assessment system to identify a pedigree of a vertebrate animal model of a disease or condition which exhibits ameliorated symptoms of the disease or condition comprising:

(i) assessing the symptoms of the disease or condition in the vertebrate animal model and/or its progeny wherein the animal model also comprises mutations induced by random or non-random mutagenesis;

(ii) selecting animals in which the symptoms of the disease or condition are ameliorated and determining that the responsible mutation is heritable.

In another aspect, the present invention provides a physiological assessment system to identify genetic or proteinaceous drug targets associated with amelioration of symptoms of a disease or condition comprising:-

(i) assessing the symptoms of a disease or condition in an animal model of said disease or condition and its progeny wherein said animal model also comprises mutations induced by random or non-random mutagenesis;

(ii) selecting animals in which the symptoms of the disease or condition are ameliorated and determining that the responsible mutation is heritable; and

(iii) positional cloning to identify the genetic sequences surrounding the mutation and any encoded product.

Progeny are preferably GI, G2 or G3 progeny and subsequent progeny generated by breeding GI, G2 or G3 progeny. Sequencing of target molecules may be required. However, if the target molecule has been previously sequenced it may not be necessary to sequence the entire target. In order to determine the precise nature of a mutation in the drug target, sequencing of this region is performed. The identification of genetic sequences includes recognising homologous or potentially hybridising sequences in a nucleotide database (for example by BLAST searching) and also includes pin pointing the location of the target genetic sequences between closely linked genetic markers. The identification of a genetic sequence also includes identity by name, for example when the drug target molecule is a known molecules, or by sequence which is generally confirmed or derived directly by sequencing. The encoded products of the instant genetic sequences include nucleic acid molecules such as RNA such as miRNAs and siRNA or polypeptide molecules such as short peptides or larger proteins.

Mutagenesis with an alkylating agent such as ethylnitrosourea (ENU) is preferred for the production of mutants in rodents however other efficient mutagens may be used such as, for example, chlorambucil. The mutations recovered after ENU mutagenesis are mainly point mutations. Many of the mutations caused by ENU will therefore be hypomorphic partial loss of function mutations although gain of function and complete loss of function mutations are also contemplated. Protocols are established which allow very efficient mutagenesis rates in several mice strains. An advantage of ENU treatment is that it results in random mutations being introduced into premeiotic spermatogonial stem cells and eventually sperm cells. These mice are mated to untreated females to produce first generation (GI) progeny which are heterozygous for a set of random mutations inherited from their father. These GI progeny are scored phenotypically and a large number of progeny are analysed in order to review the effects of the induced mutations on the mouse model. The progeny analysed in accordance with the present invention may be GI, G2, G3 or subsequent progeny generated by breeding GI, G2 or G3 mice.

When mice are identified which exhibit an ameliorated disease or condition, they are bred further to determine whether the phenotype/mutation is inheritable. Further breeding experiments may be conducted to compare the phenotype of the homozygous and heterozygous form of the mutation against various genetic backgrounds.

Positional cloning of the target molecule in confirmed candidates may be achieved following a number of alternative routes as known to those of skill in the art. Linkage studies employing sequence length polymorphisms such as SSLP markers is a standard preferred strategy for genetic mapping of mouse loci via an outcrossing/backcross mating system between the candidate mouse strain and a marker strain. In F2 progeny, genetic markers not linked to the mutation exhibit no correlation between genotype and phenotype. Further markers may be identified by standard procedures (Sambrook et al, Molecular Cloning, A Laboratory Manual CSH Press, Cold Spring Harbour USA, 1989) to further focus on the genetic sequences encompassing the mutation of interest. The genetic sequences between flanking markers are amplified from DNA samples and sequenced in order to confirm the sequence of the region and identify the mutated sequences. A candidate gene approach may be available which simplifies the identification of the responsible mutation. Specifically, genes or sequences considered to be involved in a particular disease or condition and which are detected in a candidate region identified by positional cloning are preferentially sequenced or assessed geneotypically for mutations. Homologous sequences may be identified in the available databases or by hybridisation- based strategies.

Where the solution or crystal structure of the target molecule is known, this facilitates the rational design of interacting compounds.

Diseases and conditions to which the present invention can be best applied relate to those diseases for which current treatments are inadequate and for which new targets for pharmaceutical development are required, and for which there are good model animals. The present invention is described using mouse models of human disease however, any laboratory animal can be employed which is commonly used in genetic studies and for which a desired therapeutic or physiological effect can be assessed. Rodents are preferred model animals. The mouse is a most preferred model animal because of the large number of available mouse models of human disease. Table 2 and 2a provides non-exhaustive list of examples of suitable mouse models of human disease.

The present invention extends to the use of vertebrate animal models in which genetic studies are feasible and to the use of random or non-random mutagenesis. To facilitate understanding however the following description concentrates on describing embodiments of the invention which use murine animal models with random point mutation created by infecting male mice with ENU. Clearly, however, these are exemplary embodiments and should be considered in no way limiting on the clearly broader applicability of the present invention.

In some embodiments the disease or condition is caused by a single transgene or an engineered or random mutation in a single gene. In the latter case the disease may be manifest in a recessive manner or a dominant manner. In simplest case, mice with the disease remain viable and fertile; where as in more complicated situations the disease may be lethal or render mice infertile. In other embodiments the situation may require mutations in two or more genes or a combination of a transgene and an engineered or random mutation before the disease is manifest.

Using the scenarios outlined below, the physiological assessment system can be employed by one skilled in the art to enable the identification of targets for therapeutic intervention for these many classes of heritable disease. Some of the breeding strategies also lead to gains of efficiency, in that the proportion of mice that is informative is increased over more conventional approaches.

In some embodiments (Scenario 1) male mice that are homozygous for the disease causing mutation are injected with ENU and bred to females also homozygous for the disease causing mutation. The first generation (GI) progeny are then screened to determine whether the disease is ameliorated relative to non-mutated mice homozygous for the disease causing mutation. This scenario is applicable wherein, for example, a recessive disease model leaves animals fertile and viable. In other embodiments mice with ameliorated disease are then tested to determine whether the phenotype is heritable by crossing them to non-mutagenised mice homozygous for the disease-causing mutation. If approximately half of the resultant progeny have the course of the disease ameliorated then the disease suppression is likely to be heritable and to be caused by an ENU-induced- mutation that may be mapped and identified by conventional genetic and genomic methods, such as those described herein.

In other embodiments, a similar strategy may be employed to identify ENU-induced mutations that suppress disease in G3 mice. In this scenario, GI mice are intercrossed or crossed to non-mutagenised mice homozygous for the disease-causing mutation. The resultant G2 mice are then either intercrossed or backcrossed to their GI parent to yield a cohort of G3 progeny that are screened to determine whether the disease is ameliorated relative to non-mutated mice homozygous for the disease causing mutation. If mice with a suppressed phenotype are found among the G3 progeny, they are then tested for example by one or more of the following crosses to determine whether the phenotype is heritable, (i) Suppressed G3 mice will be crossed to non-mutagenised mice homozygous for the disease-causing mutation, (ii) G3 mice with suppressed disease will be intercrossed, (iii) Non-suppressed siblings will be crossed to suppressed G3 mice, (iv) Non-suppressed siblings will be intercrossed. By monitoring the proportion of mice with a suppressed disease phenotype among the various test crosses one skilled in the art should be able to determine whether phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner. Where disease suppression is found to be heritable, the relevant ENU-induced mutation is mapped and identified by conventional genetic and genomic methods, such as those described herein.

Those skilled in the art would appreciate that even for highly penetrant suppression of the disease phenotypes the proportion of mice with ameliorated disease in any particular cross will vary. However, a reduction in the penetrance of the suppression of disease does not preclude identification of the causative ENU induced mutation. Many mouse models of human disease can be utilized using the methods revealed and summarized in Scenario 1 , including Mpl^{" "} thrombocytopenia described herein and others listed in Table 2 and 2a. One skilled in the art will recognise that many others can be found in publicly available databases (e.g. http://research.bmn.com/mkmd, http://informatics.jax.org).

In yet another embodiment, animal models are used in homozygous form. This embodiment is applicable for dominant disease animal models that leave animals fertile and viable (Scenario 2). If mice homozygous for the disease causing mutation are viable and fertile then one can essentially follow Scenario 1, even though the disease is dominant. The advantage of this is strategy compared with injecting a male mouse that is heterozygous and mating it to an unaffected female or injecting an unaffected male and mating it to a heterozygous female is that all, rather than half of the GI progeny are informative, since they carry the disease causing mutation and hence can be screened for suppression of disease. In one embodiment, in a screen of GI mice, homozygous male mice are treated with ENU and mates to animals that do not carry the disease causing- mutation. Alternatively, male mice that do not carry the disease-causing mutation are treated with ENU and mated to females that are homozygous for the disease-causing mutation. In either case, all of GI progeny will be heterozygous for the disease causing mutation and hence all can be screened for ENU-induced mutations that suppress disease.

The use of mice homozygous for the disease-mutation throughout every stage of the breeding program is even more advantageous if G3 progeny are to be screened, since again, all of the G3 animals may then be screened for ENU-induced mutations that suppress disease.

Many mouse models of human disease can be utilized using the methods revealed and summarized in Scenario 2. Some are listed in Table 2 and 2a, while one skilled in the art would recognise that many others can be found in publicly available databases (e.g. http://research.bmn.com/mkmd, http://informatics.jax.org). In yet another embodiment, the present assessment uses animal models of recessive or dominant disease that reduce the viability or fertility of mice (Scenario 3). An important constraint of ENU mutagensis screens is that mice harbouring the disease-causing mutation must be crossed with mice harbouring ENU-induced random mutations. Where mice with two copies of the disease-causing mutation are healthy and fertile, this presents no difficulties (Scenarios 1 and 2); however, often modelling a disease in mice results in animals that succumb to the effect of the disease or have reduced fertility and fecundity because of the disease.

In these circumstances, where the disease is recessive, mice that carry the disease-causing mutation but do not manifest disease are used in some embodiments. Specifically, the mice are heterozygous for the disease-causing mutation. In these embodiments, male mice heterozygous for the disease causing mutation are injected with ENU and mated to female mice also heterozygous for the mutation to generate GI pups. 75% of the pups are not useful since they carry no copies or one copy of the disease-causing mutation, will not get the disease and hence cannot be screened to determine whether an inherited ENU-induced mutation is capable of suppressing the disease. Moreover, these non-diseased mice must be discriminated from the rare cases among the remaining 25% of animals which carry two copies of the disease-causing mutation but which have the onset or progression of the disease ameliorated because of an ENU-induced mutation.

In some embodiments, discrimination may be achieved by genotyping the GI mice to allow the number of disease-causing mutations in each individual to be measured. One skilled in the art would appreciate that there are many well-established methods of detecting disease-causing mutations whether they are large or small insertions and/or deletions of nucleotides or point mutations. These methods include Southern blotting following restriction enzyme digestion of genomic DNA and a range of proprietary and non-proprietary fluorescent-based techniques for discriminating single nucleotide polymorphisms (SNPs). An alternative means of discriminating between animals that carry zero and one copy of the disease-causing mutation from those that carry two copies is by genetically linking the disease-causing mutation to another mutation or polymorphism that alters an easily measured trait. In some embodiments, if the disease-causing mutation has been engineered by homologous recombination, a semi-dominant coat-colour marker such as a transgene expressing the agouti protein may be introduced as part of the gene targeting process. If the coat-colour of the parental strain is black, this may allow mice with two copies of the disease causing mutation (and hence two copies of the agouti transgene) to be identified because of their light brown or yellow coat, mice with one copy of the disease causing mutation (and hence one copy of the agouti transgene) to be identified because of their agouti/dark brown coat and mice with no copies of the disease causing mutation (and hence no copies of the agouti transgene) to be identified by their black coat. Similar use can be made of naturally occurring mutations or polymorphisms that alter an easily measured trait. For example, one might wish to execute a screen for ENU-induced mutations that will suppress the inflammatory disease that occurs in mice with two null alleles of Socsl. These animals die of overwhelming inflammatory illness at three weeks of age, making it impossible to maintain this line of animals with homozygous disease- causing mutations. Socsl lies very close to a gene mahoganoid (md), which is known to affect coat-colour. If mice that are normally agouti carry two mutant copies of mahoganoid, their coats are instead black or dark brown. By simple mating strategies the Socsl null allele was linked to a mutant mahoganoid allele. Mice that were heterozygous for this pair of linked mutations were treated with ENU and bred to females also heterozygous for this pair of linked mutations. The resultant progeny with non or one copy of the disease-causing Socsl mutation are agouti in colour and discarded. It is then possible to monitor the black mice to determine whether their disease is suppressed. If a black animal appears healthy they can be genotyped to determine whether they carry two copies of the disease-causing Socsl mutation or whether there has been a recombination between the Socsl mutation and the coat-colour marker mahoganoid. The closer the disease-causing mutation and the coat-colour mutation are, the less frequently that this recombination will occur. One skilled in the art would realise that using a similar strategy as outlined above for generating and screening GI mice, that it is feasible by selecting GI and then G2 animals of the appropriate genotype the required matings to allow G3 animals to be screened for ENU-induced mutations that suppress the onset or progression of disease.

The opportunity to overcome the problems caused by recessive diseases that affect the viability or fertility of mice by using heterozygous animal is not available if viability or reduced fertility is dominant; that is one copy of the disease-causing mutation is sufficient to cause the disease that is to be suppressed and to affect the health of the animal. One would also appreciate that while the use of heterozygous mice makes certain suppressor screens feasible; they are not highly efficient, since 75% of the GI animals will not get the disease because they do not carry two copies of the disease-causing mutation and are hence not informative. Both of these problems may be overcome as disclosed herein by novel and innovative use of well-established conditional mutation technology. The general idea of this method is to generate strains of animals which carry the disease causing mutation in a latent form and are healthy and fertile; but which when mated yield offspring that all carry the disease-causing mutation and exhibit the disease unless its course is modified by a disease-suppressing ENU induced mutation.

In some embodiments, the creation of latent disease-causing alleles may be achieved using site-specific DNA recombinases such as cre-recombinase, which recognizes lox sites and flp recombinase, which recognizes frt sites. One skilled in the art would recognize that there are many different ways of utilizing cre/lox and flp/frt to create conditional disease- causing mutations. These include creation of loss of function alleles through deletion of all or part of a gene or through insertion of foreign DNA into a gene or through expression of a transgene from an exogenous promoter. In each the principle is similar; one animal is created in which lox sites or frt sites flank the salient piece of DNA. The presence of the lox and frt sites and the other DNA associated with these manipulations are designed such that the animals do not manifest the disease and are healthy and viable. A second strain of mice is generated in which the ere recombinase or the flp recombinase is expressed either using a transgene or an endogenous promoter. Again the expression of the recombinase in this strain of mice affects neither the health nor viability of the mice. When mice of these two strains are crossed the recombinase (ere or flp) can act to delete or rearrange the DNA flanked by the relevant sites (lox or frt) to create an active disease-causing allele.

In other embodiments, were the disease is manifest in a dominant manner (i.e. only requires one active copy and hence can occur in mice heterozygous for the activated disease-causing mutation) then one strain is generated that carries two copies (i.e. is homozygous) for the latent (lox or frt flanked) disease causing mutation and an other strain is generated which has two copies (i.e. is homozygous) for the relevant transgenic recombinase construct. In some embodiments, ENU is injected into male mice of one of the strains and they are mated to females of the other strain. All of the resultant pups will inherit the recombinase transgene and hence express the recombinase protein. They will all also inherit the frt or lox flanked latent disease-causing allele, which will then be deleted in the relevant tissue or relevant time by the recombinase to create an active disease causing allele. GI animals can then be monitored to identify those individuals that do not succumb to the disease. These animals are then mated and by monitoring the proportion of mice with a suppressed disease phenotype among the various test crosses one skilled in the art should be able to determine whether phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner. Where disease suppression is found to be heritable, the relevant ENU- induced mutation may be mapped and identified by conventional genetic and genomic methods, such as those described herein.

In still further embodiments, where the disease is manifest in a recessive manner (i.e. requires two active copies of the disease-causing mutation) it is preferred to create one strain of mice that is homozygous for the latent-disease causing mutation flanked by lox sites and which is homozygous for the transgene that expresses flp recombinase and a second strain of mice which that is homozygous for the latent-disease causing mutation flanked by frt sites and which is homozygous for the transgene that expresses ere recombinase. In one embodiment, males of one of these strains are then injected with ENU and mated to females of the other strain. All of the progeny from crossing these two strains will contain one latent lox-flanked disease-mutation, which will be activated by the action of cre-recombinase and one latent copy of the frt-flanked disease-causing mutation that will be activated by flp-recombinase. GI animals can then be monitored to identify those individuals that do not succumb. By monitoring the proportion of mice with a suppressed disease phenotype among the various test crosses one skilled in the art should be able to determine whether phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner. Where disease suppression is found to be heritable, the relevant ENU-induced mutation may be mapped and identified by conventional genetic and genomic methods, such as those described herein.

This basic principle of creation of animals with latent disease-causing mutations and crossing them to animals with the required recombinase to activate the mutation can be extended to more complicated diseases that require two or more different types of mutation in order to manifest.

Many mouse models of human disease can be utilized using the methods revealed and summarized in Scenario 1 some are listed in Table 2 and 2a, while one skilled in the art would recognise that many others can be found in publicly available databases (e.g. http://research.bmn.com/mkmd, http://informatics.jax.org). It can also be appreciated that mice in which the disease-causing mutation creates a mild reduction in health or fertility might be utilized using methods described in Scenario 1 or Scenario 3 or alternatively Scenario 2 or 3.

In still further embodiments, strategies outlined as follows are contemplated in some embodiments in which the disease or condition is experimentally induced (Scenario 4). Some mouse models of human, disease rather than occurring spontaneously in mutant animals must be induced experimentally either in wild type mouse strains or genetically modified mice. Examples of induced diseases include without limitation ischaemic injury, inflammation and infection. In accordance with this embodiment, animals of the salient genetic background are treated with ENU and mated to the required mice to generate GI animals of a genetic background known to be susceptible to the induction of the disease. Once GI animals have reached the required age, the disease is induced and its course is monitored to allow those mice in which the disease course has been ameliorated to be identified. By monitoring the proportion of mice with an ameliorated physiological assessable symptom (suppressed disease phenotype) among the various test crosses, one skilled in the art is able to determine whether the phenotype of disease suppression is heritable and if so, whether suppression is inherited in a dominant, semi-dominant or recessive manner. Where disease suppression is found to be heritable, the responsible ENU-induced mutation is mapped and identified by conventional genetic and genomic methods, such as those described herein.

In one illustrative embodiment, the physiologically assessable animal model of a human disease is a mouse model of thrombocytopenia which lacks normal TPO receptor signalling through Mpl and exhibits subnormal platelet levels and/or qualitative changes in megakaryocytopoiesis. As described in the Examples, male c-Mp '^' mice were subjected to ENU mutagenesis, bred with female c-Mpl'^' mice and their progeny assessed for platelet numbers. GI mice showing elevated platelet levels were mated to untreated mice of the same genetic background and their G2 progeny assessed for platelet levels to determine if the altered phenotype is heritable.

Alternative models of thrombocytopenia include vertebrate animals in which c-Mpl is down regulated or inhibited. Such inhibition may be mediated for example using antibodies to the thrombopoietin (TPO) receptor. Mutagenesis may also be conducted on the wild type precursor of the animal model which is subsequently transformed or its progeny are transformed into a thrombocytopenia model by administration of an inhibitory anti-Mpl antibody or receptor antagonist. Inhibitory agents other than antibodies are contemplated such as antisense or co-suppression molecules or constructs which effectively down regulate Mpl activity including it's downstream signalling activity in the model.

Accordingly, another embodiment of the present invention provides a physiological assessment system to identify a pedigree of a vertebrate animal model of thrombocytopenia which exhibits ameliorated symptoms of thrombocytopenia comprising:

(i) assessing the symptoms thrombocytopenia in the model of thrombocytopenia and/or its progeny wherein the rodent model also comprises mutations induced by random or non-random mutagenesis; and

(ii) selecting animals in which the symptoms of thrombocytopenia are ameliorated and determining that the responsible mutation is heritable.

Progeny are preferably GI, G2 or G3 progeny and subsequent progeny generated by breeding GI, G2 or G3 animals.

Various rodent models of thrombocytopenia are contemplated exhibiting altered platelet numbers compared to normal levels, or qualitative changes in megakaryocytopoiesis. Such pedigrees are useful for defining further members of the TPO/Myb pathway as defined herein and other pathways involved in megakaryocytopoiesis. Preferably, models include mutants with, inter alia, recessive thrombocytopenia on a Mpf ⁺ background (Pltl, Figure 10a), a mutant with recessive thrombocytosis on a Mp ^/+ background (Plt2), a mutant with recessive exacerbation of thrombocytopenia (mldl), and a mutant with dominant alterations in platelet granularity.

A preferred rodent model of thrombocytopenia is a Mpl'^' rodent or a rodent in which Mpl is down regulated or inhibited. Regulation may be achieved using inducible promoters to inhibit or knock out all or part of Mpl. Inhibition may be mediated for example using antibodies to the thrombopoietin (TPO) receptor.

In accordance with one aspect of the present invention one or more of Plt3, Plt4 and Plt6 rodent pedigrees are disclosed each exhibiting elevated platelet levels.

The animal models of the present invention may be in the form of the animals or may be, for example, in the form of embryos stem cells or gamete for transplantation. The embryos satem cells or gametes are preferably maintained in a frozen state and may optionally be sold with instructions for use.

The present invention provides in one embodiment an assessment system to identify genetic or proteinaceous drug targets associated with elevating platelet levels or ameliorating the symptoms of thrombocytopenia comprising:

(i) assessing symptoms of thrombocytopenia in a vertebrate model of said disease or condition and its progeny wherein the model animal also comprises mutations induced by random or non-random mutagenesis;

(ii) selecting animals in which the symptoms of thrombocytopenia are ameliorated and determining that the responsible mutation is heritable: and

(iii) positional cloning to identify the genetic sequences flanking the mutation and any encoded product.

The present invention also provides an assessment system to identify genetic or proteinaceous drug targets associated with elevating platelet levels or ameliorating the symptoms of thrombocytopenia comprising:-

(i) subjecting a Mpl^"7" rodent or a rodent in which Mpl is, or will be, down regulated or inhibited to mutagenesis;

(ii) assessing platelet levels or symptoms of thrombocytopenia in the mutated rodent and its progeny wherein if a wild type rodent is used in step (i) these animals are subjected to inhibition or down regulation of Mpl activity prior to assessment;

(iii) verifying that the phenotype of the affected progeny is hereditable; and (iv) positional cloning to identify the genetic sequences flanking the mutation and any encoded product.

Accordingly, the present invention provides a rodent or mouse model with reduced Mpl levels when used to screen for drug targets to treat thrombocytopenia or other conditions characterised by low platelet levels.

Progeny are preferably GI, G2 or G3 progeny and subsequent progeny generated by breeding GI, G2 or G3 mice.

The present invention further provides an assessment system to identify genetic or proteinaceous targets associated with elevating platelet levels or ameliorating the symptoms of thrombocytopenia comprising:

(i) subjecting a Mpl-/- mouse or a mouse in which Mpl is down regulated or inhibited to mutagenesis;

(ii) assessing platelet levels or symptoms of thrombocytopenia in the mutated mouse and its GI progeny;

(iii) verifying that the phenotype of the affected GI progeny is heritable; and

(iv) positional cloning to identify the genetic sequences flanking the mutation and any encoded product.

Any convenient and well characterized rodent strain or intercross, backcross or outbred derivatives of such strains may be used to develop the model of thrombocytopenia such as, for example, C57B1/6 or C3H strains. A wide range of mouse strains is described in, for example, The Jackson Laboratory website. Accordingly, the present invention provides genetic or proteinaceous drug targets identified using the instant physiological assessment method, or derivatives, variants, functional derivatives, or homologs thereof as a drug target for use or when used, or to screen for or develop agonists or antagonist compounds useful, in the treatment of the symptoms of a particular disease of condition. Where down regulation of a drug target ameliorates a disease or condition, agonists of inhibitory interacting molecules in the genetic net work of the target will also be therapeutic or targets for development of therapeutics and are expressly encompassed, where appropriate.

Drug target are particularly contemplated for use, or when used, to screen for or develop interacting compounds useful in the treatment and/or prophylaxis of thrombocytopenia or other conditions characterized by low platelet levels.

The present invention particularly provides a genetic or proteinaceous form of Myb transcription factor signaling pathway and other interacting members of the genetic network comprising Myb or derivatives thereof, variants, functional derivatives, homologs thereof as a drug target for use in screening or developing interacting compounds useful in the treatment of thrombocytopenia or other conditions characterized by low platelet levels.

Without intending to be bound by any particular theory or mode of action, as shown herein the inhibition or down regulation of Myb expression or activity may be a critical function of the TPO signalling pathway which is necessary for effective megakaryocytopoiesis. Specifically, mutations in Myb cause a myeloproliferative syndrome and supra- physiological expansion of megakaryocyte and platele production in Λ ^^'mice.

SEQ ID NO: 1 represents the nucleotide sequence encoding murine Myb. SEQ ID NO: 2 represents the amino acid sequence of murine Myb. SEQ ID NO: 3 represents the nucleotide sequence encoding human Myb. SEQ ID NO: 4 represents the amino acid sequence of human Myb. The term "Myb" is used herein to refer to nucleic acid and/or proteinaceous forms of Myb as the context in which the term is used makes clear. In its broadest form, the term encompasses Myb from any organism but preferably a vertebrate organism.

Functional derivatives of target molecules in nucleic acid form include nucleic acid molecules comprising a nucleotide sequence capable of hybridising to the target molecule or its complementary form under low stringency conditions.

In one aspect the present invention contemplates a drug target for use in the identification of antagonists or agonists which effectively up regulate platelet levels or ameliorate the symptoms of thrombocytopenia in a subject. As previously stated, the functional activity of a target molecule may be modulated by agonising or antagonising the target in genetic or proteinaceous form.

Where down regulation of Myb ameliorates thrombocytopenia, agonists of inhibitory interacting molecules in the Myb genetic network will also be therapeutic or targets for the development of therapeutics and are expressly encompassed.

The present invention also provides methods of screening for agonists or antagonists of the identified drug targets comprising contacting the drug target with a compound and assaying for (i) the presence of a complex between the drug target and a compound or (ii) for the presence of complex between the drug target and a ligand, by methods well known in the art. In such competitive binding assays the drug target or the ligand is labelled in order to assess the activity of the compound.

The present invention also provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target to a compound and assaying for:-

(i) the presence of a complex between the compound and the drug target; or (ii) a change in the interaction between the drug target and a ligand, binding partner or other interacting molecule; or

(iii) a change in the level of an indicator of the activity of the drug target.

The ability of a compound to modulate the activity of a target molecule may be tested in in vitro assays. Target molecule may be expressed recombinantly or occur naturally or be upregulated in cells or cell lines which are useful in in vitro screens for agonists or antagonists.

Natural products, combinatorial synthetic/peptide, polypeptide or protein libraries or phage display technologies are all available in the art for screening for modulators. A huge choice of high throughput screening methods are also available. Natural products include those from coral, soil, plant or the ocean or antarctic environments.

Two-hybrid screening is another useful method for identifying other members of a genetic network associated with or comprising a drug target. Target interactions and screens for inhibitors can be carried out using the yeast two-hybrid system, which takes advantage of transcriptional factors that are composed of two physically separable, functional domains. The most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA binding domain and a transcriptional activation domain. Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and if interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable phenotype. In the present case, for example, S. cerevisiae is co-transformed with a library or vector expressing a cDNA GAL4 activation domain fusion and a vector expressing a target pathway component fused to GAL4. If lacZ is used as the reporter gene, co- expression of the fusion proteins will produce a blue colour. Small molecules or other candidate compounds which interact with a target will result in loss of colour of the cells. Reference may be made to the yeast two-hybrid systems as disclosed by Munder et al, (Appl. Microbiol Biotechnol 52(3): 311-320, 1999) and Young et al, (Nat. Biotechnol 16(10): 946-950, 1998). Molecules thus identified by this system are then re-tested in animal cells.

In another aspect, the present invention provides a method for the treatment or prophylaxis of a disease or condition comprising administering a therapeutic amount of a compound which modulates the activity of a herein described target molecule in genetic or proteinaceous form.

In another aspect the present invention provides a method for the treatment or prophylaxis of thrombocytopenia comprising administering a therapeutic amount of a compound which modulates the activity of herein described target molecule.

The present invention provides a method for identifying compounds useful in the treatment or prophylaxis of thrombocytopenia comprising screening and/or developing compounds for their ability to modulate the functional activity of the herein disclosed target molecules.

In accordance with the present invention there is provided a drug target comprising a sequence of amino acids set forth in SEQ ID NO: 2 or SEQ ID NO: 4 or a functional derivative or homolog thereof having at least 60% similarity thereto or a sequence of nucleotides encoding SEQ ID NO: 2 or SEQ ID NO: 4 or as set forth in SEQ ID NO:l or SEQ ID NO: 3 or a functional derivative or homolog having at least 60% similarity thereto or a sequence of nucleotides capable of hybridising to SEQ ID NO: 1 or SEQ ID NO: 3 its complement under conditions of low stringency hybridisation when used to screen for antagonists or antagonists which elevate platelet levels or ameliorates the symptoms of thrombocytopenia.

In a further aspect, domains of Myb which contribute to its functional effect on megakaryocytopoiesis are specifically targeted. For example, the Plt3 mutation occurs in the sequence of nucleotides encoding the DNA binding domain and the Plt4 mutation occurs in the sequence of nucleotides encoding the leucine zipper domain. As will be apparent to one of skill in the art, the subject antagonists or agonists compounds elevate platelet levels in vivo and/or in vitro.

Accordingly, the present invention provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to modulate the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.

Accordingly, the present invention provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to antagonise the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.

Loss of Myb is lethal in fetal life resulting in a dyplastic state in which platelet formation is excessive but red cell and B-lymphocytic formation is reduced. Advantageously, the phenotypic abnormalities associated with antagonising the functional activities of Myb specifically in the DNA binding domain and leucine zipper domain are recessive except those in the platelet/megakaryocyte lineage.

The present invention provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to agonise the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.

The present invention also provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to antagonise the functional activity of genetic or proteinaceous forms of c-Myb.

The present invention also provides a method for identifying compounds useful in the treatment of thrombocytopenia or conditions characterized by low platelet levels comprising screening compounds for their ability to agonise the functional activity of genetic or proteinaceous forms of c-Myb.

The target molecules are usefully in isolated or recombinant form for screening purposes. Accordingly, the present invention further provides recombinant nucleic acids including a recombinant construct comprising all or part of the drug target in nucleic acid form. The recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosonal DNA of the host cell. Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semi-synthetic or synthetic origin which, by virtue of its origin or manipulation: (i) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (ii) is linked to a polynucleotide other than that to which it is linked in nature; or (iii) does not occur in nature. Where nucleic acids according to the invention include RNA, reference to the sequence shown should be construed as reference to the RNA equivalent with U substituted for T. Reporter constructs are useful where the target molecule comprises promoters or enhancers.

The isolated or recombinant drug targets of the instant invention are used to identify or engineer agonists and antagonists. Such modulators may be used directly or they may be further modified by methods well known in the art in order to improve their effectiveness as pharmaceutical, diagnostic or other reagents. Other considerations for an active compound include formulation and method of delivery.

An agonist or antagonist includes molecules determined by all or part of the drug target or a variant of the drug target such as antibodies, mimetics or antisense molecules.

Antibodies including anti-idiotypic antibodies, chaemeric antibodies and humanised antibodies are useful in this regard and their generation is now routine to those of skill in the art. Peptide or non-peptide mimetics can be developed as agonists of the drug targets by identifying those residues of the target molecule which are important for function. Modelling can be used to design molecules which interact with the target molecule and which have improved pharmacological properties.

Antisense polynucleotide sequences are another useful example of a therapeutic agent which can modulate the activity of target molecules, as will be appreciated by those skilled in the art. Polynucleotide vectors, for example, containing all or a portion of Myb sequences or other sequences from an Myb region (particularly those flanking a Myb gene locus) may be placed under the control of a promoter in an antisense orientation and introduced into a cell. Expression of such an antisense construct within a cell will interfere with gene transcription and/or translation. Furthermore, co-suppression and mechanisms to induce RNAi (i.e. siRNA) may also be employed. Such techniques may be useful to inhibit genes which positively promote target molecule gene expression and particularly Myb gene expression. Alternatively, antisense or sense molecules may be directly administered. In this latter embodiment, the antisense or sense molecules may be formulated in a composition and then administered by any number of means to target cells.

A variation on antisense and sense molecules involves the use of morpholinos, which are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages (for example, Summerton and Weller, Antisense and Nucleic Acid Drug Development, 7:187-195, 1997). Such compounds are injected into embryos and the effect of interference with mRNA is observed.

In one embodiment, the present invention employs compounds such as oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding a target molecule, i.e. the oligonucleotides induce transcriptional or post- transcriptional gene silencing. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding the transcription factor. As used herein, the terms "target nucleic acid" and "nucleic acid molecule encoding a transcription factor" have been used for convenience to encompass DNA encoding a target proteinaceous molecule, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of the subject invention with its target nucleic acid is generally referred to as "antisense". Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as "antisense inhibition." Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of a target gene. In the context of the present invention, "modulation" and "modulation of expression" mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are "DNA-like" elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals.

While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocychc base. The two most common classes of such heterocychc bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphoro-thioates, phosphoro-dithioates, phosphotri-esters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates, 5'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3' -amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3 '-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having inverted polarity comprise a single 3' to 3' linkage at the 3 '-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

Many of the preferred features described above are appropriate for sense nucleic acid molecules.

The goal of rational drug design is to produce structural analogs of biologically active polypeptides of interest or of small molecules with which they interact (e.g. agonists, antagonists, inhibitors or enhancers) in order to fashion drugs which are, for example, more active or stable forms of the polypeptide, or which, e.g. enhance or interfere with the function of a polypeptide in vivo. See, e.g. Hodgson (Bio/Technology, 9:19-21, 1991). In one approach, one first determines the three-dimensional structure of a protein of interest by x-ray crystallography, by computer modeling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al, Science, 249: 527-533, 1990). In addition, target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol, 202: 2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide 's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide. It is also possible to isolate a target-specific antibody, selected by a functional assay and then to solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore.

Analogs contemplated herein include but are not limited to modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogs.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH .

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 2b.

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of C_α and N _α-methylamino acids, introduction of double bonds between C_α and C_β 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 provides methods for screening for agonists or antagonists of the identified drug targets comprising contacting the drug target with a compound and assaying for (i) the presence of a complex between the drug target and a compound or (ii) for the presence of complex between the drug target and a ligand, by methods well known in the art. In such competitive binding assays the drug target or the ligand is labelled in order to assess the activity of the compound.

The present invention also provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target to a compound and assaying foπ-

(iv) the presence of a complex between the compound and the drug target; or

(v) a change in the interaction between the drug target and a ligand, binding partner or other interacting molecule; or

(vi) a change in the level of an indicator of the activity of the drug target.

The present invention further provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target comprising all or part of Myb or a nucleotide sequence encoding same to a compound and assaying for:-

(i) the presence of a complex between the compound and said drug target; or

(ii) a change in the interaction between said drug target and a ligand, binding partner or other interacting molecule; or

(iii) a change in the level of an indicator of the activity of said drug target. The present invention furthermore provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target comprising all or part of the DNA binding domain of Myb or a nucleotide sequence encoding same to a compound and assaying for:-

(i) the presence of a complex between the compound and said drug target; or

(iii) a change in the level of an indicator of the activity of said drug target.

The present invention provides a method for screening for agonists or antagonists of drug targets identified herein comprising exposing the drug target comprising all or part of the leucine zipper domain of Myb or a nucleotide sequence encoding same to a compound and assaying for:-

(i) the presence of a complex between the compound and said drug target; or

Natural products, combinatorial, synthetic/peptide polypeptide or protein libraries or phage display technologies are all available for screening for modulators. A huge choice of high through put screening methods are available. Natural products include those from coral, soil, plant or the ocean or antarctic environments.

Two-hybrid screening is also useful in identifying other members of a genetic network associated with or comprising a drug target. Target interactions and screens for inhibitors can be carried out using the yeast two-hybrid system, which takes advantage of transcriptional factors that are composed of two physically separable, functional domains. The most commonly used is the yeast GAL4 transcriptional activator consisting of a DNA binding domain and a transcriptional activation domain. Two different cloning vectors are used to generate separate fusions of the GAL4 domains to genes encoding potential binding proteins. The fusion proteins are co-expressed, targeted to the nucleus and if interactions occur, activation of a reporter gene (e.g. lacZ) produces a detectable phenotype. In the present case, for example, S. cerevisiae is co-transformed with a library or vector expressing a cDNA GAL4 activation domain fusion and a vector expressing a Myb pathway component fused to GAL4. If lacZ is used as the reporter gene, co- expression of the fusion proteins will produce a blue colour. Small molecules or other candidate compounds which interact with a target will result in loss of colour of the cells. Reference may be made to the yeast two-hybrid systems as disclosed by Munder et al, (supra, 1999) and Young et al, (supra, 1998). Molecules thus identified by this system are then re-tested in animal cells.

Another aspect of the subject invention provides a method for the treatment or prophylaxis of thrombocytopenia comprising administering a therapeutic amount of a compound which modulates a drug target identified by the herein disclosed physiological assessment system.

In a preferred embodiment the present invention provides a method for the treatment or prophylaxis of thrombocytopenia comprising administering a therapeutic amount of a compound which modulates one or more components of the Myb transcription factor signalling pathway to effectively modulate the activity of Myb. As TPO appears to be a part of the Myb signally pathway and is already proposed for use in ameliorating thrombocytopenia this use of TPO is not proposed herein to part of the present invention.

The subject invention presents a method for the treatment of thrombocytopenia or conditions characterized by low platelet numbers comprising administering a therapeutic amount of a compound which antagonises or agonises one or more components of genetic or proteinaceous forms of a Myb transcription factor signalling pathway to effectively down regulate the activity of Myb.

The subject invention further presents a method for the treatment of thrombocytopenia or conditions characterized by low platelet numbers comprising administering a therapeutic amount of a compound which antagonises or agonises one or more components of the Myb transcription factor signalling pathway to effectively up-regulate the activity of Myb.

The present invention provides pharmaceutical compositions comprising recombinant synthetic or isolated forms of the present drug targets and one or more pharmaceutically acceptable carriers, diluents or excipients and furthermore contemplates methods of their use in vitro or in vivo in methods for the treatment or prophylaxis of subjects and in particular humans with or likely to develop the symptoms of thrombocytopenia.

The present invention contemplates the use of a modulator of the Myb transcription factor signaling pathway in the manufacture of a pharmaceutical composition for the treatment of thrombocytopenia.

In another embodiment the present invention provides the use of an antagonist of Myb in the manufacture of a pharmaceutical composition for the treatment of thrombocytopenia.

It should also be understood that the present invention is directed to the treatment of the symptoms of thrombocytopenia or the elevation of platelets in any organism of commercial or humanitarian interest including, primates, livestock animals including fish and birds, laboratory test animals, companion animals, or captive wild animals. The present invention extends to the elevation of platelet levels in vitro or ex vivo.

The polypeptides, nucleic acids, antibodies, peptides, chemical analogs, agonists, antagonists or mimetics of the present invention can be formulated in pharmaceutic compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18^th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.). The composition may contain the active agent or pharmaceutically acceptable salts of the active agent. These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.

For oral administration, the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions. In preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, suspending agents, and the like in the case of oral liquid preparations (such as, for example, suspensions, elixirs and solutions); or carriers such as starches, sugars, diluents, granulating agents, lubricants, binders, disintegrating agents and the like in the case of oral solid preparations (such as, for example, powders, capsules and tablets). Because of their ease in administration, tablets and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. If desired, tablets may be sugar-coated or enteric-coated by standard techniques. The active agent can be encapsulated to make it stable to passage through the gastrointestinal tract while at the same time allowing for passage across the blood brain barrier. See for example, International Patent Publication No. WO 96/11698. For parenteral administration, the compound may dissolved in a pharmaceutical carrier and administered as either a solution or a suspension. Illustrative of suitable carriers are water, saline, dextrose solutions, fructose solutions, ethanol, or oils of animal, vegetative or synthetic origin. The carrier may also contain other ingredients, for example, preservatives, suspending agents, solubilizing agents, buffers and the like. When the compounds are being administered intrathecally, they may also be dissolved in cerebrospinal fluid.

The active agent is preferably administered in a therapeutically effective amount. The actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, supra.

Administration of the modulatory agent, in the form of a pharmaceutical composition, may be performed by any convenient means. The modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about O.lmg, 0.2mg, 0.3mg, 0.4mg, 0.5mg, 0.6mg, 0.7mg, 0.8mg. 0.9mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, com starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant.

Alternatively, targeting therapies may be used to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable for a variety of reasons, e.g. if the agent is unacceptably toxic or if it would otherwise require too high a dosage or if it would not otherwise be able to enter the target cells.

Instead of administering these agents directly, they could be produced in the target cell, e.g. in a viral vector such as described above or in a cell based delivery system such as described in U.S. Patent No. 5,550,050 and International Patent Publication Nos. WO 92/19195, WO 94/25503, WO 95/01203, WO 95/05452, WO 96/02286, WO 96/02646, WO 96/40871, WO 96/40959 and WO 97/12635. The vector could be targeted to the target cells or expression of expression products could be limited to specific cells, stages of development or cell cycle stages. The cell based delivery system is designed to be implanted in a patient's body at the desired target site and contains a coding sequence for the target agent. Alternatively, the agent could be administered in a precursor form for conversion to the active form by an activating agent produced in, or targeted to, the cells to be treated. See, for example, European Patent Application No. 0 425 731 A and International Patent Publication No. WO 90/07936.

The genetic or proteinaceous targets of the present invention may also have a range of diagnostic utilities. For example, the detection or level of the target molecule or an aberrant form thereof is indicative a subjects propensity to develop thombocytopenia or response to therapy.

Any number of methods may be employed to detect target molecules. Immunological testing is one particular method. Accordingly, the present invention extends to antibodies and other immunological agents directed to or preferably specific for the mammalian transcription factors or a fragment thereof. The antibodies may be monoclonal or polyclonal or may comprise Fab fragments or synthetic forms.

Specific antibodies can be used to screen for the subject target molecules and/or their fragments. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies referred to above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti- immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of the mammalian transcription factors.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the subject target molecules or antigenic fragments thereof and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of subject polypeptide, or antigenic parts thereof, collecting serum from the animal and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates, therefore, a method for detecting a target molecule such as Myb or a member of the Myb transcription factor signalling pathway or fragment thereof in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for said Myb or a member of the Myb transcription factor signalling pathway or fragment thereof or its derivatives or homologs for a time and under conditions sufficient for an antibody-polypeptide complex to form, and then detecting said complex.

The presence of the instant target molecules or their fragments may be detected in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Patent Nos. 4,016,043, 4,424,279 and 4,018,653.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabeled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labeled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labeled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labeled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain a subject target molecule including blood, bone marrow, spleen . The sample is, therefore, generally a biological sample comprising biological fluid. The Myb transcription factor for example is likely to be in blood.

In the typical forward sandwich assay, a first antibody having specificity for the instant polypeptide or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or where more convenient, overnight) and under suitable conditions (e.g. for about 20°C to about 40°C) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labeled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labeled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

The present invention also contemplates genetic assays such as involving PCR analysis to detect RNA expression products of a genetic sequence encoding a target molecule. The genetic assays may also be able to detect nucleotide polymorphisms or other substitutions, additions and/or deletions in the nucleotide sequence of a mammalian transcription factor. Changes in levels of target molecule expression such as following mutations in the promoter or regulatory regions or loss of target activity is proposed to be indicative of a disease condition or a propensity for a disease condition to develop. For example, a cell biopsy could be obtained and DNA or RNA extracted. Alternative methods which may be used alone or in conjunction with other methods include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP) as well as specific oligonucleotide hybridization, denaturing high performance liquid chromatography, first nucleotide change (FNC) amongst others.

The present invention extends to polymorphisms in Myb genetic sequence genes which leads to alleviation of the symptoms of thrombocytopenia. For example a mutation at about nucleotide A455 or Al 151 in the Myb gene leads to amelioration of the symptoms of thrombocytopenia.

The present invention further contemplates kits to facilitate the rapid detection of the target molecules or their fragments in a subject's biological fluid. A biological fluid includes a cell extract such as a DNA/RNA extract.

The present invention is further described by the following tables and non-limiting Examples. TABLE 1 Summary of sequence identifiers

Table 2

Table 2a Mouse Models Of Human Disease that are amenable to the suppressor screen strategy

Disease Mouse Model Scenario Reference typel von Willebrand disease Vwf+/- 2 Denis C., et al. type 3, von Willebrand disease Vwf-/- l and 3 Denis C, et al. retinitis pigπxeπtosa Pdeb rd/rd 1 Hafezi F. et al. obesity The Pomcl(-/-) 1 and 3 Yaswen L. et al. mucopolysaccharidosis l. type VII Gus mps2J/mps2J 3 Birkeorneier E. H. et a

Alport syndrome Col4a3-/- 1 and 3 Cosgrove D. et al anxiety-related disorder Htrla-/- 1 Ramboz S. etal anxiety-related disorder Htrla+/- 2 Ramboz S. et al mucopolysaccharidosis type VI (Maroteaux-Lamy Asrb-/- l and 3 Strauch O. F. etal syndrome). atherosclerosis ApoE-/- l and 3 Weng S. et l Glanzraann Itgb3-/- l nd 3 Weng S. et l thrombasthenia Gerstmann-Straussler- Pmp l and 3 Barron R. M. et al Scheinker syndrome tmlEdin/tmlEdin severe congenital malformation and Gonzalez-Iriarte M. et Hspg2 -/- 3 coronary artery al. abnormalities Table 2a continued

Disease Mouse Model Scenario Reference obsessive-compulsive Htr2c-/- disorders l and 3 Chou-Green J. M. et al lymphedema-distichiasis Foxc2 +/- 2 and 3 Kriederman B. M. et al Pompe disease Gaa -/- 1 and 3 Bijvoet A. G. et al. multiple sclerosis Ptprz -/- 1 Harroch S. et al hereditary hemorrhagic telangiectasia type 2 Acvrl +/- 2 and 3 Srinivasan S. e/ α/. disorder neuronal degeneration Ttpa -/- 1 Yokota T. et /., type II Bartter's syndrome Kcnjl -/- 3 Lorenz J. N. et al Tuberous sclerosis Tscl-/- l and 3 Kwiatkowski D. J. et al split hand/foot Dlx5-/- D 6-/- 3 Merlo G. R. et al. malformation type I

TABLE 2b

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine N arg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbomyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-meώylornithine Nmom

D-histidine Dhis L-N-methylphenylalanine N phe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-metWonine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dom L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle TABLE 2b continued

Non-conventional Code Non-conventional Code amino acid amino acid

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-memylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-me ylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dragln N-(2-aminoethyl)glyc ne Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Nom

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap

D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbarnylethyl)glycine Ngln

D-α-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanirie Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-me ylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-memylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-me ylarginine Dnmarg N-cycIopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund TABLE 2b continued

Non-conventional Code Non-conventional Code amino acid amino acid

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine Nbhe

D-N-methylglutamine Dnmgln N-(3-g^anidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dmnhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyI))gIycme Nbis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu

N-methylcyclohexylalanine Nmchexa D-N-memylmemionine Dnmmet

D-N-me ylomitib πe Dnmom N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-memylphenylalanine Dnmphe

N-methylarainoisobutyrate Nmaib D-N-raethylproline Dnmpro

N-(l-methylρropyl)glycine Nile D-N-methylserine Dπmser

N-(2-methylpropyl)glycine Nleu D-N-methylthxeonine Dnmthr

D-N-methyltryptophan Dnmtxp N-(l-methyle hyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-memyla-ωpΛylalanine Nmanap

D-N-methylvaline Dnmval N-raethylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-r-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenyl anine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L- -methylasparagine Masn

L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu TABLE 2b continued

Non-conventional Code Non-conventional Code amino acid amino acid

L-α-methylhistidine Mhis L-α-memylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet

L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-raethylmethionine Mmet L-α-methylnorieucine Mnle

L-α-methylnorvaline Mnva L-α-memylormthine Morn

L-α-methylphenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr

L-α-metøyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethy glycine carbamylmethyl)glycine

1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylarnino)cyclopropane

Table 3 SSLP Markers Used For Localization Of The W Mutation

c/> c/>

m

C/> m m

m σ>

TJ O

Table 3 continued

C/> m m

m σ>

TJ O

Table 3 continued

C/> m m

m σ>

TJ O

Table 3 continued

C/> m m

m σ>

TJ O

Table 3 continued

C/> m m

m σ>

O

Table 3 continued

C/> m m

m σ>

O

Table 3 continued

C/> m m

m σ>

O

Table 4 SSLP Markers Used For Localization Of The PU3 Mutation

C/> m m

m σ>

O

Table 5 Primers used to PCR Amplify and Sequence the Exons Of The Myb Gene

C/> m m

m σ>

O

Table 6 continued

m σ>

TJ O

Table 7. Enumeration of hematopoietic progenitor cells

Table 7 continued

Table 7 continued

Table 7 continued

Table 8. Megakaryocyte Counts

Genotype Number of megakaryocytes per high power field Bone Marrow Spleen Mpl* Myb⁺⁺ 0.8 ± 0.4 0.05 ± 0.08

Mpl-^ΛMyb^pl,3p'^t3 7.0 ±1.0 1.1 ±0.1 Mpr-Myb^p,t3+ 1.7 ±0.5 0.05 ± 0.07

M_Pr^/-Myb^p,,4/PU4 12.3 ± 3.8 4.3 ±2.2 M_Pr-Myb^PU4/+ 3.6 ±1.2 0.2 ±0.2

Mpl⁺⁺ Myb ⁺⁺ 6.2 ± 2.0 0.3 ±0.1

Mpl⁺ Myb^PU/p,t4 7.7 ±1.7 6.5 ±3.1

Table 9. Bone Marrow Megakaryocyte Ploidy

Genotype Percentage of megakaryocytes in ploidy class 2N 4N 8N 16N 32N Mpr Myb"* 7.26 ± 3.43 6.51 ±1.64 25.28 ±1.98 51.3713.39 9.0013.39

M r'-M b ™ 8.9 11.61 11.64 ±0.28 47.64 ± 4.52 29.90 ± 2.47 3.13 ± 0.28 Mpl^"'^" Myb^M,3+ 6.24 ±1.87 7.5412.14 41.3014.38 40.25 ±3.91 3.92 ± 2.09

Mpl-^/-M ^p,t4^^rt 9.56 ±2.47 18.47 ±0.92 47.46 ±2.78 22.1713.20 2.5410.55 Mpl^"'- M * 7.57 ±1.01 7.39 ± 0.78 29.03 ± 2.47 41.87±4.13 7.57 ±1.46

9.69 ±3.29 6.8911.66 10.44 ±0.98 50.76 ± 6.66 20.63 ± 2.27

Mpl^Myb^plt4Λ"^M 10.58 18.64 46.33 21.90 2.81

Table 10. CFU-s in Pit mutants

Genotype Number of bone marrow CFU-s

MprMyb⁺⁺ 4.3 ± 0.9 Mpl--Myb^Plt3/p,t3 13 Mpl--Myb^p,t3+ 10.25 M_Pr^/Myb^p,t4PU4 8.5 ± 0.7 Mpr Myb^p,t4+ 10.4±1.7

Mpl⁴ Myb ^+/+ 11.9±L0 Mpl⁺⁺ Myb^p 11.1 ±0.4

Table 14. Increased erythroid cells (Terll9+) in Pit mutant spleens Genotype Proportion of Cells Terll9+ Bone Marrow Spleen M_Pr-Myb^+/+ 36.2 ±4.3 9.4 ±5.9

M_Pr-Myb^rø/pω 32.7 ±1.1 35.9 ±3.1 Mpl^ yb"⁰* 30.8 ±1.7 10.4 ±1.6

M_Pr^ΛMyb ^plt4 40.9 ±5.9 38.6 ±14.6 Mpr^ΛMyb^p,,4/+ 31.6 ±3.5 8.5 ±2.9 Mpl^+/+Myb^+/+ 28.5 ±3.9 8.8 ±4.8

Mpl^+/+Myb^p 30.0 ±1.2 35.6 ±7.7

Table 16. Elevated CFU-e in spleens of Pit mutants

cu-e per 10 cells Bone Marrow Spleen

Mpl+/+Myb+/+ 259 ± 73 17 ± 23

Mpl -/- Myb +/+ 231 ± 76 3 ± 3

Mpl -/- Myb Plt3/Plt3 232 ± 122 155 ± 143 Mpl -I- Myb Plt3/+ 268 ± 79 5 ± 9

Mpl-/-MybPlt4Plt4 212 ± 109 154 ± 92 Mpl -/- Myb PU4/+ 308 ± 117 7 ± 9

Mpl+/+MybPlt4Plt4 176 ± 86 61 ± 63

Table 17. Hematopoietic stem cells in Pit mutant spleens

Genotype Frequency of CRU in Bone 95% Confidence Interval Marrow Mpl^"'" Myb⁺'⁺ 1 in 124,825 1 in 58,102 to l in 268,173 Mpl^7" Myb^{p,M plt4} 1 in 142,762 1 in 70,688 to 1 in 288,322 Mpl^"'^" Myb¹ 1 in 71,207 1 in 34,583 to 1 in 146,616 Mρl^w My +/+ 1 in 29,005 1 in 14,920 to 1 in 56,389

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EXAMPLE 1 MpT'^~ mice as a model for Thrombocytopenia

The Mpl^1' mouse model for thrombocytopenia has a deletion of the receptor, termed Mpl, for thrombopoietin which is the major platelet producing hormone. Mpl'^' mice have reduced levels (5 to 10% of the normal number) of circulating platelets (1.2xl0⁵+/- 5xl0⁴ /μl n=l 120 compared with 1.2xl0⁶ +/- 2.04xl0⁵ /μl, n=55; see Figure 1A and IB). This is an excellent model of human thrombocytopenia since families with mutations in the Mpl gene have also been found (van den Oudenrijn S., et al, British Journal of Haematology, 2000).

EXAMPLE 2 ENU Treatment and Generation of Mutant Mice

Male Mpl'^' mice were treated with N-Ethyl-N-Nitrosourea (ENU) according to the method of Bode (Bode, 1984). Briefly, ENU (N3385, Sigma Chemical Company) was dissolved in 5 ml of ethanol and diluted with 50 mM sodium citrate pH 5.0 and was used within four hours. The concentration of the ENU was determined spectrophotometrically at 395 nm. Male mice were then injected intraperitoneally with one dose of 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg or 400 mg/kg, two weekly doses of 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg or 200 mg/kg or three weekly doses of 66 mg/kg, 83 mg/kg, 100 mg/kg, 116 mg/kg or 133 mg/kg. Four weeks after the final injection, ENU-treated mice were mated with one or two female Mpl'^' mice. Following a period of sterility, the length of which increases as the total dose of ENU increases, first-generation (GI) progeny were produced. At 7 weeks of age, blood from the retro-orbital plexus using a capillary tube was collected into tubes containing potassium EDTA (Sarstedt, Nϋmbrecht, Germany) and the number of platelets in the peripheral blood was determined using the Advia 120 automated haematological analyser (Bayer, Tarrytown, NY).

GI mice, which had platelet numbers that were greater than 3.00x10⁵/μl (three standard deviation or more the mean value) were tested to determine whether their phenotype is heritable. GI mice were mated to untreated mice of the same genetic background and their G2 progeny were bled at 7 weeks of age and circulating platelet numbers were determined using the Advia Automated Analyser. Initially 10 G2 mice were analysed from each GI mouse. For a fully penetrant dominant phenotype one would expect approximately half of the mice to be affected. Given the likelihood of erroneously discarding a pedigree is less than 1 in 1000 (i.e. 0.5¹⁰ = 0.00098), if none of the 10 progeny exhibited the mutant phenotype then it was concluded that the trait was not heritable and no further experiments were carried out. If one or more mice exhibited the outlying phenotype then additional mice were generated, both by continuing to generate progeny from the original GI mouse or by mating affected G2s to mice of the same genetic background. If consistent heritability of the phenotype was observed then the mutant strain was maintained and expanded for further study.

EXAMPLE 3 Derivation of mice with elevated platelet levels

Mpl'^' mice were treated with the ENU as described above, which results in random mutations being introduced into the DNA of the spermatogonial stem cells and ultimately the sperm (Ranchik Trends in Genetics 7:15-21, 1991). These mice were mated to untreated female Mpl'^' mice to produce first generation (Gi) progeny, which are heterozygous for a set of ENU-induced mutations inherited from their father. At 7 weeks of age GI mice were bled and their peripheral blood platelet count was determined. GI mice (2037 mice) were examined for their platelet levels and of these, 7 exhibited platelet counts of more than 3.00xl0⁵ /μl, that is they had platelet counts of more than 3 standard deviations from the mean of untreated Mpl'^' mice and were hence candidates which might carry an ENU-induced mutation that ameliorated thrombocytopenia (Figure IC).

To determine whether the increase in platelet levels was heritable GI mice with platelets of more than 3.00xl0⁵ /μl were bred to untreated Mpl'^' mice. Of the 7 GI mice with more than 3.00x10⁵ platelets /μl two died prior to producing offspring and three clearly did not carry a mutation capable of reducing the severity of thrombocytopenia since all of their progeny had very low platelet counts characteristic of Mpl'^' mice (data not shown). The remaining two mice (985.34 and 1118.11) did however appear to have heritable suppression of thrombocytopenia since approximately half of their progeny exhibited platelet counts of more than 3.00x10⁵ /μl, while the remaining mice exhibited the very low platelet counts characteristic of Mpl'^' mice (Figure ID); the resulting pedigrees were named Plt3 and Plt4.

EXAMPLE 4 Phenotype ofPU3 and PU4 homozygous mice

Mice heterozygous for the Plt3 and P 4 mutations (Plt3/+ or Plt4/+) exhibited an amelioration of thrombocytopenia to approximately 30% of normal levels. To determine whether Plt3 and Plt4 homozygous mice had platelet levels closer to those seen in wild type Mp '⁺ mice or even higher (Figure 2) Plt3/+ Mpl'^' mice and PU4/+ Mpl'^' mice were intercrossed. Their progeny were bled at 7 weeks of age and their platelet levels were compared with the progeny derived from crossing PU3/+ Mpl'^' or PU4/+ Mpl'^' mice with +/+ Mpl'^' mice. While the latter fell into two populations with respect to platelet numbers, three populations were observed when the progeny of heterozygotes were examined. The first population corresponded to +/+ Mpl'^' mice and exhibited very low platelet counts (< 3.00x10⁵ platelets/μl). The second was consistent with the phenotype seen for heterozygous P 3/+ Mpl'^' mice and PU4/+ Mpl'^' mice (3.00x10⁵ to 1.00x10° platelets /μl). The third exhibited a dramatically elevated platelet count (2xl0⁶ to 8xl0⁶ platelets/μl), far higher than observed in wild type mice and these were hypothesized to be the homozygous PU3/PU3 Mpl'^' mice and the PU4/PU4 Mpl'^' mice (Figure 2). This genotypic assignment was confirmed by breeding the putative homozygotes with +/+ Mpl '^' mice and showing that all the resultant progeny had the mildly elevated platelet number expected of Plt4/+ Mpl'^' ice (Figure 2D). EXAMPLE 5 Haematology and Histology

Mature megakaryocyte numbers were determined by microscopic examination of hematoxylin and eosin-stained histological sections of sternal bone marrow and spleen. Megakaryocytes were readily recognisable by their large size and distinctive morphology.

Numbers of megakaryocyte progenitor cells were determined in clonal cultures. 2.5xl0⁴ bone marrow or 10⁵ spleen cells were plated in 0.3% agar in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% batch-selected fetal or newborn calf serum, and stimulated with a final concentration of lOOng/ml murine SCF, lOng/ml murine IL-3 (PeproTech, Rocky Hill, NJ) and 4 units/ml human erythropoietin (EPO, Amgen, Thousand Oaks, CA) and incubated for 7 days at 37°C in a fully humidified atmosphere of 5% CO2 in air. Cytokines were obtained from the commercial sources indicated or produced in our own laboratories by expression of recombinant proteins in Pichia pastoris or E.coli and purified prior to use. Agar cultures were fixed in 2.5% glutaraldehyde, sequentially stained for acetylcholinesterase, Luxol fast Blue and hematoxylin, and the cellular composition of each colony determined by microscopic examination at 100 to 400- fold magnification. These conditions allowed optimal stimulation of neutrophil, neutrophil- macrophage, macrophage, eosinophil, megakaryocyte, erythroid, multilineage and blast cell colony-forming cells (CFC).

Analysis of general effects on hematopoiesis were conducted by measurements of hematocrits as well as total peripheral blood white cell counts, the latter by performing manual counts using hemocytometer chambers and via automated analysis. The relative numbers of morphologically recognisable precursor cells in hematopoietic organs were assessed by manual 100 to 400 cell leukocyte differential counts of peripheral blood, bone marrow, liver and spleen following preparation of smears or cytocentrifuge preparations stained with May-Grunwald-Giemsa. In addition, the relative numbers of hematopoietic cells expressing lineage-specific cell-surface markers were measured. Single cell suspensions of bone marrow, spleen and thymus from adult mice of each genotype were incubated with saturating amounts of 2.4G2 anti-Fc8 receptor antibody to reduce background staining, then with specific monoclonal antibodies to murine cell surface antigens: anti CD4 and CD8, IgM, Ly5-B220, Mac-1, F4/80, Gr-1, Ter-119, and Thyl.2 (Pharmingen, Torrey Pines, CA). Antibodies may be directly coupled to fluorescein isothiocyanate (FITC) or biotin, the latter being visualised with R-phycoerythrin- streptavidin. Flow cytometric analyses were performed on a FACScan analyser (Becton- Dickinson, Franklin Lakes, NJ) with dead cells and eiythrocytes excluded by propidium iodide (lmg/ml) staining and gating of forward angle and side scatter of light.

Histological sections of all major organs were also prepared by standard techniques, stained with hematoxylin and eosin and examined by light microscopy for evidence of abnormality.

CFU-s were enumerated by intravenous injection of bone marrow cells into recipient mice that had been irradiated with l lGy of γ- irradiation given in two equal doses given three hours apart from a 137c_s source (Atomic Energy, Ottawa, Canada). Transplanted mice were maintained on oral antibiotic (l .lg/L neomycin sulfate; Sigma, St. Louis, MO). Spleens were removed after 12 days, fixed in Carnoy's solution (60% ethanol, 30% chloroform, 10% acetic acid), and the numbers of macroscopic colonies were counted.

Flow cytometry

Single-cell suspensions of spleen and bone marrow cells were depleted of erythrocytes by lysis with 156mM ammonium chloride (pH 7.3). Cells were stained with a saturating concentration of IgM-FITC and B220-PE, or Terl l9-PE and CD71-FITC (BD Pharmingen, San Diego, CA). Dead cells were excluded based on propidium iodide (PI) staining. EXAMPLE 6 Increased production of platelets in mice harbouring the Plt3 andPlt4 mutations

In order to determine whether there is increased production of platelets in mice harbouring the Plt3 and Plt4 mutations, the megakaryocytes, megakaryocyte, progenitor cells (Meg- CFCs) and primitive multipotential haemopoietc cells (blast-CFCs) in the bone marrow and spleen were measured. These cells are the precursors of platelets. The presence of the Plt3 and Plt4 mutations increased the numbers of all of these cells suggesting that the effect of these mutations is to elevate the output of platelets by increasing the production of their cellular precursors (Figure 3 A and 3B).

EXAMPLE 7 Test For Linkage Between PU3 and Plt4

A key step in using the information gleaned from generation and analysis of Plt3 and Plt4 mice in the development of agents to treat thrombocytopenia is the discovery of the gene or genes that are mutated in these animals. In order to determine whether the two mutations, Plt3 and Plt4, are linked, heterozygous Plt3/+ Mpl'^' and PU4/+ MpX^1' mice on a C57BL/6 background were crossed and the mice that were putatively heterozygous for both genes were identified at 7 weeks of age because of their extremely high platelet counts (> 2 x 10⁶/μl).

These mice were then bred with Mpl'^' mice on a C57BL/6 background and the platelet numbers of progeny were determined at 7 weeks. For tightly linked mutations one would expect that all of the progeny from this cross would be heterozygous for either PU3 or PU4 and therefore exhibit platelet counts that were elevated (>300xl0⁵//μl) compared with Mpl '^' mice.

More specifically, if these mutations are in the same gene or very closely linked genes then the progeny will be either compound heterozygotes (PU3/PU4 Mpl'^'), single heterozygotes

(Plt3/+ Mpl'^' or PU4/+ Mpl'^') or wild type (+/+ Mpl'^') at the mutant loci. If the Plt3 and P 4 genes are not linked then the progeny will either be double heterozygotes (Plt3/+ Plt4/+ Mpl'^'), single heterozygotes (Plt3/+ +/+ Mpl'^' or +/+ PU4/+ Mpl'^') or wild type (+/+ +/+ Mpl'^').

To distinguish compound heterozygotes from double heterozygotes mice with the highest platelet levels were mated to +/+ Mpl'^' mice (Figure 4C). In this mating compound heterozygotes yield progeny, all of which will be heterozygote (Plt3/+ Mpl'^' or Plt4/+ Mpl'^'), where as double heterozygotes yield double heterozygotes (Plt3/+ Plt4/+ Mpl'^'), single heterozygotes (PU3/+ +/+ Mpl'^' or +/+ Plt4/+ Mpl'^') or wild type (+/+ +/+ Mpl'^' ). In the case of PU3 and Plt4, the mutations are clearly in the same gene or closely linked since the progeny of the above mating all have the phenotype of heterozygotes (Figure 4C).

EXAMPLE 8 Genetic Mapping and Sequencing

The mutant gene responsible for the platelet phenotype in P 4 is identified by a process of genetic mapping and sequencing.

Affected heterozygous PU4/+ Mpl'^' mice on a C57BL/6 background were crossed to Mpl^+/' mice on a 129Sv background. Plt4/+ FI animals were then identified at 7 weeks of age because of their elevated platelet counts and intercrossed to produce approximately 100 mice in the N2 generation. At 7 weeks of age N2 mice were bled and their peripheral blood platelet number was determined. Mice were sacrificed and DNA was prepared from a piece of liver according to described methods (Laird et al, Nucleic Acid Research, 79(15): 4293, 1991).

Simple sequence length polymorphisms (SSLPs) throughout the genome (Table 3) were amplified by touchdown PCR (Don et al, Nucleic Acid Research 25:4008, 1991) using forward primers labelled with FAM, HEX, or NED fluorescent dye (Applied Biosystems Custom Oligo Synthesis Service, Foster City, CA). PCRs contained 5ng/μl genomic DNA, Taq polymerase, rabbit antiserum to Taq polymerase, 50 mM KC1, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgC12, 0.125 mM deoxynucleoside triphosphates, and 0.1 μM of the forward and reverse oligonucleotide primers. Reaction products with compatible allele sizes were pooled, separated on an ABI 3700 and genotyped using the ABI Genotyper program.

The mutant gene responsible for PU3 was shown to be closely linked to Plt4 using the breeding test described above. To confirm this, Plt3/+ Mpl'^' mice on a C57BL/6 background were crossed with PU3/+ Mpf^' mice on a 129Sv background. PU3/+ Mpl'^~Fl animals were then identified at 7 weeks of age because of their elevated platelet counts and were backcrossed to +/+ Mpf^' mice on a 129Sv background to generate N2 mice. N2 mice that were Mpl'^' were identified and their platelet counts were determined at 7 weeks of age. Mice were then sacrificed and their livers were removed and genomic DNA was made as described (Laird et al, supra, 1991). SSLPs across chromosome 10 (Table 4) were then amplified and analysed as described for Plt4.

Since the Plt3 and Plt4 mutations were closely linked, an initial chromosomal localization of the mutations was obtained using Plt4. PU4/+ Mpl'^' mice on a C57BL/6 background mated to +/+ Mpl'⁺ on a 129Sv background. The PU4/+ Mpl'^' (C57BL/6xl29Sv) F, progeny were selected because of the elevated platelet numbers compared with +/+ Mpl'^' controls. These Fj mice were then intercrossed to generate an F2 generation, which were bled at 7 weeks of age and had platelet count that varied from very low levels expected of +/+ Mpl'^' mice, intermediate levels characteristic of Plt4/+ Mpl'^' mice and exceptionally high levels characteristic of P 4/PU4 Mpl'^' mice. These mice were sacrificed and their livers were removed and genomic DNA was produced. Using a set of sequence length polymorphisms across the genome (Copeland et al, Science, 267:57-88, 1993) it was determined, for each F2 mouse, whether a marker was homozygous C57BL/6, heterozygous C57BL/6-129Sv or homozygous 129Sv. For markers not linked to the Plt4 mutation one would expect no correlation between genotype and phenotype, whereas for markers very closely linked to the mutation, one would expect animals with the highest platelet counts (usually Plt4/Plt4 Mpl'^') to be homozygous C57BL/6, animals with an intermediate platelet count to be heterozygous (usually PU4/+ Mpf^') and animals with the lowest platelet counts (usually +/+ Mpl'^') to be homozygous 129Sv. The only region of the genome to follow this pattern was at the centromeric end of chromosome 10 (Figure 5) between marker D10Mit80 and D10Mit38. Within this region there were several genes, including the IFNgR, Cited2 and Myb, which were possible candidates based on their actions in signal transduction or transcription factor complexes or their effect within the hemopoietic system. Importantly, none of these had been described to suppress thrombocytopenia. Using additional markers, the interval was narrowed further and the only strong candidate remaining was the Myb gene (Figure 5).

To confirm that the Plt3 mutation was also liked closely to this region of chromosome 10 PH3/+ Mpl'^' mice on a C57BL/6 background were mated to +/+ Mpt^1' mice on a 129 background. The Plt3/+ Mpl'^' (C57BL6xl29Sv) Fj mice were then backcrossed to +/+ Mp?'^~ mice on a 129Sv background and the resulting PU3/+ Mpl'^' and the +/+ Mpl'^' (C57BL/6xl29Sv)N₂ mice were bled at 7 weeks to determine their platelet phenotype and genotyped using the markers to the region of chromosome 10 to which the Plt4 gene was gene was localized. Unlike the F₂ cross used to map Plt4 no mice with exceptionally high platelets would be expected. Consistent with this, Figure 5B shows mice in the N2 generation with the highest platelet levels (PH3/+ Mpl'^') were most likely to be heterozygous in this genomic interval, whereas the mice with the lowest platelet numbers (usually +/+ Mpl'^') were more likely to be homozygous 129Sv, confirming the co- localization of the Plt3 and PU4 mutations (Figure 6).

EXAMPLE 9 DNA Sequencing

Mice generated by intercrossing either PU3/+ Mpl'^' mice or Plt4/+ Mpl'^' mice were typed as Plt3/Plt3, Plt3/+, P 4/PU4, PU4/+ or +/+ at 7 weeks of age by determining their peripheral blood platelet number. Using a tail biopsy taken at approximately 3 weeks of age, DNA was prepared by incubation of the tissue sample with 750 μl extraction buffer (50 mM TrisHCl, 100 mM EDTA, lOOmM NaCl, 1% SDS) and 40 μl of 20 mg/ml proteinase K overnight at 55°C, with rocking. 310 μl 5 M NaCl was added and the sample was centrifuged for 10 minutes at 13,000 rpm to pellet debris. 900 μl supernatant was mixed with 600 μl isopropanol and the sample was centrifuged at 13,000 rpm for 15 minutes to pellet the genomic DNA. The DNA pellet was washed with ethanol, dried and resuspended in lOmM Tris, ImM EDTA.

PCR reactions comprised 0.5 μg genomic DNA, 1 μl Taq polymerase (Roche, Mannheim, Germany), 2 μl 10 mM deoxynucleotide triphosphates (dNTPs), 10 μl lOxPCR buffer (Roche), 200 ng of each oligonucleotide (SigmaGenosys, Australia). The final volume was made up to 100 μl with H₂O. The PCR reaction involved heating to 96°C for 2 minutes, 30 cycles of 96°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds followed by incubation at 72°C for 10 minutes. To confirm that products had been amplified, a 20 μl aliquot of the reactions were electrophoresed on a 1% TAE agarose gel which was stained with ethidium bromide. A 10 μl aliquot of the reaction was then treated ExoSAPit exonuclease (USB Corporation, Ohio, USA) according to the manufacturer's instruction to remove remaining primers and dNTPs and sequenced with the primers used to generate the PCR products (Table 5). Sequencing reactions were carried out using BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Products were electrophoresed on a 3730XL (Applied Biosystems) and analysed with EditView 1.0.1 software (Applied Biosystems).

To determine whether the Myb gene was mutated in Plt3 and P 4 mice, primers were designed to amplify the exons and flanking intronic DNA from genomic DNA of PU4/PU4 Mpl'^', PU4/+ Mpl'^', PU3/PU3 Mpl'^', PU3/+ Mpl'^', +/+ Mpl'^' and +/+ Mpl+/+ mice all on a C57BL/6 background. In the case OΪPU3 a mutation in which nucleotide A455 is altered to T, leading to a substitution of Val for Asp at amino acid 152 was observed. For PU4 a A1151 is altered to T, leading to a substitution of Val for Asp at amino acid 384 was observed (Figures 7 and 8).

This suggests that mutation of Myb leads to suppression of thrombocytopenia and that the pharmaceutical interference with Myb activity, for example by disrupting its interaction with DNA, disrupting its interaction with other proteins, altering its half-life or its localization within the cell may be a good treatment for thrombocytopenia.

EXAMPLE 10 Genetic and Biological Analysis ofPU3, Plt4 and Pltβ Mutant Mice

Three mutant mice with ameliorated thrombocytopenia were identified (Plt3, Plt4 and Plt6, Figure 10b). Breeding experiments were conducted to precisely define their genetic inheritance patterns. Each of the phenotypes displayed a semi-dominant inheritance pattern. Offspring from heterozygous (Plt4/+) inter-crosses displayed either the low platelet counts typical of Mpl'^' mice, mild amelioration of thrombocytopenia or supra-physiological platelet levels (Figure lc). These phenotypes were shown to correspond to +/+ Mpl'^', Plt4/+ Mpl'^' and PU4/PU4 Mpl'^' genotypes by breeding experiments (Figure 10c). The proportions of these 3 phenotypes were approximately 1 :2:1 for P 4, consistent with full penetrance of the phenotype and viability of the homozygotes. Progeny from Plt3/+ Mpl'^' and Plt6/+ Mpl'^' inter-crosses also demonstrated supra-physiological platelet counts but the proportion of these was lower than for the Plt4/+ Mpl'^' crosses (Figure 10c). Breeding experiments showed this to be due to embryonic or neonatal death of a proportion of the homozygous mice rather than a lack of penetrance of the phenotype (data not shown).

EXAMPLE 11 Test for Linkage Between Plt6 and Plt3 and PU4

To determine whether the Plt3, PU4 and Pltό mutations were genetically linked compound heterozygotes were produced and mated to Mpl ^''^' mice. In order to determine whether Plt3, Plt4 and Plt6 are linked, heterozygous Plt3/+ Mpl'^', Plt4/+ Mpl'^' and PU6/+ Mpl'^', mice on a C57BL/6 background were crossed and the mice that were putatively heterozygous for both genes were identified at 7 weeks of age by their high platelet counts (> 1500 x 10⁶/ml). These mice were then bred with +/+ Mpl'^' mice and the platelet numbers of progeny were determined at 7 weeks. For tightly linked mutations would expect that all of the progeny from this cross would be heterozygous for one of the Pit mutations and therefore exhibit platelet counts that were elevated (>300xl0⁶/μl) compared with Mpl'^' mice. All progeny of the P 3-PU4 compound heterozygotes exhibited mild amelioration of thrombocytopenia, typical of PU3/+ Mpl'^' and PU4/+ Mpl'^' mice, demonstrating that the Plt3 and Plt4 mutations are tightly linked and could be alleles of the same gene (Figure 11 A). In contrast when PU3-PU6 and PU4-PU6 compound heterozygotes were mated to Mpl'^' mice, the progeny included mice with supra-physiological platelet counts (compound heterozygotes), mice with mild suppression of thrombocytopenia (single heterozygotes) and mice with unaltered thrombocytopenia (+/+), demonstrating that the Plt6 mutation is not linked to Plt3 and PU4 (Figure 11a).

EXAMPLE 12 Genetic Mapping and Sequences

To obtain a chromosomal localisation for the closely linked P 3 and Plt4 mutations PU4/+ Mpl'^' mice (C57BL/6 background) were mated with +/+ Mpf^' on a 129Sv background and then PU4/+ Mpl'^' (C57BL/6xl29Sv)F! progeny were inter-crossed to produce an F2 generation. Platelet counts in the F2 mice varied from the very low levels expected of +/+ Mpl'^' mice, intermediate levels characteristic of P 4/+ Mpl'^' mice and exceptionally high levels characteristic of Plt4/Plt4 Mpl'^' mice. Using a set of approximately 148 simple sequence length polymorphisms, the only region of the genome in which linkage was observed was at the centromeric end of chromosome 10 between marker D10Mitl24 and D10Mit214 (Figure l ib). Analysis of 120 similarly generated backcross progeny demonstrated that this interval was also closely linked to the Plt3 mutation (Figure l ib).

Within this chromosomal region the c-Myb locus represented a compelling candidate for the location of the Plt3 and Plt4 mutations, since c-Myb mutations have been found to elevate platelet numbers (Emambokus N. et al, supra, 2003; Kasper L. H. et al, et al, supra, 2002). The coding regions of the entire c-c-Myb gene from Plt3/+, PU3/PU3, PU4/+ and PU4/PU4 Mpl'^' mice were sequenced and compared with that from +/+ Mpl'^' and +/+ '^{+ +} mice. A single A to T transversion in the c-c-Myb coding sequence was discovered in both mutants resulting in substitution of the valine for an aspartic acid codon at residue 152 of the c-Myb DNA binding domain in the Plt3 allele and at residue 384 in the leucine-zipper domain of the Plt4 allele (Figure 1 lc). EXAMPLE 13 Northern Blot Analysis

Northern blot analysis revealed that c-Myb^p"³ and c-Myb^PM mRNAs were expressed in hemopoietic tissues at normal levels and, upon transfection of expression vectors into 293T cells, the PU3 and PU4 c-Myb proteins were produced at levels similar to wild type c-Myb (data not shown).

EXAMPLE 14 Transactivation Assays

To compare the activity of c-Myb^plt3 and c-Myb^plt4 with that of wild type c-Myb and a constitutively active truncation mutant (CT3) of c-Myb (Ramsay R. G. et al, supra, 1991), the respective cDNAs were cloned into the pEF-BOS (Mizushima S. et al, Nucl. Acids Res, 18:5322, 1990). The c-Myb expression constructs were then co-transfected with a reporter construct containing 5 consecutive high affinity c-Myb binding sites from the chicken c-Mim promoter upstream of the CAT gene (a gift of Prof. Joe Lipsick, Stanford University) and transactivation activity was measured as described (Chen R. H. et al, supra, 1993). The expression of the various c-Myb alleles was measured by Western blotting using the Mabl.l and Mab 5.1 antibodies as described (Ramsay R. G. et al, Oncogene Res, 4:259-269, 1989). Compared to wild type c-Myb and a constitutively active c-Myb mutant, there was a profound reduction in the activity c-Myb^plt4 and a more modest reduction of c-Myb^plt3 proteins in a transactivation assay using a c-Myb-responsive reporter gene (Figure 2d). The conclusion that partial loss of function of c-Myb can ameliorate thrombocytopenia was confirmed by taking advantage of a previously generated mouse (Mucenski M. L. et al, Cell, (55:677-689, 1991) with deletion of the c-Myb gene (c-Myb^') and showing that, similar to c-Myb^p"^3/+ Mpl'^' and c-Myb^p"^4/+ Mpl'^' mice, c-Myb^'/+ Mpl'^' mice had significantly higher numbers of platelets than c-Myb^{+ +}Mpl'^' mice (Figure 12a).

EXAMPLE 15 Analysis of Megakaryocytopoiesis in My 6 Mutants

To investigate the biological basis for the amelioration of Mpl'^' thrombocytopenia resulting from loss of function of c-Myb, megakaryocytopoiesis was analysed in Plt3 and Plt4 mutant mice. Large increases in the numbers of megakaryocyte progenitor cells and megakaryocytes in the bone marrow and spleen of heterozygous and, to a greater extent, homozygous Plt3 and Plt4 mice were observed (Figure 12a), consistent with the increase in platelet numbers in these mice being the result of expanded cellular production within the megakaryocyte lineage. The numbers of progenitor cells committed to other hematopoietic lineages were not significantly altered in heterozygous P 3 or Plt4 mutants; however, in homozygous Plt3 and PU4 mice, the numbers of all progenitor cells were elevated compared with control Mpl^1" mice (Table 6). Perturbations in the stem cell compartment seem likely to account for this observation, since significant increases in the numbers of spleen colony-forming cells accompanied these changes (Figure 12a). While the numbers of CFU-s in the bone marrow of homozygous Plt3 and Plt4 mutants were elevated, the spleen colonies derived from them were smaller than those observed in mice transplanted with heterozygous or Mpl'^' cells.

With the exception of megakaryocytopoiesis, this multi-lineage myelodysplastic state did not result in widespread excessive production of mature blood cells in homozygous mutants. The proportions of morphologically recognisable cells in the granulocyte series were slightly elevated in bone marrow and spleen of Plt3 and PU4 mutants, but this was likely to be at least in part an indirect effect of reduced lymphoid cell production (see below) and monocyte numbers were relatively unchanged (data not shown). Published studies (Emambokus N. et al, supra, 2003; Kasper L. H. et al, supra, 2002) have found that reduced expression of c-Myb results in defective production of cells in the lymphoid and erythroid lineages. Our analysis of the effects of reduced c-Myb function in Plt3 and P 4 mutants supports and extends these observations. Histological examination of the spleen revealed that abnormally small lymphoid follicles and expanded red pulp with atypical architecture accompanied the greatly expanded numbers of megakaryocytes (Figure 12c). Significantly reduced numbers of B-lymphoid cells were evident in the bone marrow and spleen (Figure 12b and data not shown) and homozygous Plt3 and Plt4 mice were leukopenic, due largely to reduced numbers of circulating lymphocytes (Table 6). Similarly, a large increase in the numbers of colony-forming unit-erythroid (CFU-e) was observed in the spleens of homozygous mutant mice (Mpl'^' c-Myb^p"^3/p"³ : 155±143; Mpl'^' c-Myb^PMIPM: 154 ±92; Mpl'^' c-Myb^+/+: 3±3 per 10⁵ cells), defects in erythroid maturation were evident in the bone marrow (Figure 12b) and the animals were mildly anaemic (Table 6), consistent with full c-Myb function being necessary for normal terminal erythroid differentiation.

EXAMPLE 16 Analysis ofEpistasis

An advantage of modifier screens is their capacity to order genes in a pathway using analyses of epistasis. The phenotype of homozygous Plt4 mutants was assessed on a wildtype background (c-Myb^p"^4/p"⁴ Mpt'⁺) in comparison with that of wild-type mice (c-Myb^+/+Mpl^+/+) mice and c-Myb^p"^4/p"⁴ Mpl'^' mice. PU4/PU4 Mpl'^' mice were mated to +/+ Mpl^+/+ mice to produce Plt4/+ Mpf^' animals, which were then intercrossed. The Mpl genotype of the progeny was determined by Southern blot (Alexander, W. S. et al, supra, 1996) and the genotype of the Plt4 locus was inferred from one or two generation progeny testing. The phenotype of PU4/P 4 Mpl'^', PU4/PU4 Mpl^+/+ and +/+ Mpl'^' mice was then compared in order to assess the epistatic relationship of the genes. Remarkably, the supra-physiological production of platelets, megakaryocytes and megakaryocyte progenitors observed in c-Myb^p"^4/p"⁴ mice was independent of the Mpl genotype (Table 6 and Figure 12) suggesting that c-Myb and Mpl lie in the same pathway rather than contributing independently to platelet formation. These data show that down-regulation of c-Myb expression or function is a key outcome of the TPO signalling pathway that is necessary for effective megakaryocytopoiesis. Comprehensive genome-wide and targeted mutagenesis screens using wild type mice have been reported recently (Herron B. J. et al, Nature Genetics, 30:185-189, 2002; Hrabe de Angelis, M. H. et al, Nature Genetics, 25:444-447, 2000; Nolan P. M. et al, Nature Genetics, 25:440-443, 2000; Kile B. T. et al, Nature 425:81-86, 2003). This paper describes the first large-scale modifier screen in vertebrates and demonstrates that the strategies that have proven so valuable in yeast, worms and flies (St Johnston D., Nature Reviews Genetics, 5:176-188 2002; Jorgensen E. M. et al, Nature Reviews Genetics, 5:356-369, 2002; Forsburg S. L., Nature Reviews Genetics, 2:659-668, 2001) are also applicable in higher organisms. Several ENU-induced mutations affecting platelet number in mice were isolated and two of these were shown to carry independent, partial loss of function mutations in the c-Myb gene. Previous studies have implied a complex role for c-Myb in regulation of megakaryocytopoiesis (Emambokus N. et al, supra, 2003) and have suggested that the co-activator p300 is a crucial interaction partner of c-Myb in regulating this process (Kasper L. H. et al, supra, 2002). As demonstrated herein down-regulation of c-Myb activity ameliorates the thrombocytopenia caused by the absence of TPO signalling. Moreover, this loss of c-Myb activity in the homozygous state also results in the production and maintenance of supra-physiological numbers of megakaryocytes and platelets in the absence of the actions of TPO, the primary physiological regulator of platelet production.

As shown herein, suppressor screens in vertebrates are of value for the identification of targets for drug discovery. Just as most ENU-induced mutations cause loss of function, most small molecule therapeutics also reduce the function of proteins to which they bind. Accordingly, screens for genes that, when mutated, lead to amelioration of disease, such as c-Myb or other genes that will emerge from our Mpl'^" suppressor screen, should provide genome-wide access to novel in vivo validated targets for pharmaceutical discovery. EXAMPLE 17 Summary of the PU3 and Plt4 Phenotypes

Platelet counts Platelet counts have been collected from mice of several different genotypes. These are presented graphically in Figure 12a and tabulated in Table 6. • Mice heterozygous (Mpl^'/" Myb^{Plt3 +}, Mpl^"''^' Myb^plt4/+) or homozygous (Mpl^"/" _MybPlt3/Plt3. _Mpj-/- _Myb ' Plt4_{) for} pjg _Qr p_{k4 Qn ft MpJ} fc^ou,. background. The data from these mice, when compared to Mpl^"'^" Myb^{+ +} mice demonstrate that mutation in the Myb gene can ameliorate the thrombocytopenia caused by lack of TPO signalling through its receptor Mpl. • Mice homozygous for Plt4 on a wild type background (Mpl^+/+ Myb^{plt Plt4}). The data from these mice, when compared with wildtype mice (Mpl^{+ +} Myb ^{+ +}) and Mpl^"7" Myb^{plt4 plt4} mice demonstrate that the expansion of megakaryocyte and platelet production cause by Myb mutation occurs independently of TPO signalling through Mpl and suggests that down- modulation of Myb may be an important action of Mpl signalling. • Mice heterozygous for a knockout of the Myb gene on a Mpl knockout background (Mpl^{" "} Myb^{+ '}). Only platelet data has been collected from these mice to date (shown in Figure 12a only, counts were Mpr^A Myb^+/' 710 ± 55, Mpl^'/' Myb^{+ +} 162 ± 72 xlO^'6 platelets/ml, n= 4 mice of each genotype) - it demonstrates that lack of one functional Myb allele elevates platelet counts in Mpl^{" '} mice. This provides supporting evidence that the Plt3 and Plt4 mutations in Myb cause partial loss of function.

Megakaryocyte and Megakaryocyte Progenitor Counts

During megakaryocytopoiesis platelets are produced as the end-products of a process of commitment of multipotent hematopoietic stem cells to the megakaryocyte lineage, production and proliferation of megakaryocyte progenitor cells, differentiation and maturation of megakaryocytes and ultimate release of platelets from mature megakaryocytes into the circulation. • Counts of megakaryocyte progenitor cells (by clonal assay of bone marrow and spleen cells in vitro using cytokines to stimulate colony formation) revealed that numbers of megakaryocyte progenitor cells were elevated in the bone marrow and especially the spleens of Plt3 and Plt4 homozygotes (and to a lesser extent heterozygotes) on a Mpl-/- background compared with Mpl -/- mice, and of Plt4 homozygotes on a wildtype background when compared with wild-type mice. This data is presented graphically in Figure 12a and tabulated for one cytokine stimulus (SCF + IL-3 + EPO) in Table 6. • Other stimuli have also been used to enumerate megakaryocyte progenitor number in bone marrow and spleen, namely Multi-CSF (IL-3), EPO and GM-CSF - all reveal elevations in megakaryocyte progenitor cell number in of Plt3 and Plt4 heterozygotes and homozygotes on a Mpl-/- background compared with Mpl-/- mice, and of Plt4 homozygotes on a wildtype background compared with wildtype controls. These data are shown in Table 7. • Counts of megakaryocytes (by enumeration of morphologically recognizable megakaryocytes in histological sections of bone marrow and spleen) revealed elevated numbers in the bone marrows of Plt3 and Plt4 heterozygotes and both the bone marrow and spleens of homozygotes on a Mpl-/- background compared with Mpl-/- mice and in spleens of Plt4 homozygotes on a wildtype background compared with wildtype controls. These data are presented graphically in Figure 12a. The data are in Table 8.

These megakaryocyte progenitor and megakaryocyte counts provide evidence that the increased platelet production caused by Myb mutation occurs as a result of a large expansion of all stages of megakaryocytopoiesis. This process occurs at an increased rate than normal in vivo. o As they mature, megakaryocytes increase their DNA content and become polyploid; thus a measure of maturation is DNA content. In experiments where megakaryocytes in bone marrow were examined for DNA ploidy, megakaryocytes from homozygous Plt3 and Plt4 mutants on a Mpl-/- background displayed a modal ploidy of 8N versus 16N for Mpl-/- mice themselves. Similarly, megakaryocytes from one homozygous Plt4 mutant on a wildtype background showed modal ploidy of 8N versus 16N for wildtype controls. Heterozygous Plt3 and Plt4 mutants on a Mpl-/- background shown a less dramatic trend towards lower ploidy of the megakaryocyte population. The full data is shown in Table 9. It is well established that megakaryocyte ploidy is usually inversely proportional to platelet count. These data suggest that reduced megakaryocyte ploidy in mice with mutation of Myb occurs independently of TPO signalling.

Other Hematopoietic Progenitor Cells

The data in Table 7 also indicate that for most stimuli tested, the total numbers of bone marrow hematopoietic progenitor cells committed to granulocyte, granulocyte/macrophage and macrophage formation were generally elevated in Plt3 and Plt4 homozygotes on a Mpl-/- background compared with Mpl-/- mice. This was also the case for progenitor cells in the spleens of Plt3 and Plt4 homozygotes on a Mpl-/- background and of Plt4 homozygotes on a wildtype background. These data indicate that mutation of Myb, in addition to expanding megakaryocytopoiesis, also causes a myelodysplastic state involving amplification of other myeloid progenitor cells. Other noteworthy aspects of Pit mutant bone marrow progenitor cell behaviour revealed by these data include: • Stimulation of megakaryocyte colony formation by GM-CSF, which is not normally observed except at very high concentrations. • Absence of multicentric blast colonies with IL-3, SCF + G-CSF or SCF + IL-3 + EPO but presence of very large dispersed colonies of blast cells. • High frequency of mature (smaller) megakaryocyte colonies, indicating that megakaryocyte formation is occurring actively in vivo. • Unresponsiveness of granulocytic clonogenic cells to IL-6 - many plates have no colonies, but in the same cultures IL-6 stimulating some megakarayocytic colony formation. • Quantitative responsiveness to GM-CSF, M-CSF and IL-3 are normal in Plt3 and 4 cells. • The Plt3 and 4 mouse cells exhibited no response to thrombopoietin and have not developed pseudo-TPO receptors.

Hematopoietic Stem Cell Compartment • Cells capable of forming blast colonies in culture are a measure of progenitor cells with multi-lineage potential. As discussed above, the numbers of these progenitors was markedly elevated in the bone marrow and spleen of Plt3 and Plt4 homozygotes on a Mpl-/- background compared with Mpl-/- mice, and evident in the spleens of Plt4 homozygotes on a wildtype background compared with wildtype controls. This data is shown in Table 7. • More primitive cells are the CFU-s, which are multipotential cells assayed by their capacity, upon intravenous injection, to form colonies in the spleens of myeloablated hosts. The numbers of these mature stem cells was markedly elevated in the bone marrow of Plt3 and Plt4 heterozygotes and homozygotes on a Mpl-/- background compared with Mpl-/- mice, but apparently unaltered in Plt4 homozygotes on a wildtype background compared with wildtype controls. These data are shown graphically in Figure 12a and tabulated in Table 10. • The definitive assay for hematopoietic stem cells in the competitive repopulation assay in which stem cells from a test bone marrow are required to compete with a standard dose of normal stem cells for the long-term and multi-lineage reconstitution of the blood forming system of a transplanted, myeloablated host. Using this assay, the number of stem cells, or competitive reconstituting units (CRU) was not found to be significantly different between Plt4 heterozygotes and homozygotes on a Mpl-/- background compared with Mpl-/- mice (Table 17). Mpl-/- mice are known to have defects in the multipotential stem cell pool relative to wild type mice, manifesting as reduced CRU, CFU-s and blast colony-forming cells as well as committed progenitor cells of multiple lineages and this was confirmed in the studies above. The novel finding here is that mutation of Myb ameliorates this deficiency at the levels of CFU-s, blast colony-forming progenitors and committed progenitor cells. The numbers of CRU are unaffected suggesting that the action of Myb in this regard may not occur in the most primitive stem cells but may be manifest in more mature multipotential cells.

Mature Hematopoietic Cells

Despite the expansion of progenitor cells of multiple hemtopoietic lineages in Plt3 and Plt4 mutants, with the exception of the dramatic proliferation of megakaryocytes and platelet numbers, this multi-lineage myelodysplastic state did not result in widespread excessive production of mature blood cells in Plt3 and Plt4 mutants. The data from enumerating morphologically identifiable blood cells in cytospin preparations from the bone marrow, spleens and peritoneal cavity of Plt3 and Plt4 mutants is shown in Table 11. This data was supplemented by analysis of hematopoietic tissues from mutant mice by flow cytometry using lineage-specific cell surface antigens/markers. The proportions of morphologically or antigenically recognisable cells in heterozygous Plt3 and Plt4 mutants on a Mpl-/- background were unremarkable, although changes as outlined below, were evident in homozygous Plt3 and Plt4 mutants on a Mpl-/- background as well as Plt4 homozygotes on a wildtype background. • Granulocytes and macrophages: the proportions of cells in the granulocyte series were slightly elevated in bone marrow and spleen of Plt3 and PU4 mutants, but this was likely to be at least in part an indirect effect of reduced lymphoid cell production (see below) and monocyte numbers were relatively unchanged (Table 11). • Lymphocytes: Decreased numbers of lymphocytes were observed in cytospin preparations the spleens, bone marrow and peritoneal cavity of homozygous Plt3 and Plt4 mutants on a Mpl-/- background as well as Plt4 homozygotes on a wildtype background (Table 11). This was confirmed by flow cytometric studies showing reduced B lineage cells (B220+) in bone marrow, spleen, lymph node, peritoneal cavity and peripheral blood of homozygous Plt3 and Plt4 mutants on a Mpl-/- background and Plt4 homozygotes on a wild-type background (an example of this is shown graphically in Figure 3b of the manuscript; all the data is in Table 12). The unconventional B lineage cells in the peritoneal cavity of mice (CD5+ IgM+) were also affected. Early T lymphocyte development can be measured using the cell surface markers c-kit and CD25 on CD4-CD8- thymocytes, with differential expression of these markers denoting progression through the so-called DN1 to DN4 maturation series. As shown in Table 13, homozygous Plt3 and Plt4 mutants on a Mpl-/- background and

Plt4 homozygotes on a wildtype background displayed a partial block in progression of thymocytes through this series. Erythroid cells: Increased numbers of nucleated erythroid cells were a common feature of the spleens of homozygous Plt3 and Plt4 mutants on a

Mpl-/- background and Plt4 homozygous mutants on a wild-type background (Table 11) and this was confirmed in flow cytometric analyses of spleen cells from homozygous Plt3 and Plt4 mutants on a Mpl-/- background and Plt4 homozygotes on a wild-type background using the erythroid specific marker Terl l9 (Table 14). The bone marrow was marginally, if at all affected. Erythroid maturation was examined using the cell surface markers CD71 and Terl 19 which mark a window of maturation that progresses from stage I (CD71hi Terl l9med) through stages II 9CD71hi Terl l9hi), III (CD71med Terl l9hi) and IV (CD711o Terl l9hi). The proportions of cells at these stages is anomalous in homozygous Plt3 and Plt4 mutants on a Mpl-/- background and Plt4 homozygotes on a wildtype background with accumulation of immature (ie stage I and II) cells at the expense of the more mature (stage III and IV) cells, both in bone marrow and in spleen (An example of this is shown in Figure 12b; all the data are shown in Table 15 in this document). Accumulation of increased numbers of colony-forming units-erythroid (CFU-e), which are erythrocyte precursors assayed as erythropoietin-responsive cluster-forming cells in semi solid culture in vitro, was also evident in the spleens of homozygous Plt3 and Plt4 mutants on a Mpl-/- background and Plt4 homozygotes on a wildtype background (Table 16). As a result of defective erythroid maturation, homozygous Plt3 and Plt4 mutants on a Mpl-/- background were mildly anemic, as were Plt4 homozygous mutants on a wildtype background (Table 6).

Organ Histopathology • All major organs in Plt3 and Plt4 mutants were examined histologically and in most cases no consistent anomalies were observed. The two exceptions were the bone marrow, where excess numbers of megakaryocytes were evident (see above) and the spleens of homozygous Plt3 and Plt4 mutants on a Mpl-/- background as well as Plt4 homozygous mutants on a wild-type background. The spleen histology in homozygous Plt3 and Plt4 mutants on a Mpl-/- background as well as Plt4 homozygous mutants on a wild-type background also revealed excess megakaryocyte numbers (see above) but also the presence of only poorly developed lymphoid follicles (consistent with the findings on B lymphocyte numbers above) and an expanded red pulp with atypical architecture including large apparently acellular areas (Figure 12c). Finally, Howell-Joly bodies, a histological feature, were observed in erythroid cells prepared from the spleens of homozygous Plt3 and Plt4 mutants on a Mpl-/- background as well as Plt4 homozygous mutants on a wild-type background - this is a diagnostic feature of spleen hypofunction.

Together these analyses of mature cells and organ histopathology define the myelodysplastic state observed in Myb mutants in the absence of TPO signalling. EXAMPLE 18 Methods

+/+ Mpl^"/", Plt4/+ Mpl"'^" and Plt4/Plt4 Mpl^{" "} mice were injected IP with 250 microlitres of saline (Figure 13 A) or 100 mg/kg carboplatin in approximately 250 microlitres saline (Figure 13B). Likewise, +/+ Mpl^{+ +}, PU4/+ Mpl^{+ +} and Plt4/Plt4 Mpl^{+ +} were also injected with the same dose of carboplatin (Figure 13C). All mice were on an inbred C57BL/6 background and were maintained in conventional clean animal facilities. At the indicated time points following injection mice were bled and the numbers of platelets were analyzed using an Advia Automated Haematological Analyser.

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. It is also to be understood that unless stated otherwise, the subject invention is not limited to specific formulation components, manufacturing methods, dosage regimes, or the like, as such may vary.

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Claims

CLAIMS:

1. A physiological assessment system to identify a pedigree of a vertebrate animal model of a disease or condition which exhibits ameliorated symptoms of the disease or condition, the system comprising:

(i) assessing a physiologically assessable symptom of the disease or condition in the vertebrate animal model and/or its progeny comprising one or more of GI, G2, G3 or subsequent generations, wherein the animal model also comprises mutations induced by random or non-random mutagenesis; and

(ii) selecting animals in which the symptom of the disease or condition is ameliorated and wherein the responsible mutation is heritable.

2. The physiological assessment system of claim 1, when used to identify genetic or proteinaceous forms of a target for therapeutic and/or prophylactic intervention associated with amelioration of symptoms of a disease or condition, wherein the system further comprises:

(iii) positional cloning to identify the genetic sequences comprising the mutation and any encoded product.

3. The system of claim 1 or 2, where in step (i) thereof random or non-random mutations are induced in a normal or susceptible animal which is bred to an animal which is normal or susceptible and the progeny, comprising one or more of GI, G2, G3 or subsequent generations, are exposed to conditions which induce the disease or condition.

4. The system of claim 1 or 2, where in step (i) thereof random or non-random mutations are introduced into one of two breeding partners wherein one or both breeding partners comprise a conditional latent disease or condition causing mutation which is activated in progeny by the action of one or more expression products encoded thereby derived from one or both parents which activate the latent mutation derived from the other parent.

5. The system of claim 4, wherein the expression product is a site specific DNA recombinase and the conditional latent disease or condition causing mutation is operably linked to a sequence recognised by a site specific DNA recombinase.

6. The system of claim 5, wherein one breeding partner comprises the conditional latent disease or condition causing mutation operably linked to lox or fit sites in homozygous form and the other partner encodes the appropriate site specific recombinase (ere or flp) in homozygous form.

7. The system of claim 5, wherein one partner comprises a conditional latent disease causing mutation operably linked to lox sites and further comprises a sequence of nucleotide expressing a flp recombinase in homozygous form and the other partner comprises a conditional latent disease causing mutation operably linked to frt sites and further comprises a sequence of nucleotides expressing a ere recombinase in homozygous form.

8. The system of claim 5, wherein one partner comprises a conditional latent disease causing mutation operably linked to lox sites in homozygous form and further comprises a sequence of nucleotide expressing a flp recombinase in homozygous form and the other partner comprises a conditional latent disease causing mutation operably linked to frt sites in homozygous form and further comprises a sequence of nucleotides expressing a ere recombinase in homozygous form.

9. The system of claim 1 or 2, where in step (i) thereof random or non-random mutations are induced in one of two breeding partners, one being homozygous for a disease or condition causing mutation which leaves the animal viable and fertile and the other also the homozygous mutation or being substantially isogenic thereto but without the homozygous mutation, and wherein the progeny of the breeding partners comprising one or more of GI, G2, G3 or subsequent generations are assessed for physiologically assessable symptoms indicating that the disease or condition is ameliorated.

10. The system of claim 9, wherein the animal model comprises a recessive disease or condition causing mutation.

11. The system of claim 9, wherein the animal model comprises a dominant disease or condition causing mutation.

12. The system of claim 9, wherein the GI progeny are assessed.

13. The system of claim 9, wherein the G3 progeny are assessed.

14. The system of claim 1 or 2, wherein step (ii) comprises one or more of the following steps to determine whether the responsible mutation or phenotype is heritable:

(i) breeding the selected animal to non-mutagenised animals homozygous for the disease or condition causing mutation and assessing progeny to determine the proportion exhibiting ameliorated symptoms;

(ii) inter-breeding the selected animals and assessing progeny to determine the proportion exhibiting ameliorated symptoms;

(iii) breeding the selected animals to animals of step (i) which do not exhibit ameliorated symptoms and assessing progeny to determine the proportion exhibiting ameliorated symptoms; and (iv) interbreeding animals of step (i) which do not exhibit ameliorated symptoms and assessing progeny to determine the proportion exhibiting ameliorated symptoms.

15. The system of claim 1 or 2, where in step (i) thereof the animal model is heterozygous for a disease or condition causing mutation and where random or non-random point mutations are induced in one of two breeding partners of the animal model and the progeny comprising one or more of GI, G2, G3 or subsequent generations, are assessed for physiologically assessable symptoms indicative that the disease or condition is ameliorated.

16. The system of claim 15, wherein step (ii) further comprises selecting animals which are homozygous for the disease or condition causing mutation and in which the symptom of the disease or condition is ameliorated.

17. The system of claim 16, wherein the step of selecting animals which are homozygous for the disease or condition causing mutation comprises:

(i) genotyping to detect a disease or condition causing mutation; or

(ii) detecting a assessable marker that is genetically linked to the disease or condition causing mutation.

18. The physiological assessment system of claim 1 or 2, wherein the vertebrate animal model is a physiologically assessable model for a disease or condition of one or more tissues, organs or systems such as, for example, heart, liver, kidney, lung, eye, brain, reproductive system, vascular system, haematopoietic system, nervous system, immune system, skin system, ear and muscle.

19. The physiological assessable system of claim 17, wherein the disease or condition is selected from; an ataxia, a developmental abnormality, a cancer, leukaemia, infectious disease, autoimmune disease, obesity, diabetes, premature aging, or a inflammatory, allergic or immune mediated condition.

20. The assessment system of claim 1 or 2, wherein the vertebrate animal model is a non-human mammalian, rodent or mouse animal model.

21. The assessment system according to claim 20, wherein the animal model is a mouse animal model.

22. The assessment system of claim 21, wherein the mouse animal model is selected from the group consisting of a mouse model for leukaemia, haemophilia, thrombocytopenia, Chediak-Higashi Syndrome, Fanconi's anaemia, allergic airway disjunction, Crohn's disease, diabetic nephropathy, Alexander disease, multiple sclerosis, inherited retardation, Huntington's chorea, rheumatoid arthritis, AIDS cardiomyopathy, Werner syndrome; Alzheimer's disease, hereditary hemochomatosis, Hirschspnmg disease, chronic wasting disease, typel von Willebrand disease, type 3, von Willebrand disease, retinitis pigmentosa, obesity, mucopolysaccharidosis type VII, Alport syndrome, anxiety-related disorder, mucopolysaccharidosis type VI (Maroteaux-Lamy syndrome), atherosclerosis, Glanzmann thrombasthenia, Gerstmann-Straussler-Scheinker syndrome, severe congenital malformation and coronary artery abnormalities, obsessive-compulsive disorders, lymphedema-distichiasis, Pompe disease, hereditary hemorrhagic telangiectasia type 2 disorder, neuronal degeneration, type II Bartter's syndrome, Tuberous sclerosis and split hand/foot malformation type I.

23. The assessment system of claim 1 or 2, wherein the vertebrate animal model is an animal model of a disease or condition which occurs in humans and/or other subjects of interest.

24. A use of a physiologically assessable vertebrate animal model of a disease or condition in a physiological assessment system to identify genetic or proteinaceous targets for therapeutic and/or prophylactic intervention wherein the system comprises:

(i) assessing the symptoms of the disease or condition in the vertebrate animal model and/or its progeny, wherein the animal model also comprises mutations induced by random or non-random mutagenesis;

(ii) selecting animals in which the symptoms of the disease or condition are ameliorated and wherein the responsible mutation is heritable;

25. The non-human animal identified in the assessment system of any one of claims 1 to 23, comprising a physiologically assessable mutation useful in the identification of genetic or proteinaceous targets for therapeutic and/or prophylactic intervention for a disease or condition.

26. The animal of claim 25, in the form of embryos, stem cells or gametes, for transplantation.

27. A physiological assessment system to identify genetic or proteinaceous targets for therapeutic and/or prophylactic intervention for elevating platelet levels or ameliorating the symptoms of thrombocytopenia comprising:

(i) assessing symptoms of thrombocytopenia in a vertebrate model of said disease or condition and/or its progeny wherein the model animal also comprises mutations induced by random or non-random mutagenesis;

(ii) selecting animals in which the symptoms of thrombocytopenia are ameliorated and determining that the responsible mutation is heritable; and (iii) positional cloning to identify the genetic sequences comprising the mutation and any encoded product.

28. The assessment system of claim 27, wherein the vertebrate model of thrombocytopenia is a Mpl-/- rodent or a rodent in which Mpl activity is, or may be, down regulated or inhibited.

29. The assessment system of claim 1, 24 or 27, wherein random mutagenesis is ENU mutagenesis.

30. An isolated or recombinant genetic or proteinaceous target for therapeutic and/or prophylactic intervention identified the physiological assessment system of any one of claims 2 to 3 or 27 to 29, or a derivative, variant, functional derivative or homolog thereof.

31. The isolated or recombinant genetic or proteinaceous target for therapeutic and/or prophylactic intervention of claim 30 for use in the identification of agonists or antagonists thereof.

32. Use of an isolated or recombinant form of a genetic or proteinaceous target for therapeutic and/or prophylactic intervention identified as a target using the physiological assessment system of any one of claims 27 to 29, or a derivative, variant, functional derivative or homolog thereof, to screen for or develop interacting compounds useful in the treatment and/or prophylaxis of thrombocytopenia or other conditions characterised by low platelet levels.

33. A Myb transcription factor, or a derivative, variant, functional derivative or homolog thereof as a drug target for use in screening for or developing interacting compounds for use in the treatment and/or prophylaxis of thrombocytopenia or other conditions characterised by low platelet levels.

34. A use of genetic or proteinaceous forms of Myb in the manufacture of a medicament for the treatment and/or prophylaxis of thrombocytopenia and/or for modulating platelet levels.

35. The use of claim 34, wherein genetic forms of Myb are used which induce specific antisense or iRNA mediated reduction in the functional activity of Myb.

36. A use of claim 35, wherein the antisense or iRNA form of Myb comprises all or part of the nucleotide sequences set forth in SEQ ID NO: 3 encoding human Myb or a complementary form or a nucleotide sequence encoding a homolog thereof comprising at least 60% nucleotide sequence similarly thereto or capable of hybridising thereto under conditions of low stringency.

37. A use of isolated or recombinant Myb or an other member of the network of interacting molecules to which Myb belongs as a target or substrate for the identification or development of antagonists or agonists which effectively up regulate platelet levels or ameliorate or prevent the symptoms of thrombocytopenia in a subject.

38. A method of identifying compounds useful in the treatment of thrombocytopenia or conditions characterised by low platelet levels, comprising screening compounds for their ability to agonise or antagonise the functional activity of genetic or proteinaceous forms of a Myb transcription factor signalling pathway.

39. A method for screening for agonists or antagonists of targets for therapeutic and/or prophylactic intervention identified herein comprising exposing the drug target to a compound and assaying for:

(i) the presence of complex between the compound and the drug target; or (ii) a change in the interaction between the drug target and a ligand, binding partner or other interacting molecules; or

(iii) a change in the level or an indicator of activity of the drug target.

40. A method of claim 39, wherein the target comprises all or part of the DNA binding domain of Myb or a nucleotide sequence encoding same.

41. A method of claim 39, wherein the target comprises all or part or part of the leucine zipper domain of Myb or a nucleotide sequence encoding same.

DATED this 27th day of August, 2004

THE WALTER AND ELIZA HALL INSTITUTE OF MEDICAL RESEARCH by DA VIES COLLISON CAVE Patent Attorneys for the Applicant(s)