FIELD

The subject specification relates to the field of therapeutic targets and provides targets, model systems, screening assays and compositions for modulating targets useful in the treatment or prevention inter alia of conditions associated with deficiencies in haematopoiesis and especially myelopoiesis. In particular, the specification discloses a teleost model of ZBTBI l dysregulation neutropenia and central nervous system degradation.

BACKGROUND

Bibliographic details of references in the subject specification are also listed at the end of the specification.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

One complex medical condition that is still poorly understood and controlled despite intensive world wide research over a number of years is cancer and in particular cancer of the blood or bone marrow, broadly referred to as leukemia. Leukemia is estimated to comprise approximately 5% of deaths from cancer.

Treatment for most forms of cancer include radiation treatment and/or chemotherapy. Bone marrow transplantation may also be used to repopulate the hematopoietic system after cytotoxic treatments. Advances in understanding the genetic and molecular basis of cancer in the last decade are enabling the rational design of cancer therapeutics. In particular, antibodies and small-molecule tyrosine kinase inhibitors have increased the efficacy and reduced the side effects of treatment. One example of an antibody therapeutic is Herceptin which binds to the HER-2 growth factor receptor (c- erbB2) for use in the treatment of breast cancer, another is Rituximab that targets the CD20 receptor for use in the treatment of B-cell lymphoma. Small molecule therapies include Imatinib (Gleevec) that targets the Bcr-Abl fusion protein for use in the treatment of chronic myeloid leukemia, and Bortezomib (Velcade) that targets the proteosome for use in the treatment of multiple myelomas. It is proposed that combinations of single target agents will provide more effective treatments. Other approaches include stem-cell therapy, gene therapy, oncolytic viruses and antibody-directed drugs and radionuclids and many such agents are undergoing clinical trials. Thus, there is a growing precedent for the targeted design and development of effective anti-cancer treatment. However, one of the difficulties in small molecule development is target selection and many potential targets lack suitability due to their pleiotropic nature and/or due to the level of redundancy in a particular pathway.

SUMMARY

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.

As used herein the singular forms "a", "an" and "the" include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a single cell, as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; and so forth. Each embodiment in this specification is to be applied mutatis mutandis to every other embodiment unless expressly stated otherwise.

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>l (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.

Genes and other genetic material (eg mRNA, nucleic acid constructs etc) are represented herein in italics while their proteinaceous expression products are represented in non-italicised form. Thus, for example "zinc finger and BTB (5road complex, Tramtrack, Bήc a brae) domain containing 11" (herein after "ZBTBH") polypeptide is the expression product of ZBTBl 1 nucleic acid sequences.

The scientific nomenclature for genes distinguishes between human, teleost, rodent and other vertebrate organisms. In the Examples, zebrafish genes are in lower case italicised and zebrafish proteins are first letter capitalised and unitalicised. In the description herein of methods of screening or testing, genes and their expression products are referred to broadly in capitalised form to encompass human, teleosts, rodent and other vertebrate forms including, in some embodiments, parts and variants thereof. The skilled artisan will select ZBTBIl or ZBTBIl sequences of human, teleost, rodent or other vertebrate origin or functional homologs thereof as required by the product or method to be practised or the exigencies of the situation. In some embodiments where zebrafish genes are employed, genes are referred to in lowercase italicised form. In other screening or testing embodiments, human or rodent sequences or functional homologs thereof are employed. In embodiments contemplating or relating to treatment or prophylaxis of humans, sequences based upon human forms for use in human subjects are expressly contemplated.

Accordingly, in one embodiment the present specification discloses the use of ZBTBl 1 or ZBTBIl in the manufacture of a medicament for the treatment of a condition associated with ZBTBI l dysregulation. The term "manufacture" includes design or selection.

As shown herein, the zebrafish marsanne {man) mutant is characterised structurally by a loss of function mutation in the ZBTBIl gene, and phenotypically by defects inter alia in neutrophil numbers as well as cerebral defects including retinal and other central nervous system degeneration.

ZBTBI l is a DNA binding transcriptional repressor often dysregulated in malignant diseases. The human homolog has been shown to bind the human metallothionein HA (MTIIA) promoter initiation sequence (Tang et al, Molecular and Cellular Biology, 19(1): 680-689, 1999).

In a particular embodiment, the present invention provides a method for identifying or assessing a variant or analog of a ZBTBIl nucleic acid or protein or an agent which modulates a condition associated with ZBTBI l dysregulation (deficiency) in a subject, the method comprising (i) contacting the agent or variant or analog with a teleost model of ZBTBl 1 deficiency; and (ii) assessing the ability of the agent or variant or analog to rescue (complement) the phenotype of the teleost model.

The phenotype of the teleost model and conditions associated with ZBTBI l dysregulation (dysfunction) are identified herein as including, without limitation, neutrophil mediated tissue injury, neutropenia, neutrophil inflammation or hyperplasia, hydrocephalus and haematological malignancies and myelopoietic disorders such as lymphoma and myeloid leukemia. Accordingly, in some embodiments, ZBTBIl dysregulation is associated with a haematological malignancy or myelopoietic disorder. In other embodiments, ZBTBIl dysregulation is associated with neutropenia or immunodeficiency. In other embodiments, ZBTBIl dysregulation is associated with neutrophil inflammation or hyperplasia. In other embodiments, ZBTBIl dysregulation is associated with retinal and central nervous system degeneration, including hydrocephalus. The phenotype of the marsanne mutation and ZBTBIl deficiency is further described in the Examples.

In some non-essential embodiments, the assessment step comprises assessing the number, location or activity of myeloid cells such as neutrophils or the presence of retinal or other central nervous system degeneration such as hydrocephalus. In an illustrative embodiment, myeloid cell numbers are assessed by detecting mpx expression or the expression of other genes whose expression is associated with myeloid cells. In an illustrative embodiment, neutrophil numbers are assessed by detecting the presence or level of mpx or spil expression or the expression of other genes whose expression is associated with neutrophils. The phenotype of the teleost model, such as low myeloid cell numbers or central nervous system degeneration such as hydrocephalus may be conveniently detected by visual or instrument mediated review of embryos at the appropriate stage or stages.

In some embodiments, the teleost model comprises the marsanne mutation. In other embodiments, the model comprises an agent that down regulates the level or activity of ZBTBl 1 in the teleost. Down regulation may be transient or stable as known to those of skill in the art. Conveniently, agents comprise an antisense oligonucleotide or nucleic acid that suppresses ZBTBIl gene expression to downregulate the level of ZBTBIl polypeptide in the teleost. In some embodiments, teleosts are modified to express antisense agents.

In another aspect, the present invention provides an isolated cellular agent, nucleic acid or vector comprising a polynucleotide encoding all or part of a polypeptide having ZBTBI l polypeptide activity wherein the polypeptide comprises an amino acid sequence set out in SEQ ID NO: 2 or 4 or a sequence having conservative substitution or at least 85% sequence identity thereto.

In some embodiments, the isolated cellular agent, nucleic acid or vector comprising or encoding a ZBTBI l polynucleotide that inhibits expression of endogenous ZBTBI l polynucleotide wherein the polynucleotide comprises a contiguous sequence of nucleotides set forth in SEQ ID NO: 1 or 3 or a complementary form thereof or a polynucleotide having at least 85% sequence identity thereto.

ZBTB 11 probes, primer, and antisense nucleic acids are specifically contemplated and the specification provides a ZBTBI l polynucleotide encoding a polypeptide having ZBTBIl polypeptide activity wherein the polynucleotide comprises a contiguous sequence of nucleotides set forth in SEQ ID NO: 1 or 3 or a complementary form thereof or a polynucleotide having at least 85% sequence identity thereto. In related embodiments, the invention provides a ZBTBI l polynucleotide comprising or complementary to all or part of a contiguous sequence of nucleotides set out in SEQ ID NO: 1 or 3 or of a variant thereof having at least 85% sequence identity thereto. For in vitro applications, the ZBTBIl polynucleotide is attached to a molecule that serves directly or indirectly as a reporter for the presence or quantification of the polynucleotide. Polypeptide or peptide forms are also contemplated and in some embodiments, the invention provides a ZBTBI l polypeptide comprising all or part of an amino acid sequence set out in SEQ ID NO: 2 or 4 or a sequence having one or more conservative substitutions or at least 85% sequence identity thereto. In a related embodiment, the specification provides an antibody or antibody fragment determined by a protein having ZBTBI l activity wherein the protein comprises all or part of an amino acid sequence set out in SEQ ID NO: 2 or 4 or a sequence having one or more conservation substitutions or at least 85% sequence identity thereto.

In another aspect, the specification describes a method of treating or preventing a condition associated with ZBTBI l dysregulation in a subject, said method comprising administering a composition that comprises an agent that modulates the level or activity of ZBTBIl polypeptide or a complex comprising same or the level or activity of ZBTBIl polynucleotide in the subject.

In some embodiments, the condition is a haematogical malignancy or myelopoietic disorder. In other embodiments, the condition is retinal or central nervous system degeneration or hydrocephalus. In further embodiments, the condition is neutropenia or immunodeficiency. In still further embodiments, the condition is neutrophil inflammation or hyperplasia. In yet still further embodiments, the condition is a myeloid leukemia or a lymphoma.

As illustrated in the Examples, in some embodiments, the agent increases the level or activity of ZBTBl 1 polypeptide or of a complex comprising same or the level of ZBTBIl polynucleotide in the subject. Conveniently, in some embodiments the composition may comprise a polynucleotide encoding a polypeptide having ZBTBIl activity.

In another exemplified embodiment, the agent decreases the level or activity of ZBTBIl polypeptide or ZBTBIl polynucleotide in the subject. In this embodiment, the composition may conveniently comprise a small molecule, antibody, protein, peptide, or inhibitory polynucleotide molecule that effectively down regulates ZBTBIl activity by preventing ZBTBI l polypeptide function or by preventing ZBTBIl expression, including transcriptional, translational or post-translational expression.

The present invention also provides an isolated cell or non-human vertebrate comprising such cells, wherein the activity of ZBTBl 1 is modified. In some embodiments, the modified cells or organisms are useful as animal models to facilitate the assessment of the role of ZBTBIl or modulators thereof. In some embodiments, agents are tested in animal models to determine their ability to modulate myelopoiesis or conditions associated with ZBTB 11 dysfunction. In some embodiments, the cell or modified vertebrate comprises a temperature sensitive loss of function mutation in ZBTBIl gene. Most conveniently, the vertebrate is a teleost, such as a zebrafish. Temperature sensitive mutations permit the evaluation of the effect of ZBTBIl dysregulation and its reversal at various hours post fertilisation (ages) and stages of development. Methods of screening for ZBTBl 1 agonists and antagonists are provided. In some embodiments, the methods comprise contacting a candidate drug with a model system comprising ZBTBIl or ZBTBIl or a complex comprising same and screening for the presence of binding between the drug and ZBTBIl or a complex comprising same, or screening for a change in function of ZBTBl 1 or a complex comprising same or the ability to form a complex.

Model systems include cellular model systems and animal model systems wherein the ZBTBIl comprises a temperature sensitive mutation.

In some embodiments, the ZBTBI l is zebrafish Zbtbll or zbtbll. In other embodiments the ZBTBIl comprises a variant or mutation responsible for impairing the functionality of the gene product. In some embodiments, the variant comprises one or more substitutions selected from A747T, Al 130T, A1266G, A1588T, C1709G, C2351T, A2483G and C2519T. In other embodiments, the variant comprises the 346T to A mutation.

In another embodiment, a method of identifying an agent that modulates haematopoiesis and especially myelopoiesis is provided. In some aspects, the agents is capable of treating a condition identified herein as associated with ZBTBIl dysregulation

(dysfunction). In some embodiments, the method comprises: (i) contacting the agent with a transgenic teleost such as zebrafish that expresses a reporter protein under the control of a

ZBTBIl expression sequence (promoter), and (ii) assessing the level or distribution of

ZBTB 11 -reporter protein in the teleost compared to controls. In some embodiments, an enhanced level or distribution of ZBTBI l -reporter expression relative to controls is indicative that the agent enhances neutrophil levels and a reduced level or distribution of

ZBTBI l -reporter expression relative to controls is indicative that the agent depresses neutrophil levels. As mentioned above, in some embodiments the ZBTBI l is human, murine, teleost or other vertebrate origin. In other embodiments, the ZBTBl 1 is zebrafish zbtb 11 or Zbtbll.

Combination therapy including the use of a ZBTBl 1 agonist or antagonist and the use of one or more cytokines or bone marrow or stem cell transfusion or other therapeutic modalities also form part of the present invention.

Pharmaceutical compositions comprising an agonist or antagonist of ZBTBI l as described herein are useful in the treatment or prevention of myelopoietic disorders are also contemplated herein.

In another embodiment, the present invention provides a diagnostic or prognostic method comprising assessing ZBTBIl or ZBTBIl in a sample from a subject. In some embodiments, a subject with one or more conditions identified herein as causally linked to a loss of function mutation in ZBTBl 1 is tested for a mutation in ZBTBIl or for a change in the level or activity of ZBTB 11 or a complex comprising same.

The above summary is not and should not be seen in any way as an exhaustive recitation of all embodiments of the present invention. BRIEF DESCRIPTION OF THE FIGURES

Some figures contain colour representations or entities. Coloured versions of the figures are available from the Patentee upon request or from an appropriate Patent Office. A fee may be imposed if obtained from a Patent Office. Figure 1 is a photographic representation depicting haploid (+ or man) zebrafish embryos which have undergone WISH with probes to markers expressed in mesodermal and hematopoietic tissues. The data show represents (A) Dorsal view of a representative 12 hours post fertilisation (hpf) embryo demonstrating normal expression of the mesoderm marker deltaC in 34/34 embryos examined from a single clutch (B) Antero-lateral view of a representative 16hpf embryo demonstrating normal expression of the early myeloid marker pu.1 in 28/28 embryos (C) Lateral view of a representative WT(+) embryo at 40hpf demonstrating normal expression of the mature myeloid marker mpx in 20/38' embryos (D) Lateral view of a representative man embryo at 40hpf demonstrating reduced expression of the mature myeloid marker mpx in 18/38 embryos. Figure 2 is a photographic representation depicting the effect of the man mutation in diploid embryos on markers of various hematopoietic cell types. The data shown represents (A) Lateral view of a representative 20hpf embryo demonstrating normal expression of the hemangioblast marker scl in 29/29 embryos examined in a single clutch (B) Lateral view of a representative 19hpf embryo demonstrating normal expression of the early myeloid marker pu.1 in 22/22 embryos (C) Lateral view of a representative 20hpf embryo demonstrating normal expression of the erythroid marker gatal in 16/16 embryos (D) Lateral view of +/? or man/man 48hpf embryos showing reduced numbers of mpx expressing cells in man/man embryos (E) Lateral view of +/? or man/man 48hpf embryos showing reduced numbers of Sudan black cytochemical stained neutrophils (F) Blood cytospins of erythrocytes from +/? or man/man 48hpf embryos showing normal morphological appearance of man/man erythrocytes.

Figure 3 is a graphical representation of number of mpx expressing cells (assessed by WISH) at 48hpf in +/? and man/man embryos demonstrating a reduction to approximately 38% of normal numbers in man/man embryos. The data show represents means ± standard deviations of 17 embryos (+/?) and 7 embryos (man/ man). Figure 4 is a photograph] cal representation of the gross morphology phenotype of manlman embryos. The data shown represents (A) Lateral view of +/? and man/man embryos at 48hpf demonstrating the abnormalities of the eye, brain and yolk (B) high power view of (A) demonstrating the reduction in eye size, brain abnormality and expansion of the 4 ^th ventricle (hydrocephalus) in man/man embryos (arrowhead).

Figure 5 is a photographical representation showing the temperature sensitive nature of the man allele demonstrating the phenotypic spectrum of the man allele when the temperature is manipulated. The data shown represents (A) Tails of 48hpf embryos which have undergone mpx WISH showing the increase in mpx cell number in man embryos when the temperature is shifted from 28 to 21°C; (B) histograms of myeloid cell numbers as in (A); and (C) histograms of myeloid cell numbers at 28 ⁰ C and 33 ⁰ C enumerated by counting fluorescent spots in Tg(mpx:EGFP) embryos showing further exacerbation of leukopenia in man at 33 ⁰ C and 28°C.

Figure 6 is a graphical representation showing that the man mutation is predicted to cause a cysteine to serine substitution at amino acid 116 (nucleotide 346 T-A substitution). The data shown represents electropherograms showing the nucleotide sequence of exon 2 of zbtb 11 from a +/+ and manlman embryo along with the predicted amino acid sequence. Residue 346 is denoted by an arrowhead.

Figure 7 is a photographical representation of the results of transiently knocking down zbtbll in \g(mpx:GΫP ^' ) zebrafish embryos using a morpholino oligonucleotide (MO) targeted to the start site of zbtbll (ATG MO). The data shown represents (A) gross morphology phenotypes at 48hpf of uninjected embryos or embryos injected at 1 cell stage with control morpholino or zbtbll ATG MO at different doses. Injection of ImM ATG MO produces a highly penetrant phenotype which phenocopies man/man (B) nearly all embryos injected with ImM ATG MO have <50 mpx expressing cells per embryo at 48hpf (C) representative 48hpf embryos injected with (i) control MO showing no effect on development, (H) ImM zbtbll ATG MO or (iii) uninjected manlman embryos showing the phenocopy of the hyprocephalus seen in man/man (arrowheads) and the marked reduction in mpx expressing cells (leukopenia) in the ATG MO injected embryos. Figure 8 is a photographic representation of the results of transiently overexpressing WT or man mRNA in embryos derived from a manl+ tg(mpx:GFP) x man/+ tg(mρx:GFP) cross. The data represent (A) numbers of morphologically WT or man/man embryos at 48hpf in uninjected, man injected or WT injected groups demonstrating the marked reduction in numbers of morphologically man/man embryos when WT RNA is injected (B) numbers of mpx cells per embryo at 48hpf in genotyped +/? (white columns) or man/ man (grey columns) embryos not injected or injected with man or WT mRNA, demonstrating that in embryos genotypically man/man injection of WT mRNA returns mpx cell numbers to +/? levels. Columns represent means, bars standard deviations (C) representative 48hpf genotyped man/man embryos showing gross morphology (left panels) and mpx'-GFJ* expression (right panels) in (i) uninjected (ii) man mRNA injected and (Ui) WT mRNA injected embryos showing normal morphological appearance and mpx cell numbers in a man/man embryo injected with WT mRNA.

Figure 9 is a representation of information regarding the source of publicly available database showing ZBTBIl sequences in different species.

Figure 10 is a graphical representation illustrating the effects of truncation mutants of the Zbtbl l protein in a man rescue assay. Various truncation mutants were compared to zbtbll WT RNA in their ability to rescue the man phenotype when over- expressed in embryos derived from a man/+ tg(mpx:GFP) x man/+ tg(mpx:GFP) cross. The Δ657-1146 mutant was still able to rescue man, however the Δ172-1146 mutant was not.

Figure 11 is a graphical representation illustrating the result of man rescue assays in embryos derived from a manl+ tg(mpx:GFP) x man/+ tg(mpχ-.GFP) cross with putative HHCC motif variants. Functional analysis of residues within the putative HHCC motif in Zbtbl l. The man rescue assay was used to assess the bioactivity of constructs created by site-directed mutagenesis on 4 residues in the HHCC motif (grey shading). The C116S, Cl 19S and H86A proteins were biologically inactive. The changed residues are boxed. BRIEF DESCRIPTION OF THE TABLES Table 1 provides a description of the SEQ ID NOs provided herein. Table 2 provides an amino acid sub-classification. Table 3 provides exemplary amino acid substitutions. Table 4 provides a list of non-natural amino acids contemplated in the present invention.

Table 5 presents data showing the genetic interval containing the man locus. Markers z27232, DC26, zl2094 and zlO183 are simple sequence polymorphism (SSLP) Vi markers. Markers rpl5b exorώ, DC7, zbtbll exon2 and apt exon 4 are single nucleotide polymorphism (SNP) markers which are restriction fragment length polymorphisms (RFLPs). The shaded grey area represents the genetic interval defined by single flanking recombinant embryos which contains the man locus.

Table 6 presents primer sequences and PCR conditions for the markers described in Example 4 and the cloning described in Example 5. Table 7 presents RNA rescue data demonstrating that human ZBTBI l RNA can rescue the man phenotype.

DETAILED DISCUSSION OF PREFERRED EMBODIMENTS

The subject invention is not limited to particular screening procedures for agents, specific formulations of agents and various medical methodologies, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

The term "agent" or "modulatory agent" refers to a compound that induces the 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 "agent" is not to be construed narrowly but extends to proteinaceous molecules such as peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and mimetics and chemical analogs thereof as well as cellular agents. Thus, the term "agent" extends to nucleic acid constructs including vectors such as viral or non- viral vectors, expression vectors and plasmids for expression in and secretion in a range of cells.

The term "derived" does not necessarily mean that the cells are directly obtained from a particular source.

By "biologically active portion" or "part" is meant a portion of a full-length parent peptide or polypeptide or nucleic acid which portion retains an activity of the parent molecule, such as DNA or protein binding or specific hybridisation to a complementary nucleic acid molecule. As used herein, the term "biologically active portion" or "part" includes fragments, deletion mutants and peptides, for example of at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 2O ₅ 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, 350 contiguous amino acids or nucleotides (and every integer in between), which comprise an activity of a reference molecule. One example of a biologically active portion is a form of the polypeptide without a signal or leader sequence. Portions of this type may be obtained through the application of standard recombinant nucleic acid techniques or synthesized using conventional liquid or solid phase synthesis techniques. Reference to a "cell" includes a system of cells such as a particular tissue or organ. Depending upon the context and as will be appreciated by the skilled artisan the

"cell" may be prokaryotic or eukaryotic. Screening methods may employ prokaryotic or eukaryotic cells while treatments and cellular models preferably employ eukaryotic cells. In relation to treating cells, the preferred cell is a human or teleost cell.

The term "complementary" refers to the capability of a single stranded form of an oligonucleotide, polynucleotide or nucleotide sequence to bind to (hybridise with) another oligonucleotide or polynucleotide or nucleotide sequence through specific pairing of bases to form a double stranded stretch of nucleic acid. Oligonucleotides or oligonucleotide subsequence can be described as "complementary" to a target sequence within a polynucleotide, and furthermore, the contact surface characteristics are complementary to each other. The term "complementary" includes base complementarity such as the binding interaction of cytosine and guanine bases or adenine and thymine or uracil bases within double stranded DNA or RNA molecules, polynucleotides or oligonucleotides. The term complementary also includes the predicted complementarity of nucleic acid molecules, oligonucleotides and polynucleotides, where those predictions are derived from the known sequences of those molecules. Base analogs, such as inosine, may also be complementary to natural base residues, and hence, base analogues may be part of a complementary sequence or oligonucleotide. Degenerate base positions within a degenerate oligonucleotide may complement several different natural bases at the same site in a target sequence. Specifically, within the mixture of oligonucleotides that constitute the degenerate oligonucleotide, at one homologous position in the sequence, the different oligonucleotides have different bases. This invention also encompasses situations in which there is non-traditional base-pairing such as Hoogsteen base pairing which has been identified in certain transfer RNA molecules and postulated to exist in a triple helix. In the context of the definition of the term "complementary", the terms "match" and "mismatch" as used herein refer to the hybridisation potential of paired nucleotides in complementary nucleic acid strands. Matched nucleotides hybridise efficiently, such as the classical A-T and G-C base pair mentioned above. Mismatches are other combinations of nucleotides that hybridise less effectively or not at all and these may be incorporated into oligonucleotides or nucleic acid molecules in order to modulate binding efficiencies. The term "gene" is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Reference herein to a "gene" is also taken to include:-a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or an mRNA or cDNA corresponding to the coding regions (i.e. exons), pre- mRNA and 5'- and 3'- untranslated sequences of the gene.

"Regulatory regions" or "expression sequences" include promoters, polyadenylation signals, transcriptional enhancers, translational enhancers, leader or trailing sequences that modulate mRNA stability and targeting sequences that direct a product encoded by a transcribed nucleic acid molecule to a particular location such as an intracellular compartment or to the extracellular environment are known in the art and are employed as appropriate.

The present invention is predicated in part upon the identification, in a teleost animal model of vertebrate development, of a critical role for ZBTB 11 in myelopoiesis (the production of myeloid cells in the bone marrow, such as granulocytes, macrophages and monocytes from myeloid precursors) and particularly the level of neutrophils (a type of granulocyte along with basophils and eosinophils). In accordance with one aspect of the invention, dysregulation of genes important in myelopoiesis or hematopoiesis underlie oncogenic processes and it is proposed that ZBTBI l has an important role in myeloid leukemia. ZBTB 11 is a member of a multigene family of transcription factors referred to as BTB-ZF (Broad complex, Tramtrack, Brie a brae domain-zinc-finger domain) or POZ- ZF (poxvirus and zinc finger domain) proteins. The N-terminus comprises a BTB protein- protein interaction domain preceded by approximately 200 amino acids, while the carboxy terminus comprises twelve Kruppel-like C ₂ H ₂ zinc finger domains, forming a presumptive DNA binding domain, providing ZBTBl 1 with the hall marks of a BTB-ZF transcriptional regulator. Several BTB-ZF genes are associated with myelopoiesis or hematopoiesis and cancer including PLZF (promyelocytic leukemia zinc finger) which is associated with poor prognosis in acute promyelocytic leukemia and ZBTB7 (leukemia related factor, pokemon) associated with regulating pi 9 ^* ^* overexpression in lymphoma. Mutations in the BTB gene, BCL6 are also be associated with cancers. BCL6 is often expressed constitutively in diffuse large B-cell lymphomas and in B-cell non-Hodgkin's lymphomas. Myeloid cells themselves are an important arm of the innate immune system and deficiencies (imbalances) in this system can result in profound immunodeficiencies and immune mediated injury.

The term "teleost" refers to a group of fish having bony skeletons and rayed fins. The group comprises zebrafish (Danio rerid), medaka, Giant rerio and puffer fish. These fish and in particular the zebrafish have proven to be useful vertebrate animal models of human and animal development. As reviewed by Carradice et ah, Blood Hl(J): 3331- 3342, 2008 zebrafish are affordable, genetically tractable vertebrates that exhibit rapid ex vivo development, high fecundity and optical transparency. Zebrafish forward genetic screens have identified several genes useful for hematology and disease modelling. As reviewed by Bennett et at, Blood 98(3): 643-651, 2008, it is established that zebrafish provide a useful animal model of normal and aberrant human myelopoiesis, not least because the molecular mechanisms including regulating transcription factors appear to be conserved. Zebrafish are particularly suited as models to study in vivo myelopoiesis because their small size also makes them suitable for high throughput screening assays (Bums et ah, Nat. Chem. Biol. 1: 263-264, 2005). Zebrafish neutrophils are detectable from about 48 hours post fertilisation and the innate immune system exists in isolated from the adaptive immune response system until approximately 4 weeks post fertilisation.

In vitro or in vivo assays can employ a wide range of markers or indicators of target activity using, for example, the methods exemplified herein. For example, myeloid cells may be monitored using fluorescent or illuminescent markers, labelled probes and in situ hybridisation techniques; transcriptional activity may, for example, be assessed by measuring the level of specific RNA produced or may be assessed via measuring the activity of a reporter molecule. Reference herein to the "activity" or "overactivity" and the like, in relation to vertebrate cells include without limitation a reference to any one or more of the following: cellular development, proliferation, cellular differentiation, cell function such as homing, engraftment, self-renewal, survival differentiation, cell number and cell survival. As the skilled artisan will appreciate, the self renewal phenotype of stem cells is tightly regulated. As stem cells divide their daughter cells maintain a critical balance between two fates: either retaining stem cell function, or alternatively differentiating into mature effector cells. There are three potential outcomes or activities of stem cell division (i) extension symmetric division where both daughter cells retain stem cell function and which results in expansion of stem cell population (ii) maintenance asymmetric division which maintains the stem cell population by producing one daughter stem cell and one daughter cell committed to differentiation and (iii) committed symmetric division where both daughter cells are committed to differentiate. Accordingly, in relation to myeloid precursors reference to "activity" includes reference to extension symmetric division, maintenance asymmetric division and/or committed symmetric division.

The activity of ZBTBI l may also be monitored using DNA or protein binding assays, reporter assays or direct or indirect assays of ZBTBl 1 activity including the use of antibodies or other proteinaceous or genetic agents in a number of assays which are well known to those of skill in the art. Antibodies, for example, may be used to detect ZBTB 11 by Western Blotting, cytometric histochemical or ELISA procedures. As discussed herein below, such agents may also distinguish between active and inactive forms of the ZBTBl 1 or between mutant and normal forms of ZBTBl 1.

In accordance with some embodiments, mutant forms of ZBTBIl are forms of ZBTBIl (found for example in a population of subjects) which are associated or linked with aberrant or insufficient myelopoiesis or in haematological malignancies. Myelopoietic disorders and haematological malignancies include both myeloid and lymphocytic leukemia, such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), chronic myelomonocytic leukaemia (CMML), chronic neutrophilic leukaemia, chronic eosinophilic leukaemia and hairy cell leukemia, and lymphoma, such as Hodgkin's disease, Non-Hodgkin lymphoma and multiple myeloma. . In other embodiments, mutant forms of ZBTBI l are associated or linked with congenital or acquired cerebral deficiencies including hydrocephalus, eye and spinal cord degeneration. In some embodiments, normal forms of ZBTBIl are forms of ZBTBI l which are not associated with these conditions. Mutant forms of ZBTBIl may also be conveniently be detected using nucleic acid based assays well known in the art and as described herein. Low levels of active polypeptide may be produced as a result of mutations in ZBTBIl leading to altered expression levels, altered transcript stability or altered functional activity. Thus, ZBTBI l activity may be monitored indirectly by monitoring RNA production and/or stability or the levels of regulatory molecules such as enhancers and repressors. The activity of ZBTBl 1 may be monitored using an in vivo teleost bioassay. In some embodiments mutant forms of mammalian ZBTBIl, or similarly mutagenesised forms of zfZBTBl l are injected into the embryo or adult at an appropriate stage or under suitable conditions to effect a phenotype where the readout for functionality is an increase in the number of neutrophils or cells expressing a neutrophil marker such as myeloperoxidase or other such marker. Alternatively, overexpression may be achieved at later timepoints by transgenesis in which ZBTBIl overexpression is driven by a later promoter or by an inducible promoter (e.g. tet-on or tet-off system), allowing screening of drug effects at later timepoints following endogenous or exogenous activation of the promoter driving ZBTBIl overexpression. Again, an expected readout for normal overexpression of ZBTBIl may be expansion of Mpx +ve cells by WISH or cytometrically, while embryos treated with a drug targeting ZBTB 11 function may show a reduction or dysfunction in myelopoiesis.

A substantially modified level or activity of ZBTB 11 is conveniently assessed in terms of a percent reduction relative to normal cells or animals or pre-treatment/pre- administration. A substantial increase includes one which results in detectable myelopoiesis (including neutrophil activity) in a subject or cell activity. Alternatively, a reduced level of gene expression of transcription targets or a reporter thereof is detected. Preferably, the modification is at least 20% enhanced or reduced compared to normal cells, more preferably about 25%, still more preferably at least about 30% reduction, more preferably at least about 40% enhanced or reduced ZBTBI l level or activity. The reduction may of course be complete loss of ZBTBI l activity in a cell or animal. A "modified" level or activity includes enhanced levels of ZBTBI l activity relative to pre- treatment levels and may equate to or exceed the level or activity of ZBTBl 1 detectable in controls.

The present invention has been exemplified with zebrafish genes as zebrafϊsh provide an ideal animal model for studying haematopoiesis and myelopoiesis and haematopoietic and myelopoietic disorders. In the light of the high level of functional and structural conservation between zebrafish and mammalian genes involved in myelopoiesis, the present invention clearly extends to homologs (orthologs) from other vertebrate groups such as murine and human and other animals. For the avoidance of doubt, in the description and claims reference to a gene or protein such as ZBTBI l or ZBTBIl, unless expressly stated otherwise, encompasses homologs or isoforms in any vertebrate species including, in particular, human homologs, murine homologs, teleost homologs and homologs of veterinary interest. Polypeptides or peptides having an activity of human ZBTB 11 polypeptide are particularly contemplated.

In one aspect the invention provides a method of treating or preventing a condition associated with ZBTBI l dysregulation in a subject, said method comprising administering a composition that comprises an agent that modulates the level or activity of ZBTBI l polypeptide or a complex comprising same, or the level or activity of ZBTBIl polynucleotide in the subject.

Reference herein to "modulate" and "modulation" includes completely or partially inhibiting or reducing or down regulating all or part of ZBTBIl functional activity and enhancing or up regulating all or part of its functional activity. Functional activity may be modulated by, for example, modulating ZBTBIl nucleic acid binding capabilities or transcriptional or translational activity, or its half-life. With regard to ZBTBIl polypeptide, its functional activity may be modulated by, for example, modulating its DNA protein or ion binding capabilities, its ability to modulate transcription of its transcriptional targets, its half-life, and/or its location in a cell. Thus, ZBTB 11 level or activity may be modulated by modulating ZBTBIl expression, transcript stability, or the activity of its regulatory molecules.

Reference to the "activity" or "functional activity" of ZBTBI l or ZBTBIl encompasses any relevant, measurable activity or characteristic of the molecule in proteinaceous or genetic form and includes ZBTB 11 -specific DNA, protein and ion binding abilities as well as the ability to produce (express) a protein having a functional activity of ZBTBI l polypeptide. As disclosed herein, ZBTBI l is critical for myelopoiesis, including the production of myeloperoxidase containing neutrophils and the presently described in vivo assays in teleosts provide a useful method of assessing ZBTBIl or ZBTBIl function. Binding or transcriptional, translational or transactivational activity are preferred ZBTBI l activities which are conveniently assessed using standard protocols known in the art as described in Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHLP, CSH, NY, 2001; Ausubel (Ed), Current Protocols in Molecular Biology, 5 ^th Edition, John Wiley & Sons, Inc. NY, 2002. For example, the ability of ZBTBl 1 to bind to promoters and modulate (drive or repress) transcription is tested using luminescence reporter assays using, for example an artificial promoter containing ZBTBl 1 consensus binding sites.

The ability of ZBTBI l to modulate cellular activities such as proliferation, development or survival can be assessed visually, spectroscopically, or using instrumentation to evaluate the presence, level or activity of a molecular marker or reporter of the activity. Neutrophil and neutrophil precursor proliferation, migration, differentiation and development can all be monitored using methods known in the art. Other activities assayed include neutrophil and neutrophil precursor presence, homing, engraftment, apoptosis and self-renewal. Functional activity, in some embodiments is assessed by analysing cells comprising ZBTBIl for expression of myeloperoxidase for example, by whole mount in situ hybridisation or by flow cytometry, or by using a commercially available myeloperoxidase assay kit. Myeloperoxidase can also be detected by fluorescence using tg(mpx:GFP).

The ability of ZBTBI l to bind DNA via its C-terminal zinc finger binding domains may also be tested. The ability of ZBTB 11 polypeptide to bind to proteins via the N-terminal BTB domain may also be also tested. ZBTBI l polypeptide stimulates a characteristic gene expression profile which serves as a useful marker of ZBTBl 1 activity. Similarly, the gene expression profile of a cell when ZBTBI l polypeptide activity is down-regulated or inhibited serves as a useful marker of lack of ZBTBI l activity. Such assays may be conveniently adapted for high throughput evaluation, for example, cytometrically such as by flow cytometry, array technology such as microarray technology, antibody technology, chromato graphic methods such as HPLC or thin layer chromatography or combinations of these. Binding is conveniently detected using antibodies. Any subject who could benefit from the present methods or compositions is encompassed. The term "subject" includes, without limitation, humans and non-human priniates, livestock animals, companion animals, laboratory test animals, captive wild animals, reptiles and amphibians, fish, birds and any other organism. The most preferred subject of the present invention is a human subject. A subject, regardless of whether it is a human or non-human organism may be referred to as a patient, individual, subject, animal, host or recipient.

Agents in accordance with this aspect of the invention may directly interact with ZBTBI l. Here, for example, small molecules, antibodies or peptides, peptidomimetics or analogs and other such molecules may be conveniently employed. Alternatively, genetic mechanisms are used to target ZBTBIl to indirectly modulate the activity of ZBTBI l. Again, various strategies are well documented and include mechanisms for pre- or post- transcriptional silencing pre-translated, translational or post-translational silencing. The use or expression of antisense molecules, dominant negative, or co-suppression or RNAi or siRNA strategies are particularly contemplated.

The modulatory agents of the present invention may be chemical agents such as small or large organic or inorganic chemical molecules, peptides, antibodies, polypeptides including dominant negative forms, modified peptides such as constrained peptides, foldamers, peptidomimetics, stapled peptides, cyclic peptidomimetics, proteins including minature proteins, lipids, carbohydrates or nucleic acid molecules including antisense or other gene silencing or activating molecules. Small molecules generally have a molecular mass of less than 500 Daltons. Large molecules generally include whole polypeptides or other compounds having a molecular mass greater than 500 Daltons. Agents may comprise naturally occurring molecules, variants (including analogs) thereof as defined herein or non-naturally occurring molecules. Gene modulatory or silencing agents (genetic agents) such as DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs ₅ mRNAs, tRNAs, rRNAs, small interfering RNAs (SiRNAs), Piwi-interacting RNA (piRNA), short hairpin RNAs (ShRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs)) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes may be employed. siRNA and mRNA are also employed to increase gene transcription by binding to parts of promoter sequences. In some embodiments, agents that modulate the activity of ZBTBI l may be derived from ZBTBI l or their encoding sequences or are variants or analogs thereof. Thus, for example, agents may be hydrocarbon-stapled peptides or minature proteins which are alpha-helical and cell-penetrating, and are able to disrupt molecular interactions (see for example, Wilder et al, ChemMedChem. _?(8):1149-1151, 2007; & for a review, Henchey et al, Curr. Opin. Chern. Sept 12, 2008). In one particular example, therapeutic antibodies having the capacity for intracellular transmission are contemplated. In some embodiments, antibodies agonise the activity of ZBTB 11 in promoting myelopoiesis or CNS formation or repair. These include antibodies such as shark antibodies, camelids and llama antibodies, scFv antibodies and intrabodies or nanobodies, e.g. scFv intrabodies and V _H H intrabodies. Such antigen binding agents can be made as described by Lui et al., BMC Biotechnol, 7: 78, 2007; Harmsen and De Haard, Appl. Microbiol. Biotechnol. 77(1): 13-22, 2007; Tibary et al., Soc. Reprod. Fertil Suppl., 64: 297-313, 2007; Muyldermans, J Biotechnol. 74: 277-302, 2001; and references cited therein.

In one embodiment, scFv intrabodies which are able to interfere with a protein- protein or protein nucleic acid interaction are used in the methods of the invention; see for example, Visintin et al., J. Biotechnol, 135: 1-15, 2008 and Visintin et al, J. Immunol. Methods, 290(1 -2): 135-53, 2008 for methods for their production. For use in the methods of the invention, such agents may comprise a cell-penetrating peptide sequence or nuclear- localizing peptide sequence such as those disclosed in Constantini et al, Cancer Biotherm. Radiopharm. 23(1): 3-24, 2008. Also useful for in vivo delivery are Vectocell or Diato peptide vectors such as those disclosed in De Coupade et al, Biochem J. 390(pt2): 407- 418, 2005 and Myer-Losic et al, 49(23): 6908-6916, 2006. Thus, the invention provides the therapeutic use of fusion proteins of the agents (or functionally active fragments thereof), for example but without limitation, where the antibody or fragment thereof is fused via a covalent bond (e.g. a peptide bond), at optionally the N-terminus or the C- terminus, to a cell-penetrating peptide or nuclear-localizing peptide sequence. Alternatively, targeting moieties may be chemically attached to the agent.

Libraries of small molecules suitable for testing are available in the art (see for example, Amezcua et al, Structure (London) 10: 1349-1361, 2002). The latter are described in International Patent Publication No. WO 2005/118629. Yeast SPLINT antibody libraries are available for testing for intrabodies which are able to disrupt protein- protein interactions (see Visintin et al (supra)). Examples of suitable methods for the synthesis of molecular libraries can be found in the art, for example in: De Witt et al, Proc. Natl. Acad. Sci. USA 90: 6909, 1993; Erb et al, Proc. Natl. Acad. Sci. USA 91: 11422, 1994; Zuckermann et al, J. Med. Chem. 37: 2678, 1994; Cho et al, Science, 261: 1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33: 2059, 1994; and Gallop et al, J. Med. Chem. 37: 1233, 1994. Thus, agents can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is suited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997; U.S. 5,738,996; and U.S. 5,807,683). Libraries of compounds may be presented, for example, in solution (e.g. Houghten, Bio/Techniques 13: 412-421, 1992), or on beads (Lam, Nature, 354: 82-84, 1991), chips (Fodor, Nature, 364: 555-556, 1993), bacteria (US 5,223,409), spores (US 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. Sci. USA, 89: 1865- 1869, 1992) or phage (Scott and Smith, Science, 249: 386-390, 1990; Devlin, Science, 249: 404-406, 1990; Cwirla et al, Proc. Natl. Acad. Sci. USA, 87: 6378-6382, 1990; and Felici, J MoI. Biol, 222: 301-310, 1991).

A potential modulator may be evaluated "in silico" for its ability to bind to a ZBTBI l active site prior to its actual synthesis and testing. The quality of the fit of such entities to binding sites may be assessed by, for example, shape complementarity by estimating the energy of the interaction. (Meng et al, J. Comp. Chem., 13: 505-524, 1992). The design of chemical entities that associate with proteins generally involves consideration of two factors. The compound must be capable of physically and structurally associating with ZBTBIl. Non-covalent molecular interactions important in the association with their interacting partners include hydrogen bonding, van der Waal's and hydrophobic interactions. Second, the compound must be able to assume a conformation that allows it to associate with a ZBTBI l polypeptide. Although certain portions of the compound will not directly participate in this association, those portions may still influence the overall conformation of the molecule. Such conformation requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the active site, or the spacing between functional groups of a compound comprising several chemical entities that directly interact. Once a binding compound has been optimally selected or designed, as described above, substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties. Generally, initial substitutions are conservative, i.e. the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should of course, be understood that components known in the art to alter conformation should be avoided.

Putative binding agents may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the one or more binding sites. Selected fragments or chemical entities may then be positioned in a variety of orientations, or "docked," to target binding sites. Docking may be accomplished using software, such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM or AMBER. Specialised computer programs may be of use for selecting interesting fragments or chemical entities. These programs include, e.g., GRID (Oxford University, Oxford, UK) 5 MCSS (Molecular Simulations, USA) AUTODOCK (Scripps Research Institute, USA) DOCK (University of California, USA) and XSITE (University College of London, UK) and CATALYST (Accelrys).

Useful programs to aid the skilled addressee in connecting chemical entities or fragments include CAVEAT (University of California, USA) ₅ 3D database systems and HOOK (Molecular Simulations, USA) De-novo ligand design methods include those described in LUDI (Molecular Simulations, USA), LEGEND (Molecular Simulations, USA), LeapFrog (Tripos Inc.,) SPROUT (University of Leeds, UK) and the like.

Structure based ligand design is well known in the art and various strategies are available that can build on structural information to determine ligands which effectively modulate the components of ZBTBIl. Molecular modelling techniques include those described by Cohen et al, J Med. Chem., 55:883-894, 1990, and Navia et al, Current Opinions in Structural Biology, 2: 202-210, 1992. Standard homology modelling techniques may be employed in order to determine the unknown three-dimensional structure or molecular complex. Homology modelling involves constructing a model of an unknown structure using structural coordinates of one or more related protein molecules, molecular complexes or parts thereof. Homology modelling may be conducted by fitting common or homologous portions of the protein whose three-dimensional structure is to be solved to the three-dimensional structure of homologous structural elements in the known molecule. Homology may be determined using amino acid sequence identity, homologous secondary structure elements and/or 1 i , homologous tertiary folds. Homology modelling can include rebuilding part or all of a three-dimensional structure with replacement of amino acid residues (or other components) by those of the related structure to be solved.

Using such a three-dimensional structure, researchers identify putative binding sites and then identify or design agents to interact with these binding sites. These agents are then screened for a modulatory effect upon the target molecule. In some embodiments, binding agents are designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably not greater than 7 kcal/mole. Computer software is available to evaluate compound deformation energy and ectrostatic interactions. For example, Gaussian 98, AMBER, QUANTA, CHARMM, INSIGHT II, DISCOVER, AMSOL and DelPhi. As illustrated in the Examples, in some embodiments, the agent increases the level or activity of ZBTBl 1 polypeptide or of a complex comprising same or the level of ZBTBIl polynucleotide in the subject. Conveniently, the composition may comprise a polynucleotide encoding a polypeptide having ZBTBl 1 activity.

Representative examples of the nucleic acid and amino acid sequences of ZBTBI l molecules are provided in the sequence listing and Figures and are further described in Table 1. Human, mouse, rat, dog, Xenopus, cow, and zebrafish Zbtbl l protein are more than 55% identical. Alignments of amino acid and nucleotide sequences of various vertebrate ZBTBI l orthologs can be routinely generated by those of normal skill in the art and show regions of conservation and identity. There is also a high level of structural conservation between BTB and zinc finger domains of ZBTBI l orthologs. In some embodiments, a ZBTBI l polypeptide has at least 60% identity to all or a reference part of the sequence of SEQ ID NO: 2 (polypeptide encoded by cDNA zebrafish zbtbll) or SEQ ID NO: 4, more preferably it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity. In other embodiments, a ZBTBI l polynucleotide encodes a polypeptide comprising at least SEQ ID NO: 2 or SEQ ID NO: 4. Suitable nucleotide sequences are set out in SEQ ID NOs: 1 and 3. Variants are also encompassed and include, of course, nucleotide substitutions that have no effect on the polypeptide sequences or substitutes that provide for conservative amino acid change.

Agents which modulate the level or activity of ZBTBI l or ZBTBIl may be derived from ZBTBI l or ZBTBIl. Such molecules may be isolated versions of naturally occurring molecules, or they may be modified, synthetic or recombinant forms that are variants of naturally occurring forms. Reference to polynucleotide sequences include reference to their complementary forms.

A ZBTBIl nucleic acid encoding a ZBTBl 1 polypeptide, including homologs and orthologs from species other than human, and complementary forms may be obtained by a process which comprises the steps of screening an appropriate library under stringent hybridisation conditions with a labeled probe having the sequence of a desired nucleic acid and isolating full-length cDNA and genomic clones containing said nucleic acid sequence. Such hybridisation techniques are well known in the art. One example of stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65 ⁰ C using a salt solution of about 0.9M. However, the skilled person will be able to vary such conditions as appropriate in order to take into account variables such as probe length, base composition, type of ions present, etc. For a high degree of selectivity, relatively stringent conditions such as low salt or high temperature conditions, are used to form the duplexes. Highly stringent conditions include hybridisation to filter-bound DNA in 0.5M NaHP04, 7% sodium dodecyl sulphate (SDS), ImM EDTA at 65°C, and washing in O.lxSSC/0.1% SDS at 68 ⁰ C (Ausubel F.M. et al, eds., Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3, 1989). For some applications, less stringent conditions for duplex formation are required. Moderately stringent conditions include washing in 0.2xSSC/0.1% SDS at 42°C (Ausubel et al., 1989 (supra)). Hybridisation conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex. Thus, particular hybridisation conditions can be readily manipulated, and will generally be chosen as appropriate. In general, convenient hybridisation temperatures in the presence of 50% formamide are: 42 ⁰ C for a probe which is 95-100% identical to the fragment of a gene encoding a polypeptide as defined herein, 37 ⁰ C for 90-95% identity and 32°C for 70-90% identity.

One skilled in the art will understand that, in many cases, an isolated cDNA sequence will be incomplete, in that the region coding for the polypeptide is cut short at the 5' end of the cDNA. Methods to obtain full length cDNAs or to extend short cDNAs are well known in the art, for example RACE (Rapid amplification of cDNA ends; e.g. Frohman et al, Proc. Natl Acad. Sci USA 85: 8998-9002, 1988). Recent modifications of the technique, exemplified by the Marathon® technology (Clontech Laboratories Inc.) have significantly simplified the search for longer cDNAs. This technology uses cDNAs prepared from mRNA extracted from a chosen tissue followed by the ligation of an adaptor sequence onto each end. PCR is then carried out to amplify the missing 5 '-end of the cDNA using a combination of gene specific and adaptor specific oligonucleotide primers. The PCR reaction is then repeated using nested primers which have been designed to anneal with the amplified product, typically an adaptor specific primer that anneals further 3' in the adaptor sequence and a gene specific primer that anneals further 5' in the known gene sequence. The products of this reaction can then be analysed by DNA sequencing and a full length cDNA constructed either by joining the product directly to the existing cDNA to give a complete sequence, or carrying out a separate full length PCR using the new sequence information for the design of the 5' primer.

Recombinant ZBTBl 1 polypeptides may be prepared by processes well known in the art from genetically engineered host cells comprising expression systems. The appropriate nucleic acid sequence may be inserted into an expression system by any variety of well known and routine techniques, such as those set forth in Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbour Laboratory Press, Cold Spring Harbour, NY, 1989. If a polypeptide is to be expressed for use in cell-based screening assays, the appropriate nuclear targeting signal should be incorporated. If the polypeptide is secreted into the medium, the medium can be recovered in order to isolate said polypeptide. If produced intracellularly, the cells must first be lysed before the polypeptide is recovered.

Polypeptides can be recovered and purified from recombinant cell cultures or from other biological sources by well known methods including, ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, affinity chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, molecular sieving chromatography, centrifugation methods, electrophoresis methods, lectin chromatography, FPLC and HPLC. In one embodiment, a combination of these methods is used. In other exemplified embodiment, the agent decreases the level or activity of

ZBTBI l polypeptide or ZBTBIl polynucleotide in the subject. In this embodiment, the composition may conveniently comprise a small molecule, antibody, peptide, or inhibitory polynucleotide molecule that effectively down regulates ZBTBI l activity by preventing ZBTBIl polypeptide function or by preventing ZBTBIl expression, including transcriptional, translational or post-translational expression.

Antisense polynucleotide sequences are useful agents in preventing or reducing the expression of ZBTBIl.

Antisense molecules may interfere with any function of a nucleic acid molecule. The functions of DNA to be interfered with can include replication, transcription and translation. 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 the ZBTBIl gene, i.e. leading to reduced levels or activity of ZBTBIl polypeptide.

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. In the context of the subject invention, the term "oligomeric compound" refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, 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 "genetic material", "genetic forms", "nucleic acids", "nucleotide" and "polynucleotide" include RNA, cDNA, genomic DNA, synthetic forms and mixed polymers, both sense and antisense strands, and may be chemically or biochemically modified or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog (such as the morpholine ring), internucleotide modifications such as uncharged linkages (e.g. methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.), charged linkages (e.g. phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g. polypeptides), intercalators (e.g. acridine, psoralen, etc.), chelators, alkylators and modified linkages (e.g. α-anomeric nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen binding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.

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 genetic agents, polypeptides, peptides or parts or compositions in accordance with this invention preferably comprise from about 8 to about 80 nucleobases or amino acid residues (i.e. from about 8 to about 80 linked nucleosides or amino acid residues). 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, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases or amino acid residues in length.

In another embodiment, the present invention provides an isolated cellular agent, nucleic acid or vector comprising a polynucleotide encoding a polypeptide having ZBTBI l polypeptide activity for use in the treatment or prevention of a condition associated with ZBTB 11 dysregulation.

In another embodiment, the invention provides an isolated cellular agent, nucleic acid or vector comprising a ZBTBIl polynucleotide that inhibits the expression of ZBTB 11 polynucleotide for use in the treatment or prevention of a condition associated with ZBTBI l dysregulation. A ZBTBIl polynucleotide is contemplated herein for use in the treatment or prevention of a condition associated with ZBTBI l dysregulation. Additionally, ZBTBIl polynucleotides are contemplated for use in the diagnosis of a condition associated with ZBTBIl dysregulation. For diagnostic applications, the polynucleotide is optionally attached to a molecule that serves directly or indirectly as a reporter for the presence or quantification of the polynucleotide.

Antibodies and aptamers provide diagnostic as well as therapeutic and preventative embodiments. In one broad embodiment, an antibody or aptamer determined by a polypeptide having ZBTB 11 activity is proposed for use in the treatment, prevention or diagnosis of a condition or susceptibility to a condition associated with ZBTBI l dysregulation.

As mentioned previously herein, the agents or compositions of the present invention may be ZBTBl 1 or parts thereof, or ZBTBIl or parts thereof or complementary forms or molecules derived or designed from ZBTBl 1 or ZBTBIl.

Thus, in some embodiments, the present invention provides a composition comprising ZBTBl I or a functional variant or ZBTBI l or an agent from which either or these is producible which substantially enhances the activity of ZBTBH. In some embodiments said compositions are for use in modulating myelopoiesis.

The terms "agonist", "antagonist", "modifier", "compound", "drug", "composition" and terms such as "agent", "medicament", "active" and "drug" are used interchangeably herein to refer to a chemical compound or cellular composition which induces a desired pharmacological and/or physiological effect. The terms encompass pharmaceutically acceptable and pharmacologically active ingredients including but not limited to salts, esters, amides, pro-drugs, active metabolites, analogs and the like. The term includes genetic and proteinaceous or lipid molecules or analogs thereof as well as cellular compositions as previously mentioned. The instant compounds and compositions are suitable for the manufacture of a medicament for the treatment and/or prevention of conditions associated with myelopoietic disorders, as described herein.

In relation to cellular compositions, the present invention extends to cellular compositions including genetically modified myeloid precursor cells which are capable of regenerating tissues and/or organs of an animal subject in situ or in vivo. Stem cells or stem cell-like cells may be multipotent or pluripotent however the ability to repopulate the myeloid system is sufficient. Other cellular compositions comprise vectors such as viral vectors for delivery of nucleic acid constructs as described herein.

In relation to ZBTBIl, the terms "functional form" or "variant", "functionally equivalent derivative" or "homolog" include molecules which selectively hybridize to ZBTBIl or a complementary form thereof over all or part of the genetic molecule under conditions of low or medium stringency at a defined temperature or range of conditions, or which have about 60% sequence identity to a nucleotide sequence encoding ZBTBIl polypeptides. Exemplary ZBTBIl nucleotide sequences include those comprising or complementary to the nucleotide sequences set forth in SEQ ID NO: 1 or 3 (Zebrafish zbtbll cDNA) or SEQ ID NO: 5 (human ZBTBI l) or their complements or polynucleotides that hybridise to all or a reference part of the sequence under conditions of medium stringency as known in the art. It should be noted that the term "ZBTBH" or "zbtbll" expressly encompasses all forms of the gene including regulatory regions and genomic forms or specific fragments and constructs comprising same, or parts thereof whose sequences are publicly available.

Medium stringency conditions may be applied where necessary and 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 _n , = 69.3 + 0.41 (G+C)% (Marmur et al., J. MoI. Biol. 5: 109, 1962). However, the T _m of a duplex DNA decreases by I ⁰ C with every increase of 1% in the number of mismatch base pairs (Bonner et al, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42 ⁰ C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 2O ⁰ C to 65 ⁰ C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65 ⁰ C.

ZBTBI l polypeptides include all biologically active naturally occurring forms of ZBTBI l as well as biologically active portions thereof and variants and derivatives of these. Biological activity as determined herein includes modifying progenitor cell activity, the development of myeloid systems, modifying early myelopoiesis as shown herein in a teleost animal model, and potentiating transcription of transcriptional targets. The terms functional form or variant, functionally equivalent derivatives or homologs include polypeptides comprising a sequence of amino acids having at least about 60% sequence identity to ZBTBl 1 or to one or more functional domains of ZBTBl 1 such that at least one functional activity of ZBTB 11 is maintained.

Derivatives and variants are molecules which exhibit at least one biologically relevant function of the naturally occurring polypeptide such as DNA binding (such as via the zinc finger domains) or protein binding (such as via the BTB domain).

Exemplary ZBTBIl amino acid sequences include those comprising sequences set forth in SEQ ID NO: 2 (Zebrafish Zbtbl 1) or SEQ ID NO: 6 (human ZBTBl 1). Representative examples of the nucleic acid and amino acid sequences of ZBTBI l molecules are provided in the sequence listing, further described in Figure. Human, mouse and zebrafish ZBTBI l protein show a substantial level of sequence identity or similarity. Accordingly, the terms ZBTBI l or ZBTBI l in the claims encompass all homologs and isoforms in any animal species including human homologs and isoforms and homologs of veterinary interest. Preferably, a homolog of ZBTBI l or ZBTBl 1 has at least 60% identity to the amino acid sequences of SEQ ID NO: 2 or 4 or 6, more preferably it has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% identity. Similar levels of identity are contemplated for the encoding polynucleotide sequences set out in SEQ ID NOs: 1, 3 and 5.

A biologically active variant of a ZBTBI l (including Zbtbl l) polypeptide may differ from that polypeptide or parts thereof generally by as much as 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6- 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.

A ZBTB 11 polypeptide/peptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a ZBTBI l polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985, Kunkel et al, Methods in Enzymol, 154: 361- 382, 1987, U.S. Pat. No. 4,873,192, Watson et al Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987 and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al., Natl. Biomed. Res. Found, 5: 345-358, 1978. Methods for screening gene products of combinatorial libraries made by point mutations or truncation, and for screening cDNA libraries for gene products having a selected property are known in the art. Such methods are adaptable for rapid screening of the gene libraries generated by combinatorial mutagenesis of polypeptides. Recursive ensemble mutagenesis (REM), a technique that enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify useful polypeptide variants (Arkin et al, Proc. Natl. Acad. Sci. USA, 89: 7811- 7815, 1992; Delgrave et al, Protein Engineering, 6: 327-331, 1993). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be desirable as discussed in more detail below. Variant ZBTBl 1 polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference amino acid sequence. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub- classified as follows:

Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.

Basic: The residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having a basic side chain include arginine, lysine and histidine.

Charged: The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium. Amino acids having a neutral/polar side chain include asparagine, glutamme, cysteine, histidine, serine and threonine.

This description also characterises certain amino acids as "small" since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity. With the exception of proline, "small" amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not. Amino acids having a small side chain include glycine, serine, alanine and threonine. The gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains. The structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the α-amino group, as well as the α-carbon. Several amino acid similarity matrices (e.g., PAMl 20 matrix and PAM250 matrix as disclosed for example by Dayhoff et ah, 1978 (supra); and by Gonnet et al, Science, 25(5(5062): 1443-1445, 1992), however, include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.

Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large. The residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not. Small residues are, of course, always nonaromatic. Dependent on their structural properties, amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 2.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamme; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting variant polypeptide. Whether an amino acid change results in a functional ZBTB 11 polypeptide can readily be determined by assaying its activity. Activities that can readily be assessed are known to those of skill and include assays to determine binding or dimerization, enzyme activity, niethylation, transcription repression detected by, for example, Biocore, kinetic, affinity chromatography and pull-down and receptor analyses. The structural impact of modifying a polypeptide may also be analysed in silico. Conservative substitutions are shown in Table 3 below under the heading of exemplary substitutions. More preferred conservation substitutions are shown under the heading of preferred substitutions. Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.

Alternatively, similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains. The first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains; the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine; and the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, Third Edition, Wm.C. Brown Publishers, 1993.

Thus, a predicted non-essential amino acid residue in a ZBTBI l polypeptide is typically replaced with another amino acid residue from the same side chain family. Alternatively, mutations can be introduced randomly along all or part of the polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity. Following mutagenesis of the coding sequences, the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined. Accordingly, the present invention also contemplates variants of the naturally- occurring ZBTBI l polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues. In general, variants will display at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identity to a reference ZBTBI l polypeptide sequence as, for example, set forth in SEQ ID NOs: 1 or 3. Moreover, sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more amino acids but which retain certain properties of the reference polypeptide are contemplated. The present variant polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to polynucleotide sequences, or the non-coding strand thereof. Particular variants of SEQ ID NO:1 include single point mutations at any one or more selected from nucleotide positions A747T, Al 130T, A1266G, A1588T, C1709G, C2351T, A2483G and C2519T.

In some embodiments, variant polypeptides differ from a ZBTBI l polypeptide sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s). In another, variant polypeptides differ from the corresponding sequence in any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. If this comparison requires alignment the sequences should be aligned for maximum similarity. ("Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a nonessential residue or a conservative substitution. A sequence alignment for ZBTBIl proteins from a range of vertebrate species can be used to demonstrate conserved residues (See Figure 9 for other vertebrate species Accession Numbers).

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities. Suitably, the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type. An "essential" amino acid residue is a residue that, when altered from the wild- type sequence of an polypeptide agent of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.

Percentage identity is a well known concept in the art and can be calculated using, for example but without limitation, the BLAST software available from NCBI (Altschul et al, J. MoI Biol. 215: 403-410, 1990; Gish and States, Nature Genet. 3: 266-272, 1993, Madden et al, Meth. Enzymol. 266: 131-141, 1996; Altschul et ah, Nucleic Acids Res. 25: 3389-3402, 1997; Zhang and Madden, Genome Res. 7: 649-656, 1997). Preferably, the percent identity between a particular nucleotide sequence and a reference sequence is at least about 65% or at least about 70% or at least about 80% or at least about 85% or more preferably at least about 90% similarity or above such as at least about 95%, at least 96%, at least 97%, at least 98%, at least 99% or greater. Percent identities between 60% and 100% are encompassed.

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, 1997 (supra). 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, Chapter 15, 1994-1998.

A percentage of sequence identity between nucleotide sequences, 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) 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 for amino acid sequences. The term "derivative" or the plural "derivatives" whether in relation to genetic or proteinaceous molecules includes, as appropriate, parts, mutants, fragments, and analogues as well as hybrid, chimeric or fusion molecules and glycosylation variants. Particularly useful derivatives retain at least one functional activity of the parent molecule and comprise single or multiple amino acid substitutions, deletions and/or additions to the ZBTBI l amino acid sequence. Preferably, the derivatives have functional activity or alternatively, modulate ZBTBI l functional activity. The term "modulate" includes up-modulate or up-regulate and down-modulate or down-regulate.

As used herein reference to a part, portion or fragment of ZBTBIl 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 to 35 as well as greater than 35 nucleotides. Thus, this definition includes nucleic acids of 12, 15, 20, 25, 40, 60, 100, 200, 500 nucleotides of nucleic acid molecules having any number of nucleotides between 500 and the number shown in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5 or a complementary form thereof. The same considerations apply mutatis mutandis to any reference herein to a part, portion or fragment of ZBTBl 1 polypeptide. Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage without the loss of other functions or properties. Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. Preferred substitutions are ones which are conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and tyrosine, phenylalanine (see Table 3).

Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules or binding sites on proteins interacting with the ZBTBI l polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence and its underlying DNA coding sequence and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et ah, J. MoI. Biol, 157: 105- 132, 1982). Alternatively, the substitution of like amino acids can be made effectively on the basis of hydrophilicity. The importance of hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Patent No. 4,554,101). The use of the hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Patent No. 5,691,198.

The term "homolog" or "homologs" refers herein broadly to functionally and/or structurally related molecules including those from other species.

Mimetics or mimics are also contemplated as agonists. Reference herein to "mimetics" includes nucleic acid or peptide mimetics and it intended to refer to a substance which has conformational features allowing the substance to perform as a functional analog. A peptide mimetic may be peptide containing molecules that mimic elements of protein secondary structure (Johnson et al, "Peptide Turn Mimetics" in Biotechnology and Pharmacy; Pezzuto et al eds Chapman and Hall, New York, 1993). Peptide mimetics may be identified by screening random peptide libraries such as phage display libraries for peptide molecules which mimic the functional activity of ZBTB 11. Alternatively, mimetic design, synthesis and testing are employed.

Nucleic acid mimetics include, for example, RNA analogs containing N3'~ P5' phosphoramidate internucleotide linkages which replace the naturally occurring RNA 03'— P5' phosphodi ester groups. Enzyme mimetics include catalytic antibodies or their encoding sequences, which may also be humanised.

Peptide or non-peptide mimetics can be developed as functional analogs of ZBTBI l 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. Rational drug design permits the production of 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 modelling or most typically, by a combination of approaches. Useful information regarding the structure of a polypeptide may also be gained by modelling 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.

As briefly described, it is possible to design or screen for mimetics which have enhanced activity or stability or are more readily and/or more economically obtained.

Analogs of ZBTBI l or other agents described herein preferably have enhanced stability and/or activity. They may also be designed in order to have an enhanced ability to cross biological membranes or to interact with only specific substrates. Thus, analogs may retain some functional attributes of the parent molecule but may posses a modified specificity or be able to perform new functions useful in the present context i.e., for administration to the nucleus, bone marrow, etc.

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 NaBH ₄ ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH ₄ . The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using A- 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 4.

Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH ₂ ) _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 and the introduction of double bonds between C _α and C _β atoms of amino acids.

The small or large chemicals, polypeptides, nucleic acids, antibodies, peptides, chemical analogs, or mimetics of the present invention can be formulated in pharmaceutical compositions which are prepared according to conventional phaiτnaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 20th ed. Williams and Wilkins, 2000. 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. Accordingly, pharmaceutical compositions are provided comprising an active agent which modulates the activity of ZBTBI l or its transcriptional targets for use or when used in modulating neutrophil activity as defined herein. In another embodiment, the use of the herein described agent is expressly contemplated in the manufacture of a medicament for the treatment of conditions associated with ZBTBI l variants. In some embodiments, the subject is tested for ZBTBl 1 variants prior to administration. 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 be 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 ^• ■ i ^• 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, 2000 (supra).

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. The agents contemplated herein may be formulated for application to a stent or other device to be introduced into the body to improve circulation, such as a stent or other support. Instead of administering these agents directly, they could be produced in the target cell, or tissue such as the heart, lung or skin, e.g. in a viral vector such as those 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 73 IA and International Patent Publication No. WO 90/07936.

Treatment or prophylaxis of myelopoietic disorders, deficiencies or genetic conditions involving them as referred to herein by gene or cell therapy is also contemplated. Specifically, expression constructs are produced comprising all or part of ZBTBIl nucleic acid sequences as described herein or variants thereof as described herein. The present invention contemplates recombinant nucleic acids including a recombinant construct comprising all or part of ZBTBIl. 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. Such constructs are useful to elevate ZBTBI l levels or to down-regulate ZBTBIl levels such as via antisense means or RNAi-mediated gene silencing. As will be well known to those of skill in the art, such constructs are also useful in generating animal models carrying modified alleles of ZBTBIl and, as pharmaceutical compositions for modulating the activity of ZBTBl 1 in a subject in vivo.

In accordance with this aspect of the present invention, the cells of a subject may be tested to determine whether gene or cell therapy with an agent comprising ZBTBIl is indicated. The provision of wild type or enhanced ZBTBl 1 function to a cell which carries a mutant or altered form of ZBTB 11 should in this situation complement the deficiency and result in an improvement in the subject. Alternatively, cells capable of providing normal or enhanced ZBTBI l activity (function) may be provided. The ZBTBIl allele may be introduced into a cell in a vector such that the gene remains extrachromosomally. Alternatively, artificial chromosomes may be used. Typically, the vector may combine with the host genome and be expressed therefrom. Gene therapy would be carried out according to generally accepted methods, for example, as described by Friedman, Ed., Therapy for Genetic Disease, Oxford University Press, pp. 105-121, 1991 or Culver, Gene Therapy: A Primer for Physicians, 2 ^nd Ed., 1996. Suitable vectors are known, such as disclosed in U.S. Patent No. 5,252,479, International Patent Publication No. WO 93/07282 and U.S. Patent No. 5,691,198. Gene transfer systems known in the art may be useful in the practice of the gene therapy methods of the present invention. These include viral and non- viral transfer methods as well known in the art.

Non-viral gene transfer methods are also known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer. Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery.

In an approach which combines biological and physical gene transfer methods, plasmid DNA of any size is combined with a poly Iy sine-conjugated antibody specific to the adenovirus hexon protein and the resulting complex is bound to an adenovirus vector. The trimolecular complex is then used to infect cells. The adenovirus vector permits efficient binding, internalization and degradation of the endosome before the coupled DNA is damaged. For other techniques for the delivery of adenovirus based vectors, see U.S. Patent No. 5,691,198. Liposome/DNA complexes are also capable of mediating direct in vivo gene transfer.

Expression vectors in the context of gene therapy are meant to include those constructs containing sequences sufficient to express a polynucleotide that has been cloned therein. In viral expression vectors, the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes ZBTB 11 , expression will produce ZBTB 11. If the polynucleotide encodes a sense or antisense polynucleotide or a ribozyme or DNAzyme, expression will produce the sense or antisense polynucleotide or ribozyme or DNAzyme. Thus, in this context, expression does not require that a protein product be synthesized. In addition to the polynucleotide cloned into the expression vector, the vector also contains a promoter functional in eukaryotic cells. The cloned polynucleotide sequence is under control of this promoter. Suitable eukaryotic promoters are routinely determined.

Receptor-mediated gene transfer may be achieved by conjugation of DNA to a protein ligand via polylysine. Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. These ligand-DNA conjugates can be injected directly into the blood if desired and are directed to the target tissue where receptor binding and internalization of the DNA-protein complex occurs. To overcome the problem of intracellular destruction of DNA, co-infection with adenovirus can be included to disrupt endosome function. Accordingly, patients who carry an aberrant ZBTBIl allele are treated with a gene delivery vehicle such that some or all of their cells receive at least one additional copy of a functional normal ZBTBl 1 allele. Preferably only specific cells are targeted.

Alternatively, peptides or mimetics or other functional analogues which have ZBTBl 1 activity can be supplied to cells which carry aberrant ZBTBIl alleles. Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors. In addition, synthetic chemistry techniques can be employed to synthesize the instant active molecules. Active molecules can be introduced into cells by microinjection or by use of liposomes, for example. Alternatively, some active molecules may be taken up by cells, actively or by diffusion. In some embodiments, supply of molecules with ZBTB 11 activity leads to enhanced myelopoiesis.

Diseases or a susceptibility to these diseases can now be diagnosed by monitoring subjects for modification in the level or activity of ZBTBI l or specific mutations or epigenetic modifications or aberrations (such a methylation events) in ZBTBIl.

One particular mutation results in a non-conservative 116 ^{Cys t0 Ser} substitution in ZBTB 11 as described herein.

A wide range of mutation detection screening methods are available as would be known to those skilled in the art. Any method which allows an accurate comparison between a test and control nucleic acid sequence may be employed. Scanning methods include sequencing, denaturing gradient gel electrophoresis (DGGE), single-stranded conformational polymorphism (SSCP and rSSCP, REF-SSCP), chemical cleavage methods such as CCM, ECM, DHPLC and MALDI-TOF MS and DNA chip technology. Specific methods to screen for pre-determined mutations include allele specific oligonucleotides (ASO) ₅ allele specific amplification, competitive oligonucleotide priming, oligonucleotide ligation assay, base-specific primer extension, dot blot assays and RFLP-PCR. The strengths and weaknesses of these and further approaches are reviewed in Sambrook, 2001, (supra).

By identifying ZBTBIl as subject to mutations which affect the level or activity of ZBTBI l, the present invention provides methods of diagnosis of conditions associated with modified ZBTBIl and further provides genetic or protein based methods of determining the susceptibility of a subject to develop these conditions. The diagnostic and prognostic methods of the present invention detect or assess an aberration in the wild type ZBTBIl gene or locus to determine if ZBTBIl will be produced or if it will be over-produced or under-produced. The term "aberration" in the ZBTBIl gene or locus encompasses all forms of mutations including deletions, insertions, point mutations and substitutions in the coding and non-coding regions of ZBTBIl. It also includes changes in methylation patterns of ZBTBIl or of an allele of ZBTBIl. Deletions may be of the entire gene or only a portion of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g. in the tumor tissue and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited. A ZBTBIl allele which is not deleted (e.g. that found on the sister chromosome to a chromosome carrying a ZBTBIl deletion) can be screened for other mutations such as insertions, small deletions, point mutations and changes in methylation pattern.

Useful diagnostic techniques to detect aberrations in the ZBTBIl gene include but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded conformational analysis (SSCA), Rnase protection assay, allele-specific oligonucleotide (ASO hybridization), dot blot analysis and PCR-SSCP (see below). Also useful is DNA microchip technology.

Predisposition to conditions associated with myelopoietic defects can be ascertained by testing any tissue of a human or other mammal for mutations in a ZBTBIl gene. This can be determined by testing DNA from any tissue of a subject's body. In addition, pre-natal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic fluid for mutations of the ZBTBIl gene. Alteration of a wild type allele whether, for example, by point mutation or by deletion or by methylation, can be detected by any number of means.

There are several methods that can be used to detect DNA sequence variation. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing, can detect sequence variation. Another approach is the single-stranded conformation polymorphism assay (SSCP) (Orita et al, Proc. Nat. Acad. Sci. USA, 86: 2776-2770, 1989). This method can be optimized to detect most DNA sequence variation. The increased throughput possible with SSCP makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis. The fragments which have shifted mobility on SSCP gels are then sequenced to determine the exact nature of the DNA sequence variation. Other approaches based on the detection of mismatches between the two complementary DNA strands include clamped denaturing gel electrophoresis (CDGE) (Sheffield et al., Am. J. Hum. Genet, 49: 699-706, 1991), heteroduplex analysis (HA) (White et al, Genomics, 12: 301-306, 1992) and chemical mismatch cleavage (CMC) (Grompe et al, Proc. Natl. Acad. Sci. USA, 86: 5855-5892, 1989). Other methods which might detect mutations in regulatory regions or which might comprise large deletions, duplications or insertions include the protein truncation assay or the asymmetric assay. A review of methods of detecting DNA sequence variation can be found in Grompe, Proc. Natl. Acad. Sci. USA, 86: 5855-5892, 1993. Once a mutation is known, an allele specific detection approach such as allele specific oligonucleotide (ASO) hybridization can be utilized to rapidly screen large numbers of other samples for that same mutation. Such a technique can utilize probes which are labeled with gold nanoparticles to yield a visual color result (Elghanian et al. , Science 277: 1078- 1081 , 1997). Other tests for confirming the presence or absence of a wild type or mutant

ZBTBIl allele include amplicon melting analysis (Wittwer et al., Clinical Chemistry, 49: 853-860, 2008) single-stranded conformation analysis (SSCA) (Orita et al, 1989 (supra)); denaturing gradient gel electrophoresis (DGGE) (Wartell et al., Nucl. Acids Res., 18: 2699- 2705, 1990; Sheffield et al, Proc. Natl. Acad. Sci. USA, 86: 232-236, 1989); RNase protection assays (Finkelstein et al, Genomics, 7: 167-172, 1990; Kinszler et al, Science, 251: 1366-1370, 1991); denaturing HPLC; allele-specific oligonucleotide (ASO hybridization) (Conner et al, Proc. Natl Acad. Sci. USA, 80: 278-282, 1983); the use of proteins which recognize nucleotide mismatches such as the E. coli mutS protein (Modrich, Ann. Rev. Genet, 25: 229-253, 1991) and allele-specific PCR (Ruano et al, Nucl. Acids. Res., 17: 8392, 1989). For allele-specific PCR, primers are used which hybridize at their 3' ends to a particular ZBTBIl mutation or to junctions of DNA caused by a deletion of ZBTBIl. If the particular ZBTBIl mutation is not present, an amplification product is not observed. Amplification Refractory Mutation System (ARMS) can also be used, as disclosed in European Patent Publication No. 0 332 435 and in Newtown et al, Nucl. Acids. Res., 17: 2503-2516, 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. Alternatively, nucleic acid sequencing and/or deep sequencing technologies can be used (e.g. as provided by Illumina Inc, San Diego, CA).

Microchip technology is also applicable to the present invention. In this technique, thousands of distinct oligonucleotide or cDNA probes are built up in an array on a silicon chip or other solid support such as polymer films and glass slides. Nucleic acid to be analyzed is labelled with a reporter molecule (e.g. fluorescent label) and hybridized to the probes on the chip. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips. Using this technique, one can determine the presence of mutations or sequence the nucleic acid being analyzed or one can measure expression levels of a gene of interest or multiple genes of interest such as genes encoding products in a biochemical pathway. The technique is described in a range of publications including Hacia et al, Nature Genetics, 14: 441-447, 1996, Shoemaker et al, Nature Genetics, 14: 450-456, 1996, Lockhart et al, Nature Biotechnology, 14: 1675-1680, 1996, DiRisi et al, Nature Genetics, 14: 457-460, 1996 and Lipshutz et al, Biotechniques, 19: 442-447, 1995. Alteration of wild type ZBTBIl genes can also be detected by screening for alteration of wild type ZBTBI l proteins. For example, monoclonal antibodies imniunoreactive with ZBTBI l can be used to screen a tissue. Lack of cognate antigen would indicate a ZBTB 11 mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant ZBTBl 1 gene product. Such immunological assays can be done in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA assays. 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 is derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation (i.e. comprising ZBTBI l) or can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981; Kohler et al., Nature, 256: 495-499, 1975; Kohler et al, European Journal of Immunology. 6: 511-519, 1976). Examples of primers used to amplify regions of ZBTBIl genetic sequence are routinely derived by the skilled addressee based on known sequences for ZBTBIl, and include those presented in Table 6.

The agents of the present invention may be identified and tested in in vitro or in vivo screens. Natural products, combinatorial, synthetic/peptide/polypeptide or protein libraries or phage display technology are all available to screening for agents. Natural products include those from coral, soil, plant, or the ocean or Antarctic environments. Small molecule libraries are particularly convenient.

Method of screening for ZBTBI l agonists and antagonists are provided. In some embodiments, the methods comprise contacting a candidate drug with a model system comprising ZBTBI l or a complex comprising same and screening for the presence of binding between the drug and ZBTBl 1 or a complex comprising same, or screening for a change in function of ZBTBI l or a complex comprising same or the ability to form a complex. Model systems may be in vitro or in silico. Model systems include cellular model systems and animal model systems wherein the ZBTBIl comprises a temperature sensitive mutation.

In some embodiments, the agent to be tested is contacted with a system comprising ZBTBl 1 or ZBTBIl. Then, the following may be assayed for: the presence of a complex between the agent and ZBTBl 1 or ZBTBIl; a change in the interaction between ZBTBI l and a target; a change in the activity of the target, or a change in the level or activity of an indicator of the activity of the target. Competitive binding assays and other high throughput screening methods are well known in the art and are described for example in International Publication Nos. WO 84/03564 and WO 97/02048).

In another embodiment, a method of identifying an agent that can be used to treat or prevent a condition associated with ZBTBI l dysregulation/dysfunction or which modulates haematopoiesis or myelopoiesis is provided. In some embodiments, the method comprises: (i) contacting the agent with a transgenic teleost such as zebrafish that expresses a reporter protein under the control of a ZBTBI l expression sequence (promoter), (ii) assessing the level or distribution of ZBTBl 1 -reporter protein in the teleost compared to controls. In some embodiments, an enhanced level or altered distribution of ZBTB 11 -reporter expression relative to controls is indicative that the agent enhances neutrophil levels or will be therapeutic and a reduced level or altered distribution of ZBTBI l -reporter expression relative to controls is indicative that the agent depresses neutrophil levels.

In another form of assay, agents that interact with (e.g. bind to) ZBTBl 1 may be identified in a cell-based assay where a population of cells expressing the polypeptide is contacted with a candidate agent and the ability of the candidate agent to interact with the complex is determined. Preferably, the ability of a candidate agent to interact is compared to a reference range or control. In another embodiment, a first and second population of cells expressing ZBTBI l or a complex comprising same are contacted with a candidate agent or a control agent and the ability of the candidate agent to interact with ZBTB 11 or complex is determined by comparing the difference in interaction between the candidate agent and control agent. If desired, this type of assay may be used to screen a plurality (e.g. a library) of candidate agents using a plurality of cell populations expressing ZBTBI l, variants thereof or a complex comprising same. If desired, this assay may be used to screen a plurality (e.g. a library) of candidate agents. The cell, for example, can be of prokaryotic origin (e.g. E. coli) or eukaryotic origin (e.g. yeast, mammalian, vertebrate). Further, the cells can express ZBTBIl endogenously or be genetically engineered to express the polypeptide.

For in vivo screens of teleosts, agents are administered by any convenient route such as for example by submerging the zebrafish stage in media in which the agent is dissolved, injection, electroportation, lipofection, ingestion or biolistically. Methods of administering agents to fish embryos is described in Westerfield, The Zebraflsh Book: Guide For The Laboratory Use Of Zebraflsh (3 ^rd Edition), 1995.

Assessment of myelopoiesis may be via visual assessment of live embryos, larve or adult fish. Alternatively, quantifiable or qualifiable signals can be generated by, for example, in situ hybridization, antibody staining of specific proteins, microangiography, terminal deoxyuridine nucleotide end labeling to detect dead or dying cells. In situ hybridisation (ISH), including whole-mount in situ hybridisation can be used for descriptive analyses of specific gene expression over a time-course of development or for detecting cell or tissue specific expression. ISH takes advantage of the optical transparency of embryos during the first few days of development, which can be prolonged using 1- phenyl-2 -thiourea. Labelled antisense RNA probes are used to provide a semi-quantitative measure of gene expression through out the entire embryo if required.

Myelopoiesis or the presence of specific myeloid cells such as neutrophils can be detected by, for example, visual inspection, flow cytometry, colorimetry, fluorescence microscopy, light microscopy, chemiluminescence, digital image analyzing, standard microplate reader techniques, fluorometry, including time-resolved fluorometry, visual inspection, CCD cameras, video cameras, photographic film, or the use of current instrumentation such as laser scanning devices, fluorometers, photodiodes, quantum counters, plate readers, epifluorescence microscopes, scanning microscopes, confocal microscopes, flow cytometers, capillary electrophoresis detectors, or by means for amplifying the signal such as a photomultiplier tube, etc. Signals can be discriminated and/or analyzed by using pattern recognition software. Changes in the distribution of a protein or cell both spatially and temporally, including a complete absence of a protein or cell, can be detected and protein expression profiles can be generated. Changes in a level of an enzyme or enzymatic activity within the intact teleost can also be detected by various means, including, e.g., myeloperoxidase detection or use of a streptavidin (avidin) conjugated reporter enzyme. Such methods may be routinely automated.

Zebrafish for use in the subject methods may be wild type zebrafish or modified zebraflsh. Various robust methods are available for producing modified zebrafish. In some embodiments, the activity of a ZBTBI l polypeptide or of other factors in the myeloid differentiation, such as without limitation, scl, pu.l and spil, or the like are reduced by generating an induced mutation in the endogenous gene or by inhibiting gene expression using nucleic acid interference (RNAi, morpholino antisense or genetic silencing). Genes can be modified using methods known in the art for producing stable or transient transgenic or genetically modified teleosts (see for example Meng et al, Methods in Cell Biology: Zebrafish Eds Detrich et al, Academic Press: 133-148, 1998; Udvadia et al., Dev. Biol, 256: 1-17, 2003; Meng et al, Nat. Biotechnology, 26(6): 650-701, 2008). Modified teleosts may be generated by random mutagenesis or insertional mutagenesis via mouse retroviral vectors or by targeting induced local lesions in genomes (TILLING) or from catalogues libraries of insertional mutants described in Wienholds et al, Genome Res i3(12): 2700-2707, 2003. Targeted mutagenesis is achievable using zinc finger nucleases (Meng et al, 2008 (supra); Doyon et al, 2008 (supra)). Heterozygous mutants can be crossed to produce carriers and homozygous mutants, where applicable. Nucleic acid inhibition of target genes produces so called "morphant" teleosts wherein translation of the target gene is inhibited. As described herein morpholino oligonucleotides are widely used to generate morphant phenotypes by binding sense mRNA and inhibiting translation (see Nasevicius et al, Nat. Genet. 26: 216-220, 2000). Genetic agents which reduce the activity of ZBTBI l polypeptides or a transcription target of ZBTBI l in a cell include genetic agents (i.e. comprising a nucleic acid molecule) which inhibit production of ZBTBI l in a cell at any stage including, for example, post-transcriptional silencing mediated by RNAi (see for example, United States Patent Publication No. 2007/0042983, International Publication No. WO 01/68836 and International Publication No. WO 03/064626). Such nucleic acids can be chemically synthesised, expressed from a vector or enzymatically synthesised as known in the art. As described in the present examples, mutants were crossed with transgenic zebrafish comprising fluorescent markers of neutrophil development.

In other embodiments, the present invention provides a transgenic teleost comprising a nucleic acid sequence encoding a reporter protein whose expression is driven by the ZBTBIl (including zbtbll promoter). The production of transgenic teleosts is described in Lawson and Weinstein, Dev Biol. 248(2): 307-18, 2002 and United States Patent Publication No. 2004/0143865. Reporter proteins are conveniently fluorescent or illuminescent such as green fluorescent protein, cyan fluorescent protein, green-reef coral fluorescent protein, red fluorescent protein and yellow fluorescent protein and others are reported in the published literature and described in International publication No. WO 03/079776.

In one embodiment, all or part of the ZBTBl 1/zbtb 11 gene promoter is operatively linked to a reporter construct and engineered into an expression construct as known to those of skill in the art. For example, a pGL3 -series reporter plasmid may be conveniently employed. Stable or transient transfection of cells may be used to generate cell lines capable of being tested with potential agents.

In a cell based approach a ZBTBI l responsive cell line is generated comprising an inducible ZBTBIl gene. Potential agents are tested for their ability to up-regulate or down-regulate expression differentiation markers when ZBTBIl is activated. For example, a ZBTBI l responsive cell line is the human cancer line K562 referred in Ceballos et at, Oncogene, 19: 2194-2204, 2000. Such techniques are well known in the art and are described, for example, in Sambrook, 2001 {supra) and Ausubel, 2002 (supra). The term "modified" in relation to cells or animals includes genetically modified but encompasses non-genetic or epigenetic modifications to affect, for example, ZBTBI l activity by, for example, the administration of an agent such as, without limitation, an organic or inorganic chemical agents, antibody, enzyme, peptide, genetic or proteinaceous molecule to effectively modulate the functional activity of ZBTBIl or ZBTBlL In some examples, teleosts are modified using antisense molecules. Morpholinos are conveniently used as described in Ekker and Landon, Genesis, 30:89-93, 2001.

The term "genetically modified" refers to changes at the genome level and refers herein to a cell or animal that contains within its genome a specific gene which has been altered. Alternations may be single base changes such as a point mutation or may comprise deletion of the entire gene such as by homologous recombination. Genetic modifications include alterations to regulatory regions, insertions of further copies of endogenous or heterologous genes, insertions or substitutions with heterologous genes or genetic regions etc. Alterations include, therefore, single of multiple nucleic acid insertions, deletions, substitutions or combinations thereof. Cells and vertebrates which carry a mutant ZBTBl 1 allele or where one or both alleles are modified can be used as model systems to study the effects of ZBTBIl in myelopoiesis and myelopoietic disorders and/or to test for substances which have potential as therapeutic or prophylactic agents when ZBTBIl function is impaired or dysregulated. Animals for testing therapeutic agents can be selected after mutagenesis, knock-down, or introduction of over expression molecules of whole animals or after treatment of germline cells or zygotes. Such treatments include insertion of mutant ZBTBI l alleles (including those carrying loxP flanking sequences), usually from a second animal of the same species, as well as insertion of disrupted homologous genes. Alternatively, the endogenous ZBTBI l gene of the animals may be modified by insertion or deletion mutation or other genetic alterations using conventional techniques. These animal models provide an extremely important testing vehicle for potential therapeutic products. The cells may be isolated from individuals with ZBTBI l mutations, either somatic or germline. Alternatively, the cell line can be engineered to carry the mutation in the ZBTBl 1 allele, as described above, or by gene modification using zinc finger nucleases (see Meng et ah, 2008 {supra); Doyon et al, Nat. Biotech. 26: 702-708, 2008). After a test substance is applied to the cells, the phenotype of the cell is determined. Any trait of the cells can be assessed. As illustrated herein, a teleost model system is particularly advantageously employed.

Thus a genetically modified animal or cell includes animals or cells from a transgenic animal, a "knock in" or knock out" animal, conditional variants or other mutants or cells or animals susceptible to co-suppression, gene silencing or induction of RNAi.

Conveniently, targeting constructs are initially used to generate the modified genetic sequences in the cell or organism. Targeting constructs generally but not exclusively modify a target sequence by homologous recombination. Alternatively, a modified genetic sequence may be introduced using artificial chromosomes. Targeting or other constructs including reporter constructs for screening potential ZBTBI l modulators are produced and introduced into target cells using methods well known in the art which are described in molecular biology laboratory manuals such as, for example, in Sambrook, 2001 (supra); Ausubel, 2002 (supra). Targeting constructs may be introduced into cells by any method such as electroporation, viral mediated transfer or microinjection. Selection markers are generally employed to initially identify cells which have successfully incorporated the targeting construct. Genetically modified mammalian organisms are generated using techniques well known in the art such as described in Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbour Laboratory Press, CSH NY, 1986; Mansour et al, Nature 336: 348-352, 1988; Pickert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press, San Diego, CA, 1994. Stem cells including embryonic stem cells (ES cells) are introduced into the embryo of a recipient organism at the blastocyst stage of development. There they are capable of integration into the inner cell mass where they develop and contribute to the germ line of the recipient organism. ES cells are conveniently obtained from pre-implantation embryos maintained in vitro (Robertson et al, Nature 322: 445-448, 1986). Once correct targeting has been verified, modified cells are injected into the blastocyst or morula or other suitable developmental stage, to generate a chimeric organism. Alternatively, modified cells are allowed to aggregate with dissociated embryonic cells to form aggregation chimera. The chimeric organism is then implanted into a suitable female foster organism and the embryo allowed to develop to term. Chimeric progeny are bred to obtain offspring in which the genome of each cell contains the nucleotide sequences conferred by the targeting construct. Genetically modified organism may comprise a heterozygous modification or alternatively both alleles may be affected.

Another aspect of the present invention provides cells or animal comprising one, two or more genes or regions which are modified. For example, the genetically modified cells or animals may comprise a gene capable of functioning as a marker for detection of modified cells. Alternatively, the instant animals may be bred with other transgenic or mutant non-human animals to provide progeny some of which exhibit one or both traits or a modified trait/s. Chimeric animals are also contemplated. Genetically modified cells or non-human organisms may be provided in the form of cells or embryos for transplantation. Cells and embryos are preferably maintained in a frozen state and may optionally be distributed or sold with instructions for use.

In a further aspect, the present invention provides a genetically modified cell, or non-human animal comprising such cells, wherein a ZBTBIl gene is modified and the cell or animal produces a substantially enhanced level or activity of ZBTBI l polypeptide, or substantially reduced level or activity of ZBTBl 1 polypeptide compared to a non-modified animal of the same species, or is substantially incapable of producing ZBTBI l polypeptide.

The genetically modified cells and non-human animals may be a non-human primate, livestock animal, companion animal, laboratory test animal, captive wild animal, reptile, amphibian, fish, bird or other organism. Preferably the genetically modified non- human animal is a teleost.

In a further aspect, the present invention provides a genetically modified cell, or non-human animal comprising such cells, wherein a zbtbll gene is modified and the cell or animal produces a substantially enhanced level or activity of Zbtbl l polypeptide, or substantially reduced level or activity of Zbtbl l polypeptide compared to a non-modified animal of the same species, or is substantially incapable of producing Zbtbll polypeptides.

In one aspect, the modified cell or non-human animal is genetically modified and produces a substantially reduced level of ZBTBI l or is substantially incapable of producing ZBTBI l or produces ZBTBI l having substantially reduced or no activity. Preferably a ZBTBIl gene is modified. Modification may be in one or both alleles and may optionally be within a regulatory region of the gene. In another embodiment, the genetic modification resulting in a cell or animal capable of exhibiting a modified level or activity of ZBTBI l comprises genetic modification outside the ZBTBIl gene to cause expression of genetic or proteinaceous molecules which effectively modulate the activity of ZBTBI l or ZBTBIl. In a preferred aspect, the modified cell or non-human animal is genetically modified and substantially overproduces ZBTBI l having normal or altered activity relative to an unmodified cell or animal of the same species.

In one aspect, the modified cell or non-human animal is genetically modified and produces a substantially reduced level of Zbtbll or is substantially incapable of producing Zbtbl l or produces Zbtbl l having substantially reduced or no activity. Preferably a zbtbll gene is modified. Modification may be in one or both alleles and may optionally be within a regulatory region of the gene. In another embodiment, the genetic modification resulting in a cell or animal capable of exhibiting a modified level or activity of Zbtbll comprises genetic modification outside the zbtbll gene to cause expression of genetic or proteinaceous molecules which effectively modulate the activity of Zbtbl l or zbtbll. In a preferred aspect, the modified cell or non-human animal is genetically modified and substantially overproduces Zbtbl l having normal or altered activity relative to an unmodified cell or animal of the same species.

In yet another aspect, the invention provides a method of screening for or testing an agent capable of complementing a phenotype shown by a cell or non-human animal comprising a modified ZBTBIl nucleic acid or ZBTBI l polypeptide and exhibiting a substantially modified level or activity of ZBTB 11 polypeptide. Preferably, the cell or animal is contacted with the agent and its effect on the activity of ZBTBIl or its transcriptional targets determined. In one aspect the method comprises screening for mutants which exhibit a complementing phenotype and then mapping and identifying the modifying gene. In another aspect the method comprises screening for agents which enhance the level or activity of ZBTBI l in a normal or modified cell. In some embodiments, small-molecule libraries are screened for agents which directly or indirectly modulate ZBTBI l polypeptide activity. One method is described by Stegmaier et ah, PLOS Medicine, 4(4): 702-714, 2007. Here, expression profiles diagnostic of a ZBTBl 1- on activity and a ZBTBI l -off activity are chosen, and the ability of small-molecules to produce either the ZBTB 11 -on or the ZBTB 11 -off profile is determined. Antisense knockdown strategies for selecting the ZBTBI l -off activity are routine in the art and include ShRNAs directed against the ZBTB 11 transcript.

In some embodiments screening can be performed as described in Lieschke and Currie, Nat Rev Genet., 8(5): 353-367, 2007. Small groups of zebrafish embryos or larvae are arrayed in multi-well microtitre plates and standard concentrations of small molecules are robotically pipetted into the raising media in individual wells. Throughput is increased if suppression can be assessed directly in the larvae using fluorescent read-outs, or if it can be made quantitative in some way, particularly if the scoring process is suited to automation. Scoring can also be coupled with an immunological or gene-expression assay to monitor cell-cycle progression. The active compounds that are identified can undergo a secondary process of validation, dose and toxicity assessment, and can be extended by exploration of analogs generated by combinatorial chemistry, before proceeding to testing in other animal models. Examples of chemical screening in zebrafish are described in Schlueter and Peterson, Circulation, 120(3):255-63, 2009; Mukhopadhyay et al, Current Opinion in Hematology, i5(3):221-227, 2008. In further embodiment, the subject invention provides a use of a cell or non- human animal comprising a modified ZBTBIl or ZBTBI l and exhibiting a substantially enhanced level or activity of ZBTBI l in screening for or testing agents for use in the treatment or prophylaxis of a conditions, states and disorders as described herein. The present invention further provides a method for identifying agents useful in the treatment or prophylaxis of conditions associated with dysregulated ZBTBIl activity or dysregulated ZBTBIl expression levels such as described herein comprising screening compounds for their ability to modulate the functional activity of ZBTBIl polypeptides or modulate ZBTB 11 activity in promoting myelopoiesis or leukemogenesis or their ability to modulate ZBTB 11 expression levels .

In a particular embodiment, the present invention contemplates a teleost bioassay to assess or identify agents which modulate a condition associated with said ZBTBI l deficiency as described herein.

In a particular embodiment, the present invention provides a method for identifying or assessing a variant or analog of a ZBTBIl nucleic acid or protein or an agent which modulates a condition associated with ZBTBIl dysregulation (deficiency) in a subject, the method comprising (i) contacting the agent or variant or analog with a teleost model of ZBTBl 1 deficiency; and (ii) assessing the ability of the agent or variant or analog to rescue (complement) the phenotype of the teleost model. Conditions associated with ZBTBIl deficiency as described herein include a haematological malignancy or myelopoietic disorder, a retinal or central nervous system degeneration or hydrocephalus, neutropenia or immunodeficiency, neutrophil inflammation or hyperplasia, or myeloid leukemia or lymphoma.

In some embodiments, contacting the agent or variant or analog includes injecting the agent or RNA encoding the agent into embryos that are the progeny of a cross between heterozygotes man mutant zebrafish. In some embodiments, the assessment step assess the ability of the agent, variant or analog to rescue the phenotype in the progeny as determined by a reduction in the proportion of embryos that exhibit the man phenotype. The man mutant is an illustration of a teleost model of ZBTBI l dysregulation or dysfunction. Another illustrative example is provided by producing phenocopies of the man mutant as described in Example 6 where antisense targeting of the ATG in zbtbl 1 produces embryos with the man phenotype.

In some embodiments, the man phenotype or the phenotype of the teleost model of ZBTBl 1 dysregulation means a reduction in the number, location or activity of myeloid cells such as neutrophils, granulocytes, T cells or leukocytes compared to a control such as a wildtype zebrafish. In other embodiments, the man phenotype means hydrocephalus or central nervous system or retinal degeneration. In some embodiments, the number, location or activity of neutrophils is determined using markers of the presence of neutrophils in zebrafish such as Sudan blue staining, myeloperoxidase activity or activity of a reporter linked to expression of genes specifically expressed by myeloid cells or cells affected by ZBTBI l dysregulation. In some embodiments, wildtype zebrafish are also crossed and injected to determine the effect of overexpressing ZBTBl 1 as a control.

In some embodiments software that processes and records signal or marker patterns, fluorescence intensity or distribution is employed to facilitate high throughput analysis.

In another embodiment, the present invention provides Zbtbl l polynucleotides and encoded polypeptides described herein, i.e. as set forth in SEQ ID NOs: 1 and 2 and 3 and 4, or variants thereof comprising conservative substitutions or nucleotide sequences having at least 98 to 99% sequence identity thereto or to a complementary sequence or that hybridises thereto or to a complementary form under conditions of at least medium stringency.

As determined herein, overexpression of ZBTBIl in the model teleost provides a new measure of ZBTBIl functional activity and a convenient model for assessing the ability of an agent either to affect haematopoiesis or myelopoiesis or, to assess the ability of an agent to provide ZBTB 11 functional activity.

In some embodiments, the invention provides a method of identifying or assessing an agent which modulates haematopoiesis or myelopoiesis, the method comprising (i) contacting the agent with a teleost model of ZBTBI l dysregulation; and (ii) determining the effect of the agent on a marker of ZBTBI l dysregulation such as neutrophil numbers or hydrocephalus in the teleost relative to controls. In some embodiments, step (ii) comprises screening the teleost for expression of one or more markers of neutrophils. In an illustrative method, the marker is selected from mpx or scl. Conveniently the teleost may be modified to underexpress or overexpress a ZBTB 11 variant or ZBTB 11 or a liomolog under the control of an inducible promoter. In some embodiments, variants of ZBTBI l are employed such as wherein the teleost overexpresses a variant of ZBTBI l or ZBTBIl or of a homolog thereof.

Teleosts for use in the subject methods may be modified to overexpress or underexpress one or more molecules expressed during myelopoiesis such as one or more of lcpl, nephrosin, lyz or MTIIA. In some embodiments, the teleost model is a model of haematopoiesis or myelopoiesis. Reference herein to an ZBTBI l polypeptide or a polypeptide having ZBTBI l activity includes polypeptides having the amino acid sequences of naturally occurring forms of ZBTBI l polypeptides or fragments (parts) thereof or of variants thereof. A large number of ZBTBI l polypeptide sequences are known and an illustrative example as set out in the sequence listing (see Table 1). Any such variant or modified (variant) versions of these or other sequences or fusion proteins comprising them may be tested or overexpressed in the present invention. Accordingly, in some embodiments, fusion proteins comprising ZBTBl 1 are overexpressed in the teleost model.

The term "overexpression" is used broadly in this specification to refer to the production or presence of ZBTB 11 polypeptide or a functional variant thereof at more than physiological levels of the protein in a particular cell, tissue or organism. Overexpression or misexpression may be produced or induced by introducing nucleic acids encoding a polypeptide having ZBTBl 1 activity or potential ZBTBl 1 activity that are "expressed" by transcription and/or translation within a cell or alternatively by direct introduction of the protein. Alternatively agents that promote or "switch on" endogenous ZBTBIl expression in particular cells, tissues or organisms are employed.

A substantially modified level or activity of ZBTBl 1 is conveniently assessed in terms of a percent reduction or increase relative to normal cells or animals or pre- treatment/pre-administration. A substantial reduction or increase includes one which results in a detectable reduction or increase in the number of neutrophils in a subject or cell. Alternatively, a reduced level of gene expression of transcription targets or a reporter thereof is detected. Preferably, the modification is at least 20% enhanced or reduced compared to normal cells, more preferably about 25%, still more preferably at least about 30% reduction, more preferably at least about 40% enhanced or reduced ZBTBl 1 level or activity. The reduction may of course be complete loss of ZBTBI l activity in a cell or animal. A "modified" level or activity includes enhanced levels of ZBTBI l activity relative to pre-treatment levels and may equate to or exceed the level or activity of ZBTBI l detectable in controls. Overexpression includes a forced expression in all tissue or more particularly specific tissue or regions. No particular level of expression is prescribed. The terms refer to expression that is not essentially normally developmentally regulated. Another embodiment provides a method of assessing the functional activity of an agent selected from the group consisting of ZBTBI l or a variant of ZBTBIl or an agonist or antagonist of ZBTBl 1 activity, or an agonist or antagonist of an ZBTBl 1 transcriptional target in myeloid or CNS tissue. In some embodiments, the method comprises: (i) contacting the agent and a model system comprising a teleost (zebrafish) and (ii) determining the effect of the agent on markers of ZBTBI l dysregulation such as neutrophil numbers, retinal degeneration or hydrocephalus in the teleost relative to controls. In some embodiments, the teleost is further contacted with an ZBTBI l antagonist or a putative ZBTB 11 antagonist. In some embodiments, the teleost is a wild type teleost. In other embodiments, the model system is sensitised or modified to determine the effect of specific molecules expressed during development. Thus, in some embodiments, a modified teleost is employed which overexpresses or underexpresses one or more molecules expressed during myelopoiesis such as one or more of zbtbll, lcpl, nephrosin, lyz or MTIIA. Reduced levels are conveniently reached using antisense, such as morpholino oligonucleotides/nucleic acids. In other embodiments, the teleost is screened for expression of one or more markers of neutrophils such as mpx expression or of detectable reporters of such expression. In some embodiments, zebrafish embryos are contacted at the 1 to 4 cell stage, the 1 to 8 cell stage, or the 1 to 16 cell stage. In other embodiments, zebrafish embryos are contacted at the 1 to 2 cell stage, the 2 to 4 cell, the 4 to 8 cell, the 8 to 16 cell, or the 16 to 32 cell stage. Preferably, the cell stage is fewer than 16, more preferably fewer than 8, and most preferably fewer than 4 or 2. In some embodiments, the agent is introduced as nucleic acid such as mRNA into one or more zebrafish embryos at the 1 to 16-cell stage, preferably the 1 to 4 cell stage. Most preferably, the cell stage is 1 to 2, 2 to 4 cell or 4 to 8 cell. In other embodiments, nucleic acid is introduced at the 16-cell stage or 32-cell stage or later. The present invention provides a convenient bioassay for assessing the ability of an agent to provide ZBTBI l functional activity. As shown described herein (see in particular Example 7) overexpression of ZBTBI l in single cell zebrafish man mutant embryos resulted in phenotype complementation that could readily be detected and measured qualitatively or quantitatively. By restricting ZBTBI l dysregulation to the mesoderm, by delaying injection slightly, a more refined effect upon myelopoiesis or retinal or central nervous system degeneration could be qualitatively or quantitatively assessed.

Accordingly, in one embodiment, the present invention provides a method of assessing the functional activity of an agent in a teleost developmental model, the method comprising (i) contacting the agent with a model system comprising a teleost; (ii) determining the effect of the agent on in the teleost relative to controls. In some embodiments, the method comprises (iii) comparing the effect of the agent in (ii) with the effect of overexpressing ZBTBl 1 or a homolog of ZBTBl 1 in the teleost. In an illustrative embodiment described in Example 9, RNA encoding a variant of ZBTBI l lacking a functional domain is injected in zebrafish embryos at the 1 to 4 cell stage and the effect on development is determined relative to the effect of overexpressing a known functional form of ZBTBl 1 in the teleost model. As previously described, while 1 to 2, 1 to 4 or 1 to 8 cell embryos are conveniently employed, embryos may be at other stages such as the 1 cell stage through stages approaching the end of embryonic development. Alternatively the effect of agents on myelopoiesis, haematopoiesis and CNS development at any life stage of the teleost may also be determined. Measurements may be made visually or by using a device, including a computer aided device. Thus developmental defects can be scored in the teleost, myelopoiesis and CNS development may be assessed visually such as by monitoring retinal or brain development, using markers of particular tissues including expression markers of different stages of myelopoiesis or CNS development as described herein or known in the art. Thus markers are assessed qualitatively or quantitatively including the position, quantity or developmental timing of expression.

In some embodiments, steps (ii) and/or (iii) comprise screening a teleost for expression of one or more markers of myelopoiesis, haematopoiesis, retinal or CNS development.. In an illustrative embodiment (see Example 6 and Figure 7) an expanded population of GFP-positive cells is observed in a Tg(mpx:EGFP). In some embodiments, the agent comprises mRNA encoding a variant of ZBTBl 1 or a variant of a homolog of ZBTBI l such as a human or mammalian homolog of ZBTBI l. In particular, human homologs, murine homologs, teleost homologs and homologs of veterinary interest are contemplated. Illustrative variants are those comprising all or part of disease alleles. Polypeptides having an activity of human ZBTBI l are particularly contemplated. There are several related isoforms known in the art each of which may be employed herein. Illustrative human ZBTBIl variants are set out in the description, sequence listing and Figures. RNA encoding fusion protein comprising ZBTBI l or ZBTBI l variants are also contemplated.

The effects of ZBTBI l overexpression over a 48 hour period in 1-cell zebrafϊsh are conveniently assessed. In particular, the graded effect of different concentrations of functional ZBTB 11 on a population of zebrafish embryos may be assessed. This assay permits a very sensitive assessment of the functional effect of the agent on development and therefore the functional effect of any modification to the agent to produce variants (such as truncations, mutations, substitutions, insertions etc).

ZBTBIl overexpression may be achieved at particular timepoints by transgenesis in which ZBTBIl overexpression is driven by a later promoter or by an inducible promoter (e.g. the tet-on or tet-off system, see Hillen and Berens, Annu. Rev. Microbiol, 48: 345- 369, 1994; Gossen and Bujadt, Proc. Natl Acad. ScI USA, 89: 5547-5551, 1992; Chiu-Ju Huang et al, Dev Dyn, 233: 4, 1294 - 1303, 2005), allowing screening of drug effects at specific timepoints following endogenous or exogenous activation of the promoter driving ZBTBIl overexpression. Again, an expected readout for overexpression of ZBTBIl may be expansion of mpx+VQ cells by WISH or observation of aforementioned transgenic lines, while embryos treated with a drug targeting ZBTBI l function may show a reduction in neutrophil cell expansion. Other inducible systems exploit heat-shock mechanisms to modulate translation (see for example, DiDomenico, Proc. Natl. Acad. Sci. USA 79: 6181-6185, 1982; Adam et al, Exp Cell Res, 256: 282-290, 2000).

Alternatively, the Cre-LoxP system can be used to provide appropriate conditional ZBTBI l levels (see Sternberg and Hamilton, J MoI Biol, 150: 467-486., 1981; Lakso et al, Proc Natl Acad Sci USA., 89(14): 6232-6, 1992; Langenau et al, Proc Natl Acad Sci USA., 102(11): 6068-73, 2005) by providing targeted activation (or inactivation).

One modification for antisense and sense molecules involves the use of morpholinos which, as touched on above, are oligonucleotides composed of morpholine nucleotide derivatives and phosphorodiamidate linkages. Morpholino nucleic acids typically comprise heterocyclic bases attached to the morpholino ring. A number of linking groups may link the morpholino monomeric units in a morpholino nucleic acid. One class of linking groups have been selected to give a non-ionic oligomeric compound. The non- ionic morpholino-based oligomeric compounds are less likely to have undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are non-ionic mimics of oligonucleotides which are less likely to form undesired interactions with cellular proteins. Morpholino-based oligomeric compounds are disclosed in U.S. Patent No. 5,034,506; WO 00024885 and WO 00045167.

One method for modifying expression of ZBTBI l is the GAL4-UAS system described for example by Fischer et al, Nature, 332: 853-856, 1998, as reviewed by Scheer et al, Mechanism of Development, 80: 153-158, 1999, this technique is based on two different kinds of transgenic strains, called activator and effector lines. In an activator line the gene for the yeast transcriptional activator GAL4 is placed under the control of a specific promoter, while in the effector line the gene of interest is fused to the DNA- binding motif of GAL4. The effector gene will be transcriptionally silent unless animals carrying it are crossed to those of an activator line. In the progeny of this cross, expression of the effector gene will reflect the pattern of expression of GAL4 in the activator, which is ultimately dependent on the promoter that has been used to control it. This, of course, allows controlled ectopic expression of the effector gene. The use of activators with different expressivities, which arise due to positional effects acting on the activator construct, allows the experimenter to exploit a relatively wide range of levels of effector gene expression.

The present invention is further described by the following non-limiting Examples.

EXAMPLE 1

Identification of the marsanne mutation in a forward genetic ethyl nitrosourea screen for myeloid failure in zebrafish

In order to screen for zebrafish myeloid failure mutants, male adult zebrafish were treated with ethyl nitrosourea (ENU) at a dose of 2.5-3mM with three exposures of 1 hr duration as described. Fl females were selected and haploid embryos generated as previously described (Hogan et al, Curr. Biol. 16: 506-511, 2006). F2 clutches of haploid embryos were separated into batches, raised at 28 or 33 ⁰ C and screened for altered expression of pu.l, myeloperoxidase (mpx), or deltaC by whole-mount in situ hybridization (WISH) with screening priority given in that order. One mutant (ES37.282) was recovered from this screen with reduced expression of mpx at 33 ⁰ C ₃ though normal expression of pu.1 and deltaC. This mutant was designated marsanne {man (official allele designation man ^%n2 )) (see Figure ID).

EXAMPLE 2

Marsanne mutant zebrafish have reduced numbers of embryonic neutrophils and die between 96-120 hours post fertilization(hpβ

The man/+ founder fish was mated to +/+ polymorphic outbred zebrafish strains SK & WIK, then recovered as a stable, heritable mutant line. In a stable diploid pedigree it behaves as a typical recessive Mendelian trait. Analysis of multiple pedigrees descended from the man founder consistently demonstrate a fully penentrant recessive phenotype with 100% embryonic lethality by 96-120hpf. Phenotypic analysis of man/man embryos by WISH demonstrates normal early expression of the hematopoietic markers scl, pu.1 and gatal (19-20hpf) and normal erythrocyte morphology at 48hpf but a reduction in mpx expressing cells by 48hpf. Embryonic neutrophil numbers are also reduced by sudan black cytochemistry (see Figure T). mpx cell numbers in man/man embryos are reduced to 38% of normal numbers at 48hpf (Figure 3). The man phenotype is also characterized by abnormalities of the brain (including characteristic hydrocephalus), eye, spinal cord, yolk and circulation by 48hpf (Figure 4). At 30 hpf there was a slightly smaller eye in mutant embryos. By 48 hpf, mutant embryos had smaller eyes, abnormal forebrain opacity and enlargement of the 4 ^th ventricle. By 72 hpf, the mutant embryos were shorter in length, with marked reduction in eye size, progression of the brain abnormalities and craniofacial abnormalities. In addition, the yolk darkened, the swim-bladder failed to inflate and the circulation was sluggish with reduction in the heart rate and oedema over the heart. By 100 hpf, the circulation in mutant embryos had further declined with marked oedema over the heart and by 120 hpf mortality was 100%.

EXAMPLE 3

The reduction in embryonic neutrophils in marsanne mutant zebrafish is dependant on rearing temperature and partially rescued at 21 °C

Zebrafish husbandry protocols dictate a standard rearing temperature for adults and embryos at 28 ⁰ C, although they exist in the wild from 21-38°C. man was recovered from the ENU screen designed to capture temperature sensitive alleles by screening at 33°C. Although man is mpx deficient at 28 ⁰ C, partial rescue of the mpx deficiency can be achieved by reducing the water temperature to 21°C (Figure 5). Conversely the deficiency can be exacerbated by raising the temperature to 33 ⁰ C (Figure 5). Thus man is a temperature sensitive conditional allele, allowing manipulation of in vivo biological function from -33% to 85% of normal function (as assessed by mpx cell numbers).

EXAMPLE 4

The man mutation exists in a 50 kilobase interval on chromosome 6

The man mutation was localized using standard zebrafish positional cloning techniques (http://l 34.174.23.167/zonrhmapper/positionCloningGuide/PositionalClo ningGuideweb Ju ne2007.pdf). Briefly, man/+ SK fish were mated to +/+ WIK fish to generate man/+ SK/WIK progeny. Positional cloning was undertaken utilizing the embryo progeny of man/+ SK/WIK in-crosses. The genotypes of the genetic markers closest to the man mutant locus will be SK/SK in man/man embryos and SK/WIK or WIK/WIK in manf+ and +/+ embryos respectively. Embryos phenotypically man/man may genotype at more distant markers as SK/WIK if recombination has occurred between this marker and the man locus. Positional cloning utilizing simple sequence length polymorphism (SSLP) markers and single nucleotide polymorphisms (SNPs) generating restriction fragment length polymorphisms (RFLPs) defined the genetic interval flanked by single recombination events as a 48 kilobase region on chromosome 6 containing 1 complete gene and 500 nucleotides of another (Table 5). Sequences for markers described in Table 5 are available from Genbank z27232 (accession G48053), zl2094 (G46534), zlO183 (G39760) or in Table 6. PCR conditions are available in Table 6.

EXAMPLE 5

The man mutation causes a cysteine to serine substitution in the N terminal region of the BTB-ZF transcription factor zbtbll zbtbll full length cDNA was amplified from RNA prepared from 48hpf man/man and +/+ embryos, sequenced and man/man cDNA cloned using standard techniques as described in Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Ed, John Wiley & Sons, Inc, NY, 2002. The coding regions of zbtbll were sequenced in man/man and +/+ embryo cDNA and a T to A nucleotide substitution in exon 2 was identified (346T- A) which in genomic DNA was located in exon 2 of the gene (Figure 6), and which was present in all man/+ fish but no +/+ fish. Further more, genotyping of the ES37.282 ENU treated founder demonstrated the 346T-A substitution was not present prior to ENU treatment. In addition genotyping of the flanking recombinant mutant phenotype embryos at this nucleotide demonstrated them to both be 346AA. The mutation lies outside the predicted functional domains but at an evolutionarily highly conserved cysteine residue (116 ^Cys→Ser ). A WT zbtbll construct was generated using site directed mutagenesis on the man construct to substitute A for T at nucleotide 346. Primer sequences and PCR conditions used to generate full length zbtbll are available in Table 6. EXAMPLE 6

In vivo transient knockdown and rescue assays demonstrates that loss of zbthl If unction phenocopies man and that the T to A substitution in exon 2 is responsible for the man phenotype An antisense morpholino oligonucleotide designed to target the ATG of zbtbll when injected into single cell embryos reliably phenocopies the man/man phenotype (Figure 7). Injection into single cell man/man embryos of capped RNA encoding WT zbtbll rescues the morphological features of man and the deficiency of mpx expression at 48hpf (Figure 8). Injection of man RNA had no effect.

EXAMPLE 7 Transient over-expression of RNA encoding wildtype Homo Sapiens ZBTBIl is able to rescue the man phenotype

Injection into single cell man/man embryos of capped RNA encoding WT Homo Sapiens ZBTBIl (HS WT cDNA) rescues the morphological features of man at 48hpf (Table 7), demonstrating the human ZBTB 11 is able to substitute functionally for zebrafish Zbtbl l.

Collectively, these data provide genetic evidence demonstrating that zbtbll is the gene product responsible for the man phenotype, and that the 346T to A mutation is responsible for impairing the functionality of the gene product.

EXAMPLE 8 Characterisation of zbtbll dysfunction in the marsanne (man) mutant man embryos have been broadly characterised by microscopy, histology, and whole mount in situ hybridisation (WISH) gene expression analysis. By WISH, early haemopoietic fate-specification genes are expressed normally (e.g. scl, gatal, pu.l/spil).

Erythroid genes show normal expression (e.g. hbbeS), and man erythrocytes show normal morphology and progressive maturation. Within myelopoiesis, man has a general loss of myelomonocytic cells, wjpx-expressing cells are reduced by 50% (81±12 for man+ll vs 42±10 for man-/- at 48 hours post fertilisation (hpf), pO.OOOl), and there are reduced numbers of cells expressing other leukocyte markers (lcpl, nephrosin, lyz). man also has other phenotypic characteristics. It is embryonic lethal at 4-5 days post fertilisation (dpf). It develops a small eye (due to apoptotic retinal degeneration and disorganisation) and central nervous system degeneration leading to pronounced hydrocephalus. The loss of mpx-expressing cells is not invariable in mutants with cerebral defects e.g. the mutant cephalophonus, a null mutation in the prpδ locus encoding a major spliceosome component, has massive cerebral apoptosis, but normal numbers of mpx- expressing cells at 30 hpf. man was recovered from a screen for mutants with defects in myeloid cell development using the myeloid markers spil and mpx. Expression of spil initiated normally in man, but in contrast, mpx was markedly reduced. Myeloperoxidase is a peroxidase enzyme expressed specifically in the azurophilic granules of neutrophil granulocytes. In zebrafϊsh, mpx transcripts are first detected at 19 hpf in the ICM but after the onset of circulation are detected in cells distributed throughout the embryo, although mainly over the surface of the yolk and in the axial vessels. By 48 hpf, mpx transcripts (and peroxidase enzyme activity) are detected in cells within the DV A/posterior blood island (PBI) region and over the surface of the yolk. In clutches of embryos from a man heterozygous in-cross, the same ¹ A of embryos that displayed the man gross morphology phenotype also demonstrated a marked reduction in mpx expressing cells by WISH.

Zebrafish granulocytes stain with the histochemical stain Sudan black B. Sudan black is a fat-soluble dye that preferentially stains the granules of neutrophil granulocytes more avidly than any other cell structure. In zebrafish embryos, Sudan black staining closely mirrors mpx expression. 48 hpf man embryos also demonstrated a marked reduction in Sudan black staining cells, similar to the reduction in mpx cell numbers.

The predominant reduction in myeloid cells in the PBI region at 48 hpf suggested an effect of the man mutation on definitive myelopoiesis, which commences in the VDA/PBI region around this time.

Other haematopoietic cells produced from the definitive wave of haematopoiesis apart from myeloid cells and erythrocytes include T and B lymphocytes, thrombocytes, eosinophil granulocytes and mast cells. The recombinase activation gene ragl is expressed in the developing zebrafish thymus from 4 dpf and marks T lymphocytes in this organ, B lymphocytes, marked by their expression of ighm, do not appear until 10 dpf initially in the pancreas. Thrombocytes first appear at 2 dpf in the PBI region and later enter circulation. They express the glycoprotein Ilb/IIIa receptor (itga2b, also known as CD41) and the thrombopoietin receptor mpll (Also known as c-mpϊ). Mast cells have recently been identified in zebrafish by their expression of caboxypeptidase A5 (cpa5) and are believed to be derived from a common granulocyte/monocyte progenitor. The eosinophil population, can be purified from adult kidney on the basis of their physical characteristics and expression of a gata2 derived GFP transgene.

Expression of ragl at 82 hpf in the thymus of man embryos was completely absent, although normal expression of ragl was observed in the olfactory epithelium at this time-point, showing that despite the significant brain and facial abnormalities seen in man by 82 hpf, ragl could still be expressed normally outside the haematopoietic system. Expression of foxnl, a marker of thymic epithelial cells was markedly reduced or absent in man embryos. This indicates a role for the man gene in both T cell specification and thymus formation, alternatively the effects seen may be secondary to the structural defects seen in this region of man embryos by this timepoint. B lymphocytes were not able to be analysed as the man mutation is embryonic lethal before the onset of ighm expression. Expression of both thrombocyte markers mpll and itgalb was completely absent from the PBI of 82 hpf man embryos.

The circulation of erythrocytes over the yolk and the trunk globin expression suggested that development of erythropoiesis in man embryos was delayed between 48 hpf and 82 hpf. Alternatively the normal down-regulation of hbae3 between 48 and 82 hpf may not have occurred in man, but this would not explain the abnormal presence of erythrocytes in circulation over the yolk at this timepoint.

The man mutation causes neuronal cell death via apoptosis. By 48 hpf man embryos had brain and eye abnormalities with reduction in eye size, opacification of the forebrain and increased brain ventricle size (hydrocephalus), man embryos at 48 hpf had striking abnormalities in the nervous system throughout the brain, eye and spinal cord. Neuronal cells within the brain and eye were undergoing apoptosis with cellular shrinkage, nuclear condensation and membrane abnormalities. Consistent with this, the vital dye acridine orange, marks apoptotic but not necrotic cells. In man, acridine orange staining demonstrated an increase in apoptotic cells at 48 hpf particularly in the midbrain and forebrain but also throughout the rest of the brain and eye.

The eye of man embryos showed complete loss of the normal ordered structure. The lens was reduced in size with disruption of the surrounding epithelial layer. The retinal ganglion cell layer, inner plexiform layer, amacrine cell layer, bipolar cell layer, outer plexiform layer, photoreceptor layer and pigmented epithelial layers of the retina could not be distinguished. The marked enlargement of the brain ventricles compared to WT embryos was confirmed by rhodamine-dextran ventriculography.

By lOOhpf man embryos exhibited slowing of the heart rate, with sluggish circulation of erythrocytes and marked oedema overlying the heart. The defect underlying the circulation was not due to an abnormality in the vasculature structure itself as examination of man embryos also carrying ihsflila:GFP transgene demonstrated that the man mutation had no effect on the formation of the major trunk vessels, nor the intersegmental vessels. Heart development did not proceed normally, expression of the cardiac gene nkx2,5 in man embryos was seen in an elongated domain reminiscent of the primitive heart tube, suggesting the complex cardiac morphogenetic movements needed to form a multi-chambered heart did not occur.

Formation of the gastrointestinal system was also abnormal. Expression of foxAl at 48 hpf, which is expressed in the developing gut and digestive organs, showed that only a primitive liver bud had formed and no pancreas.

The man mutation did not cause a global defect in embryo development. At 48 hpf, despite the embryo exhibiting significant abnormalities in several tissues, other tissues remained unaffected. In the trunk, despite marked degeneration in the spinal cord with apoptosis, muscle tissue appeared normal. The pronephric duct, a mesoderm-derived tissue like haematopoietic tissue, also appeared normal.

Marsanne mutant zebrafϊsh have defects in myeloid, thrombocyte and lymphoid lineages and in the development of specific tissues including the nervous system with brain and eye degeneration due to apoptosis of neural cells and other ectodermal tissue showing abnormalities, e.g. liver and pancreas. This suggests that zbtbl 1 has an essential role inter alia in myeloid, thrombocyte and lymphoid lineages. EXAMPLE 9

Rescue assay to determine bioactivity ofzebrafish ZBTBIl variants

The zebrafish rescue assay was used to evaluate the ability of mutants of zbtbl 1 to rescue the man phenotype. The following capped full length zbtbl 1 mKNA were constructed. Wildtype

RNA encoding (i) wildtype Zbtbl 1 or (ii) variant Zbtbl 1 lacking amino acid 657-1146 (including the C ₂ H ₂ zinc finger domain corresponding to amino acids 569-937 in man and amino acids 566-935 in mouse) or (iii) amino acids 172-1146 (including, in addition, deletion of the BTB domain, amino acids 199-278 in zebrafish, amino acids 204-282 in man and amino acids 204-282 in mouse). Also, (iv) RNA encoding Zbtbl l comprising substitutions in one of four residues in the HHCC motif within amino acids 79-119 of Zbtbl l (C116S, C119S, H86A and H79A).

In the rescue bioassay, zebrafish comprising a loss of function mutation in the zbtbll gene are injected with RNA encoding the ZBTBIl variant (i to iv) in order to determine the ability of the protein encoded by the introduced RNA to rescue the mutant phenotype. Specifically, a population (clade) of embryos resulting from a cross between man heterozygotes were injected (e.g. at the 1-4 cell stage) with test RNA encoding test protein and embryos assessed to determine whether or not the test RNA or its encoded product are able to rescue the phenotype of the man mutant. The diploid progeny (embryos) of a cross between man heterozygotes are expected to exhibit a Mendelian ratio of 25% mutant (man ^A ) and 75% wildtype (50% man ^+/" , 25% man ^+/+ ) . After injection at the 1-4 cell stage, embryos are allowed to develop and are then assessed to determine a reduction in the number or proportion of mutant embryos expressing the man phenotype. Specifically, individual embryos were screened for numbers of mpx expressing cells at 48hpf per embryo.

Figures 10 and 11 set out the constructs and results diagrammatically. Thus, in Figure 10, the wildtype zbtbll and mutant lacking a zinc finger domain were able to reduce the proportion of embryos exhibiting the man phenotype (from about 25% to about 2%). While the uninjected population or the population injected with a mutant lacking the zinc finger domain and the BTB domain, showed the expected ratio of about 25% man mutants indicating that this truncated variant was unable to rescue the man phenotype. Similarly in Figure 11, the wildtype (WT) zbtbll RNA was able to rescue the man phenotype while the HHCC mutants were biologically inactive.

Rescue of the man phenotype caused by zbtbll loss of function may be assessed by a broad range of strategies based upon detecting one or more of the phenotype changes associated with zbtbll dysfunction as determined herein. The assay described above was conducted using a transgenic zebrafish line which also expresses green fluorescent protein (GFP) in neutrophils from the mpx promoter enabling man embryos to be recognised and scored directly under fluorescence microscope, if required. The number, location or activity of neutrophils in developing embryos may be determined by any number of available techniques known to those skilled in the art. These include Sudan black cytochemistry or immunocytochemistry using agents directed to neutrophil markers or other reporters of myeloperoxidase activity. The man phenotype is characterised by a range of phenotypic changes any of which may be assessed in an embodiment of the rescue assay. For example, changes in myelopoiesis may be determined by monitoring the level number or activity of myeloid cells, neutrophils, myelomonocytic cells or leukocytes. Alternatively, zebrafish survival could be assessed as man mutants are, for example, lethal by day 4. Other forms of degeneration that could be monitored include central nervous system degeneration, retinal degeneration and hydrocephalus. The development of other tissues such as the gastrointestinal system, pancreas, heart development, brain ventricle development, thymus development etc, may be assessed.

Table 1 Summary of sequence identifiers

Table2 Amino acid sub-classification

Table 3 Exemplary and Preferred Amino Acid Substitutions Table 4 Codes for non-conventional amino acids

Non-conventional Code Non-conventional Code amino acid amino acid α-aminobutyric acid Abu L-N-methylalanme Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-metliylglutamine Nmgln carboxylate L-N-metliylglutamic 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 DgIn L-N-methylnorvaline Nmnva

D-glutamic acid DgIu L-N-methylomithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine DiIe L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyro sine 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 NIe

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

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

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine 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 NgIu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

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

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-carbamylethyl)glycine NgIn

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

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgIu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine 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-methylalanine Dnmala N-cyclooctylglyciήe Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

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-guanidinopropyl)glycine Narg

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

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine _. Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnnilys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine NaIa D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl) glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval

D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap

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

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

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-α-methylalanine Mala

L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine MgIn L-α-methylglutamate MgIu

L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe

L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine MnIe

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-α-methylρhenylalanine Mphe L-α-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan 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 carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl- Nmbc ethylamino)cyclopropane

Table 5

oo

I

Table 6 primer sequence (5'-3') PCR conditions

Cycle

Name F R Initialisation Denaturation Annealing Extension Final extension number z27232,zl2094,zl0183 95'C x 2 mins 95'C x 30 sees 72.5'C x 30 sees 72'C x 1 min 72'C x 10 mins 45 A TTTGTGCACG GCAGCATTGGC TCTGCATTG AAATATGGT rpl5b exon2 (SEQ ID NO: 7) (SEQ IDNO: 8) 98'Cx 30 sees 98'Cx 10 sees 60'Cx 30 sees 72'Cx 45 sees 72'Cx 5 mins 40 CTGACCCTCTTT TTGCTGCAGAG GCTTCCAG ATGGTTGTC

DC7 (SEQ ID NO: 9) (SEQ IDNO: 10) 95'C x 2 mins 95'Cx 30 sees 72.5'C x 30 sees 72'CxI min 72'Cx 10 mins 45 TCTACTACTATC ATAAGTACATG CGCAAGTCTTC TTGC GTACATGAG zbtbll exon2 (SEQIDNO: 11) (SEQIDNO: 12) 98'Cx 30 sees 98'Cx 10 sees 60'Cx 30 sees 72'Cx 30 sees 72'Cx 5 mins 40 oo AACAACACACT GAAATGGCAAT GAGTCATTCG CAATGCAAG dpt exon4 (SEQIDNO: 13) (SEQ IDNO: 14) 98'Cx 30 sees 98'Cx 10 sees 60'Cx 30 sees 72'Cx 30 sees 72 ¹ Cx 5 mins 40 TGCTCGATCCA TGAAACCGTTT TGTTGAACT TGCTTTCCT

DC26 (SEQ ID NO: 15) (SEQIDNO: 16) 98'Cx 30 sees 98'Cx 20 sees 65'Cx 30 sees 72'Cx 15 sees 72'Cx 5 mins 40 gsatcgat GTTCCC ggagatcΥGCAAA

CCTTGGAAAAC CATTCACACAT

ATT CCA zbtbll cDNA (SEQ ID NO: 17) (SEQ ID NO: 18) 98'C x 30 sees 98'C x 10 sees 58'Cx 30 sees 72'Cx 2 mins 72'Cx 5 mins 30

(added restriction enzyme sites in italic underline )

GGACTGCATCA GTTTGGCAATG AGGAAAGCATT AATGCTTTCCTT zbtbll mutant- WT CATTGCCAAAC GATGCAGTCC mutagenesis (SEQIDNO: 19) (SEQ ID NO: 20) 95'CxI min 95'C x 50 sees 60'Cx 50 sees 68'Cx 8 mins 68'Cx 7 mins 18

Table 7 Transient over-expression of RNA encoding WT Homo Sapiens ZBTBIl

* Values given are numbers of embryos and (% of Total).

BIBLIOGRAPHY

Adam et al, Exp Cell Res, 256: 282-290, 2000

Altschul et al, J. MoI. Biol. 215: 403-410, 1990 Altschul et al, Nucleic Acids Res. 25: 3389-3402, 1997

Amezcua ef α/, Structure (London) 10: 1349-1361, 2002

Arkin et al, Proc. Natl. Acad. ScI USA, 89: 7811-7815, 1992

Ausubel (Ed), Current Protocols in Molecular Biology, 5 ^U Edition, John Wiley & Sons,

Inc, NY, 2002 Ausubel et al, Current Protocols in Molecular Biology John Wiley & Sons Inc, Chapter

15, 1994-1998

Ausubel et al, eds., Current Protocols in Molecular Biology, Vol. I, Green Publishing

Associates, Inc., and John Wiley & Sons, Inc., New York, at p. 2.10.3, 1989

Bennett et al., Blood 98(3): 643-651, 2008 Boimeτ et al, Eur. J. Biochem. 46: 83, 1974

Burns et al, Nat. Chem. Biol. 1: 263-264, 2005

Carradice et al, Blood 111(7): 3331-3342, 2008

Carrell et al, Angew. Chem. Int. Ed. Engl. 33: 2059, 1994

Ceballos et al, Oncogene, 19: 2194-2204, 2000 Chiu-Ju Huang et al, Dev Dyn, 233: 4, 1294 - 1303, 2005

Cho et al, Science, 261: 1303, 1993

Cohen et al, J. Med. Chem., 55:883-894, 1990

Conner et al, Proc. Natl. Acad. Sci. USA, 80: 278-282, 1983

Constantini et al, Cancer Biotherm. Radiopharm. 25(1): 3-24, 2008 Cull et al , Proc. Natl Acad. Sci. USA, 5P: 1865-1869, 1992

Culver, Gene Therapy: A Primer for Physicians, 2 ^nd Ed., 1996

Cwirla et al, Proc. Natl Acad. Sci. USA, 87: 6378-6382, 1990

Dayhoff et α/., Natl. Biomed. Res. Found, 5: 345-358,1978

De Coupade et al, Biochem J. 590(pt2): 407-418, 2005 Delgrave et al, Protein Engineering, 6: 327-331, 1993

Devlin, Science, 249: 404-406, 1990 De Witt et al, Proc. Natl. Acad. Sci. USA 90: 6909, 1993

DiDomenico, Proc. Natl Acad. Sci. USA 79: 6181-6185, 1982

DiRisi et al., Nature Genetics, 14: 457-460, 1996

Douillard and Hoffman, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981

Doyon et al, Nat. Biotech. 26: 702-708, 2008

Ekker and Landon, Genesis, 50:89-93, 2001

Elghanian et al, Science 277: 1078-1081, 1997

Erb et al, Proc. Natl Acad. Sci USA 91: 11422, 1994 Erickson et al, Science 249: 527-533, 1990

European Patent Application No. 0 425 73 IA

European Patent Publication No. 0 332 435

Felici, J MoI. Biol, 222: 301-310, 1991

Finkelstein et al, Genomics, 7: 167-172, 1990 Fodor, Nature, 364: 555-556, 1993

Friedman, Ed., Therapy for Genetic Disease, Oxford University Press, pp. 105-121, 1991

Frohman et al, Proc. Natl Acad. Sci USA 85: 8998-9002, 1988

Gallop et al., J. Med. Chem. 37: 1233, 1994

Gish and States, Nature Genet. 3: 266-272, 1993 Gonnet et al, Science, 256(5062): 1443-1445, 1992

Gossen and Bujadt, Proc. Natl Acad. Sci. USA, 89: 5547-5551, 1992

Grompe et al, Proc. Natl. Acad. Sci. USA, 86: 5855-5892, 1989

Hacia et al, Nature Genetics, 14: 441-447, 1996

Harmsen and De Haard, Appl Microbiol Biotechnol. 77(1): 13-22, 2007 Henchey et al, Curr. Opin. Chem. Sept 12, 2008

Hillen and Berens, Annu. Rev. Microbiol, 48: 345-369, 1994

Hodgson, Bio/Technology 9: 19-21, 1991

Hogan et al, Curr. Biol. 16: 506-511, 2006

Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbour Laboratory Press, CSH NY, 1986

Houghten, Bio/Techniques 13: 412-421, 1992 International Patent Publication No. WO 00/024885

International Patent Publication No, WO 00/045167

International Patent Publication No. WO 01/68836

International Patent Publication No. WO 03/064626 International Patent Publication No. WO 03/079776

International Patent Publication No. WO 2005/118629

International Patent Publication No. WO 84/03564

International Patent Publication No. WO 90/07936

International Patent Publication No. WO 92/19195 International Patent Publication No. WO 93/07282

International Patent Publication No. WO 94/25503

International Patent Publication No. WO 95/01203

International Patent Publication No. WO 95/05452

International Patent Publication No. WO 96/02286 International Patent Publication No. WO 96/02646

International Patent Publication No. WO 96/11698.

International Patent Publication No. WO 96/40871

International Patent Publication No. WO 96/40959

International Patent Publication No. WO 97/02048 International Patent Publication No. WO 97/12635

Johnson et ah, Peptide Turn Mimetics in Biotechnology and Pharmacy; Pezzuto et al eds

Chapman and Hall, New York, 1993

KinszlQr et ah, Science, 251: 1366-1370, 1991

Kohler ef ah, European Journal of Immunology. 6: 511-519, 1976 Kohler et al. , Nature, 256: 495-499, 1975

Kunkel et ah, Methods in Enzymoh, 154: 367-382, 1987

Kunkel, Proc. Natl. Acad. Sci. USA, 82: 488-492, 1985

Kyte et ah, J. MoI. Biol., 157: 105-132, 1982

Lakso et ah, Proc Natl Acad Sci USA., 89(U): 6232-6, 1992 Lam, Anticancer Drug Des. 12: 145, 1997

Lam, Nature, 354: 82-84, 1991 Langonm etal., Proc Natl Acad Sci USA., 102(11): 6068-73, 2005

Lawson and Weinstein, Dev Biol. 248(2): 307-18, 2002

Lieschke and Currie, Nat Rev Genet, 8(5): 353-367, 2007

Lipshutz et al, Biotechniques, 19: 442-447, 1995 Lockhart et al, Nature Biotechnology, 14: 1675-1680, 1996

Lui et al, BMC Biotechnol, 7: 78, 2007

Madden έtf tf/., Meth. Enzymol. 266: 131-141, 1996

Mansour et al, Nature 336: 348-352, 1988

Marmur et al, J. MoI. Biol. 5; 109, 1962 Meng et al, J. Comp. Chem., 13: 505-524, 1992

Meng et al, Methods in Cell Biology: Zebrafish Eds Detrich et al, Academic Press: 133-

148, 1998

Meng et al, Nat. Biotechnology, 26(6): 650-701, 2008

Modrich, ,4«rc. Rev. Genet., 25: 229-253, 1991 Mukhopadhyay et al, Current Opinion in Hematology, 1 J(3):221-227, 2008

Muyldermans, J Biotechnol. 74: 277-302, 2001

Myer-Losic et al, 49(23): 6908-6916, 2006

Nasevicius et al, Nat. Genet. 26: 216-220, 2000

Navia et al, Current Opinions in Structural Biology, 2: 202-210, 1992 Newtown et al, Nucl. Acids. Res., 17: 2503-2516, 1989

Orita et al, Proc. Nat. Acad. Sci. USA, 86: 2776-2770, 1989

Pickert, Transgenic Animal Technology: A Laboratory Handbook, Academic Press, San

Diego, CA, 1994

Remington's Pharmaceutical Sciences, 20th Ed. Williams and Wilkins, 2000 Robertson et al, Nature 322: 445-448, 1986

Ruano et al, Nucl. Acids. Res., 17: 8392, 1989

Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbour

Laboratory Press, Cold Spring Harbour, NY, 1989

Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHLP, CSH, NY, 2001 Sambrook, Molecular Cloning: A Laboratory Manual, 3rd Ed, John Wiley & Sons, Inc,

NY, 2002 Scheer et al, Mechanism of Development, 80: 153-158, 1999

Schlueter and Peterson, Circulation, 120(3):255-63, 2009

Scott and Smith, Science, 249: 386-390, 1990

Sheffield et al., Am. J. Hum. Genet., 49: 699-706, 1991 Sheffield et al , Proc. Natl. Acad. Sci. USA, 86: 232-236, 1989

Shoemaker et ah, Nature Genetics, 14: 450-456, 1996

Stegmaier et al, PLOS Medicine, 4(4): 702-714, 2007

Sternberg and Hamilton, J. MoI. Biol, 150: 467-486., 1981

Summerton et al, Antisense and Nucleic Acid Drug Development, 7: 187-195, 1997 Tang et ah, Molecular and Cellular Biology, 19(1): 680-689, 1999

Tibary et al, Soc. Reprod. Fertil. Supph, 64: 297-313, 2007

U.S. Patent No. 4,554,101

U.S. Patent No. 4,873,192

U.S. Patent No. 5,034,506 U.S. Patent No. 5,223,409

U.S. Patent No. 5,252,479

U.S. Patent No. 5,403,484

U.S. Patent No. 5,550,050

U.S. Patent No. 5,571,698 U.S. Patent No. 5,691,198

U.S. Patent No. 5,738,996

U.S. Patent No. 5,807,683

Udvadia et al, Dev. Biol, 256: 1-17, 2003

United States Patent Publication No. 2004/0143865 United States Patent Publication No. 2007/0042983

Visintin et al, J. Biotechnol, 135: 1-15, 2008

Visintin et al, J. Immunol Methods, 2P0(l-2):135-53, 2008

Wartell et al, Nucl. Acids Res., 18: 2699-2705, 1990

Watson et a Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987

Wells, Methods Enzymol, 202: 2699-2705, 1991 Westerfield, The Zebraflsh Book: Guide For The Laboratory Use Of Zebrafish (3 ^rd

Edition), 1995

White et al, Genomics, 12: 301-306, 1992

Wienholds et al., Genome Res 73(12): 2700-2707, 2003

Wilder et al., ChemMedChem. 2(8): 1149-1151, 2007

Wittwer et al., Clinical Chemistry, 49: 853-860, 2008

Zhang and Madden, Genome Res. 7: 649-656, 1997

Zubay, G., Biochemistry, Third Edition, Wm.C. Brown Publishers, 1993

Zuckermann et al, J. Med. Chem. 37: 2678, 1994