WO2010019995A1 - Modulation du développement vasculaire par le facteur de transcription erg - Google Patents

Modulation du développement vasculaire par le facteur de transcription erg Download PDF

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WO2010019995A1
WO2010019995A1 PCT/AU2009/001057 AU2009001057W WO2010019995A1 WO 2010019995 A1 WO2010019995 A1 WO 2010019995A1 AU 2009001057 W AU2009001057 W AU 2009001057W WO 2010019995 A1 WO2010019995 A1 WO 2010019995A1
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erg
activity
cell
teleost
vascular
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Felix Ellett
Graham John Lieschke
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The Walter And Eliza Hall Institute Of Medical Research
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Definitions

  • the present specification describes the role of the Erythroblast Transformation Specific (ETS) transcription factor ERG in vertebrate development and in particular, its role in the origin and development of vascular tissue (vasculogenesis).
  • ETS Erythroblast Transformation Specific
  • the specification provides methods and agents for modulating vascular growth. In one embodiment, methods are provided for identifying agents useful in modulating the development of vascular tissue in a vertebrate subject.
  • ETS transcription factors form a large multigene family of approximately twenty-six members that share a common DNA-binding "ETS domain".
  • the family comprises nine subfamilies which are grouped by conservation of various other domains as reviewed by Oikawa et al, Gene, 303: 11-34, 2003.
  • the ETS factor, ERG contains four functional domains responsible for DNA-binding, transcriptional activation or transcriptional repression.
  • the amino-terminus comprises a "pointed domain", the specific function of which has not been determined in ERG, but which has been associated with protein-protein interaction in other ETS family members (Kim et al, EMBO J., 20: 4173-4182, 2001; Carrere et al, Oncogene, 16: 3261- 3268, 1998).
  • Near the carboxyl-terminus is an ETS DNA binding domain which is definitive for all ETS family members, and has a winged helix-turn-helix topology.
  • ETS domain binds DNA via its third helix, and is critical to the function of ERG (Carrere et al, 1998 ⁇ supra)); Kodandapani et al, Nature. 380: 456-460, 1996). ERG binds to 5'- GGA(A/T)-3' which is a core recognition motifs in enhancers and promoters to activate transcription (reviewed in Sharrocks, Nat. Rev. MoI. Cell Biol, 2: 827-837, 2001).
  • ERG is expressed in various mesodermal cells and neural crest cells during embryogenesis, T cell precursors, endothelial cells, platelets and HSCs (Vlaeminck- Guillem et al, Meek Dev., 91: 331-335, 2000; Anderson et al, Development, 126(14): 3131-1348, 1999; Rainis et al, Cancer Res., 65: 7596-7602, 2005).
  • One study showed that insertion of an ERG transgene into the developing limb buds of the chick was sufficient to maintain chondrocytes (cartilage-forming cells) in an immature state and prevent the replacement of cartilage with bone (Iwamoto et al, J. Cell Biol, 150: 27-40, 2000). This implicated ERG in the regulation of cartilage formation, consistent with its expression pattern in the embryo (Vlaeminck-Guillem et al. , 2000 (supra)).
  • ERG is a proto-oncogene with transforming capabilities (Hart et al, Oncogene, 10: 1423-1430, 1995).
  • a t(21;22) chromosomal translocation which fuses the EWS and ERG genes can be detected in 5-10% of patients with the paediatric bone cancer Ewing's sarcoma. In 80% of remaining Ewing's sarcomas, EWS is fused with FLI-I, the most closely related paralog of ERG.
  • EWS is an RNA binding protein that can associate with the RNA polymerase II complex responsible for transcription, but its cellular function is unclear.
  • the resulting EWS-ERG fusion protein consists of the N-terminal region of EWS and the C-terminal region of ERG, including the ETS domain. Carcinogenesis is thought to occur because the EWS region confers aberrantly strong transactivation activity while the ETS domain of ERG continues to bind DNA.
  • ERG has been implicated generally in physiological hematopoiesis.
  • ERG expression may influence differentiation from an erythroid to a megakaryocyte lineage.
  • Loughran et ah, Nat. Immunol, 9(7): 810-819, 2008 reported that in the mouse ERG is essential for definitive hematopoiesis, for normal HSC function and for maintaining normal numbers of mature blood cells.
  • ERG has been implicated in early hematopoiesis and a number of diseases in adults are associated with various disruptions of the ERG locus. Accordingly, there is a also a need in the art for methods for assessing the functional effect of variant forms of ERG, including naturally occurring or artificially generated forms thereof and for identifying and testing agents that modulate the pathways comprising ERG.
  • SEQ ID NO: correspond numerically to the sequence identifiers ⁇ 400>l (SEQ ID NO: 1), ⁇ 400>2 (SEQ ID NO: 2), etc.
  • SEQ ID NO: 1 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.
  • ERG polypeptide is the expression product of ERG nucleic acid sequences.
  • the specification describes a method of modulating vascular development, said method comprising contacting a subject, cell or tissue with an agent which modulates the level or activity of an ETS transcription factor or of a transcriptional target thereof in said subject, cell or tissue.
  • vascular development encompasses production or growth or proliferation of vascular tissue or precursor cells thereof in adult subjects.
  • development includes re-development of blood vessels after events such as trauma or surgery, as well as blood vessel formation associated with tumour growth or other conditions.
  • Cellular activity means growth, proliferation, migration, differentiation or development of a precursor cell towards a more fully differentiated phenotype.
  • a "cell” is preferably a vascular cell or a precursor thereof in other embodiments a suitable eukaryotic, vertebrate or mammalian cell line may be employed.
  • the agent enhances or down regulates the activity of ERG polypeptide, or a transcriptional target of ERG polypeptide or a downstream effector of ERG polypeptide activity in a cell.
  • a transcriptional target is a gene to which ERG polypeptide binds or its expression product.
  • the transcriptional target is the regulatory region of a gene to which ERG polypeptide binds to modulate transcription.
  • genes referred to in the illustrative examples distinguishes between human, murine and zebrafish genes according to art recognised protocols. Specifically, for human genes, names are capitalized and italicised and human proteins are capitalized but not italicised. Murine genes have the first letter capitalised and proteins are not italicised. Zebrafish genes are in lower case and italicised, proteins are not italicised. Although the present invention has been in part exemplified with zebrafish genes, it is not limited to zebrafish genes and extends to homologs from other vertebrate groups such as murine and human and other animals. As shown in Example 1 and Figure 1 Erg is highly conserved between species.
  • a gene or protein such as an ERG polypeptide or peptide or an ERG nucleic acid molecule, 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 having an activity of human ERG are particularly contemplated. There are several related isoforms known in the art each of which may be employed herein.
  • Illustrative human ERG variants are set out in the sequence listing (see Table 1) showing sequences for Accession Nos.: AK297807, AK300395, AK301277, AK303518, AK304662, AY204741, AY204742, BC040168, EU432099, M21535.1 and S72621.
  • the present invention is predicated, in part, on the identification of a role for ETS transcription factors in a teleost animal model of vertebrate development. More particularly, of a role for ERG in teleost vasculogenesis.
  • the term "teleost” refers to a group of fish having bony skeletons and rayed fins.
  • the group comprises zebrafish (Danio reri ⁇ ), 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 al., Blood 111(7): 3331-3342, 2008 zebrafish are affordable, genetically tractable vertebrates that exhibit rapid ex vivo development, high fecundity and optical transparency.
  • ERG has a specific activity in blood vessel formation and particularly in vascular precursors and developing vessels.
  • Erg and ERG or homologs thereof are identified herein as a useful target for modulating vasculogenesis.
  • vasculogenesis The formation of blood and lymph vessels is referred to as vasculogenesis and includes the development of vascular precursors and vascular tissue.
  • Angiogenesis which refers to the development of new blood vessels from existing blood vessels is a subsequent event in the development of vascular tissue.
  • Vascular precursors include pluripotential haemangioblasts which produce angioblasts and hematopoietic stem cells, and angioblasts that take part in blood vessel formation.
  • ERG has a role in vasculogenesis and also has a role in early angiogenesis.
  • ERG is proposed herein to be expressed and to be active specifically in vascular precursors and in endothelial cells forming vessels
  • Etsrp and TaI-I have additional roles in myoblast and erythroblast specification.
  • both cell types are proposed to develop from a common haemangioblast cell of mesodermal origin (as shown schematically in Figure 7).
  • Agents which modulate the activity of ERG may act on ERG polypeptide or alternatively they may act at the level of ERG transcription or translation.
  • a transcriptional activator of ERG binds to ERG and enhances transcription of ERG.
  • upregulation of the activity of ERG in a subject using ERG polypeptide or a functional variant thereof, or an agent from which an ERG polypeptide is producible, or variant thereof, or an agent that effectively enhances ERG activity is proposed to be useful for enhancing angioblast cell activity or for modulating vascular development.
  • agents that down regulate the functional activity of ERG in a cell are proposed for use in reducing the rate of angioblast or vascular development in a subject.
  • the subject agents bind to ERG, i.e. all or part of coding, non-coding or regulatory regions in ERG DNA or RNA and modulate gene expression at pre-transcriptional, transcriptional or post-transcriptional, including translational stages.
  • the agent binds to a transcriptional target of ERG and modulates its activity.
  • Suitable agents include gene silencing agents such as antisense, siRNA, shRNA, miRNA, ribozyme, DNAzyme, morpholino or other iRNA molecules.
  • the method comprises contacting a subject, cell or tissue with an agonist of ERG activity or of a transcriptional target thereof, wherein the ERG agonist enhances vascular precursor development and the development of pre- circulatory vascular tissue.
  • a method of reducing vascular development comprising contacting a subject, cell or tissue with an antagonist of ERG activity or of a transcriptional target thereof, wherein the antagonist reduces vascular precursor development and/or the development of pre-circulatory vascular tissue.
  • the vascular development is vasculogenesis. In other embodiments, the vascular development is vasculogenesis and/or angiogenesis. In other embodiments, the vascular development is angiogenesis whereas in other embodiments the vascular development is vasculogenesis and not angiogenesis.
  • the cell or tissue comprises vascular progenitor cells such as haemangioblasts or angioblasts.
  • the cell or tissue comprises pre-circulatory vascular structures, such as in or leading to or from organs or tissues such as the skin, endometrium, placenta, eye, brain, nervous system, pancreas, heart, lung, kidney, bladder and gut, or their precursors.
  • the tissue is ischemic or cancerous tissue.
  • the agent modulates the level or activity of an ETS transcription factor or of a transcriptional target thereof in vascular progenitor cells, such as angioblasts. In still another embodiment, the agent modulates the level or activity of an ETS transcription factor or of a transcriptional target thereof in vascular structures including pre-circulatory vascular structure.
  • the ETS factor is ERG or a complex comprising ERG or a variant thereof.
  • the ETS factor is ERG and one or more ETS factors selected from ESTRP, ETSl, ETS2, FLIlB, FLI-I, LMO2, SPI-I, FEV, DETS-6, GABP, ELG, PEA3, ELK, SAP-I, NET, ELF-I, NERF, E74A, ESE, TEL and YAN, or a homolog thereof.
  • the agent enhances ERG activity or the level or activity of transcriptional targets of ERG in angioblasts or pre-circulatory vascular structures. In other embodiments, the agent enhances ERG activity in vasculogenesis and angiogenesis.
  • the specification provides a method for modulating angioblast activity, comprising administering an effective amount of an agent which modulates the level or activity of an ERG polypeptide or a transcriptional target of ERG in said cells.
  • the agent comprises a sequence of nucleotides encoding an ERG polypeptide or a functional variant thereof.
  • the agent may be cellular, nucleic acid or a viral vector,
  • the agent reduces the level or activity of ERG polypeptide or a transcriptional target of ERG polypeptide in a cell.
  • the agent is a genetic agent which reduces the production of ERG polypeptide in a cell.
  • the agent is a cell, plasmid or viral vector.
  • the agent for use in the present methods is, for example, a small-molecule, antibody, peptide, peptidomimetic, a constrained peptide or a gene silencing molecule or the like.
  • the subject methods are for use in vitro, in utero, in vivo or ex vivo.
  • the subject methods are for use in vivo, in utero, or ex vivo treatment or prophylaxis of a condition associated with too much or too little vasculogenesis in a vertebrate subject.
  • the condition involves a vascular cell precursor (progenitor) cell defect such as an angioblast defect, or a vascular defect.
  • the condition involves a defect in vascular tissue associated with an organ or tissue in a subject or wherein the defect is ischemia.
  • the defect may be caused by suboptimal levels of vascular precursor cells.
  • a vascular defect is associated with a congenital or acquired defect, state or condition selected from the group consisting of: Alzheimer's disease, cardiovascular disease, coronary artery disease, congestive heart failure, peripheral arterial disease, ocular vascular disease, carotid artery disease, wound or burn healing, chemical or mechanical trauma, surgery, transplantation, aortic arch condition (coarctation), athersclerosis, acute myocardial infarction, unstable angina, chronic stable angina, transient ischemic attacks, strokes, preeclampsia, deep venous thrombosis, embolism, disseminated intravascular coagulation and thrombotic cytopenic purpura, thrombotic disorders, inflammatory disorders, chronic vascular disease, autoimmune disorders, transplant vasculopathy/rejection, atherosclerosis, hypertension, aneurysmal disease, vasospastic syndromes, ischemic coronary syndromes, cerebral vascular disease, angiogenic (both pro and anti)
  • the indication is prostate cancer.
  • the present specification provides an isolated cellular agent, nucleic acid or vector comprising a sequence of nucleotides encoding an ERG polypeptide or a functional variant thereof for use in the treatment or prophylaxis of a vascular defect in a vertebrate subject.
  • ERG nucleic acid for use as a marker for vasculogenesis is conveniently tagged to serve directly or indirectly as a reporter of the location, quantification or activity of ERG or ERG.
  • agents agents, agents, modulator or modulators
  • agonists agents which modulate, directly or indirectly, the activity of ERG polypeptide or one or more of its transcriptional targets in a cell.
  • modulators will be effective in modulating ERG activity or the activity of its transcriptional targets.
  • the agents and compositions of the present invention include small or large chemical molecules, antibodies, peptides, polypeptides, proteins including cell- penetrating proteins or nucleic acid molecules including antisense or other gene silencing molecules, and their precursors or derivatives.
  • overexpression of ERG in a teleost provides a new measure of ERG functional activity and a convenient model for assessing the ability of an agent either to affect vasculogenesis or vascular precursor activity, or, to assess the ability of an agent to provide erg functional activity.
  • the invention provides a method of identifying or assessing an agent which modulates vascular precursor activity or vasculogenesis, the method comprising (i) contacting the agent with a teleost model of ERG overexpression wherein the teleost overexpresses or is capable of overexpressing a polypeptide having the activity of erg or ERG or a homolog of ERG or erg; and (ii) dete ⁇ nining the effect of the agent on vascular progenitor activity or vasculogenesis induced by overexpression of erg or ERG or a homolog in the teleost relative to controls.
  • step (ii) comprises screening the teleost for expression of one or more markers of vascular precursor activity or vasculogenesis.
  • the marker is selected from one or more of fik-1 or FLK-I or a homolog, fli-1 or FLI-I or a homolog, scl or SCL or a homolog, and erg or ERG or a homolog, or a detectable reporter of such expression.
  • the teleost may be modified to overexpress erg or ERG or a homolog under the control of an inducible promoter.
  • variants of ERG are employed such as wherein the teleost overexpresses a variant of erg or ERG 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 vasculogenesis such as etsrp, fli- 1, scl/tal-1, flk-1, ets-1, ets-2, gata-1, flk-1 (vegfr2) and cdh5 or, ETSRP, FLI-I, SCL/TAL-1, FLK-1, ETS-I, ETS-2, GATA-I, FLK-1 (VEGFR2) and CDH5 or homologs thereof.
  • the teleost model is a model of vasculogenesis or vascular progenitor activity.
  • ERG polypeptide or a polypeptide having ERG activity includes polypeptides having the amino acid sequences of naturally occurring forms of ERG polypeptides or fragments (parts) thereof or of variants thereof.
  • a large number of ERG 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 ERG are overexpressed in the teleost model.
  • overexpression is used broadly in this specification to refer to the production or presence of ERG 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 erg activity or potential erg 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 ERG expression in particular cells, tissues or organisms are employed.
  • the specification contemplates a method of screening for an agent which modulates vasculogenesis in a subject, cell or tissue said method comprising screening for an agent that modulates the functional activity of ERG polypeptide or a transcriptional target of an ERG polypeptide in a cell or a downstream effector of ERG polypeptide activity.
  • the method comprises: (i) combining the agent and a cellular or animal model; and (ii) identifying a change in the activity of the cellular or animal model relative to controls indicating that ERG polypeptide activity including the activity of a transcriptional target or a downstream effector molecule has been modified.
  • the identifying step includes identifying the presence of a complex between ERG or ERG and the modulator, or a change in the level or activity of a marker of the activity of ERG polypeptide in a cell. In other embodiments, the identifying step includes assaying for one or more of the following: a change in the activity of the ERG or ERG; a change in the level or activity of a reporter of the activity of ERG or ERG; and the presence of a complex between the ERG or ERG and the modulator.
  • the animal model is a teleost or zebrafish vertebrate animal model.
  • the method involves an initial further step of introducing random or non-random mutations, or modulating expression of ERG gene and/or one or more other genes in the cellular or animal model.
  • the mutation or down-regulating agent is introduced via a mutagenesis agent or vector. The teleost may conveniently be modified.
  • the specification provides agents as defined herein for use in modulating vasculogenesis.
  • the specification contemplates a method of identifying an agent that modulates vasculogenesis, said method comprising; (i) contacting the agent with a transgenic teleost such as zebrafish that expresses a reporter protein under the control of an ERG expression sequence (such as a promoter), and (ii) assessing the level or distribution of ERG-reporter protein in the teleost compared to controls.
  • a transgenic teleost such as zebrafish that expresses a reporter protein under the control of an ERG expression sequence (such as a promoter)
  • ERG expression sequence such as a promoter
  • assessing the level or distribution of ERG-reporter protein in the teleost compared to controls an enhanced level or enhanced distribution of ERG-reporter expression relative to controls is indicative that the agent enhances vasculogenesis.
  • a reduced level or reduced distribution of ERG-reporter expression relative to controls is indicative that the agent depresses vasculogenesis.
  • the teleost is further modified to increase or decrease therein the activity of one or more factors in the angioblast vascular development pathway, such as without limitation, ERG, ETSRP, FLI-I, SCL/TAL-1, FLK-I, ETS-I, ETS-2, GATA-I, FLK-I (VEGFR2) and CDH5.
  • the reporter is a fluorescent or illuminescent protein.
  • Another embodiment provides a method of assessing the functional activity of an agent selected from the group consisting of ERG or a variant of ERG or an agonist or antagonist of ERG activity, or an agonist or antagonist of an ERG transcriptional target in vascular endothelial tissue.
  • the method comprises: (i) contacting the agent and a model system comprising a teleost (zebrafish) and (ii) determining the effect of the agent on vasculogenesis in the teleost relative to controls.
  • the teleost is further contacted with an ERG antagonist or a putative ERG antagonist.
  • the teleost is a wild type teleost.
  • the model system is sensitised or modified to determine the effect of specific molecules expressed during vasculogenesis.
  • a modified teleost is employed which overexpresses or underexpresses one or more molecules expressed during vasculogenesis such as ETSRP, ETSRP, FLI-I, SCL/TAL-1, FLK-I, ETS-I, ETS-2, GATA-I, FLK-I (VEGFR2) and CDH5, and the like.
  • Reduced levels are conveniently reached using antisense morpholino nucleic acids.
  • the teleost is screened for expression of one or more markers of vasculogenesis such as expression of FLK-I, FLI-I, SCL, and/or ERG or of detectable reporters of such expression.
  • zebrafish embryos are contacted at the 1 to 4 cell stage, the 1 to 8 cell stage, the 1 to 16 cell, the 1 to 32 cell or the 1 to 128 cell stage.
  • 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.
  • the cell stage is fewer than 16, more preferably fewer than 8, and most preferably fewer than 4 or 2.
  • zebrafish embryos are contacted at the approximately 8 to 16 cell stage or 4 to 64 or 2 to 128 cell stage or at the 1 cell stage.
  • the model is a teleost model of a human or vertebrate disease or condition such as a vascular defect or disease, transplantation, infection of the blood or lymphatic system, high blood pressure, trauma, chemical or mechanical insult, wound, burn, hypoxia, ischaemia, cancer, clonal haemopathy, or equivalent condition.
  • the agent is introduced as nucleic acid such as mRNA into one or more zebrafish embryos at the 1 to 32-cell stage, preferably the 8 to 16 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 64-cell stage or 96-cell stage or later.
  • the present invention provides a convenient bioassay for assessing the ability of an agent to provide ERG functional activity. As shown described herein (see in particular Example 4) widespread overexpression of erg in 1-2 cell zebrafish embryos resulted in major developmental defects that could readily be detected and measured qualitatively or quantitatively. By restricting erg overexpression to the mesoderm, by delating injection to the 8 to 16 cell zygote stage, a more refined effect upon vasculogenesis could be qualitatively or quantitatively assessed.
  • 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 development or vasculogenesis in the teleost relative to controls.
  • the method comprises (iii) comparing the effect of the agent in (ii) with the effect of overexpressing erg or a homolog of erg in the teleost.
  • RNA encoding a variant of erg lacking a functional domain is injected in zebrafish embryos at the 1 to 2 cell stage and the effect on development or vasculogenesis is determined relative to the effect of overexpressing a known functional form of ERG in the teleost model.
  • embryos may be at other stages such as the 1 cell stage through stages approaching the end of embryonic development.
  • the effect of agents on vasculogenesis at any life stage of the teleost may also be determined. Methods for determining the effect of agents will vary with the developmental stage assesses as known to those in the art.
  • vasculogenesis may be assessed visually such as by monitoring intersegmental vessel development, using markers of particular tissues including expression markers of different stages of vasculogenesis as described herein or known in the art. Thus markers are assessed qualitatively or quantitatively including the position, quantity or developmental timing of expression.
  • steps (ii) and/or (iii) comprise screening a teleost for expression of one or more markers of vascular precursor activity or vasculogenesis.
  • an expanded population of GFP-positive cells is observed in a Tg(/7z7 ⁇ :EGFP) strain in a dose-dependent manner.
  • Other suitable markers include flk-1, scl, etsrp, ets, gata, cdh5 or homolgs thereof.
  • the agent comprises mRNA encoding a variant of erg or a variant of a homolog of erg such as a human or mammalian homolog of ERG.
  • 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 ERG are particularly contemplated.
  • Illustrative human ERG variants are set out in the sequence listing (see Table 1) showing sequences for Accession Nos.: AK297807, AK300395, AK301277, AK303518, AK304662, AY204741, AY204742, BC040168, EU432099, M21535.1 and S72621.
  • RNA encoding fusion protein comprising ERG or ERG variants are also particularly contemplated.
  • the effects of ERG overexpression over a 24 hour period in 1-cell zebrafish are shown in Figures 9 and 10.
  • expression of erg is restricted to the mesodermal tissues and the functional activity of erg is assessed in context of vasculogenesis or vascular precursor activity.
  • erg overexpression and the activity of the agent are determined from the 8 to 16 cell stage.
  • the specification provides a method for diagnosing a defect in vasculogenesis and/or angiogenesis in a subject or tissue, said method comprising screening a sample from a subject for a loss of function mutation or modification in ERG or ERG or in the nucleotide sequence encoding a transcriptional target thereof in vascular tissue.
  • the specification provides an isolated cell or teleost comprising such cells, wherein the activity of ERG is enhanced to facilitate the assessment of vasculogenesis and/or angiogenesis in said teleost compared to a non-modified teleost of the same species.
  • the cell or teleost is modified to overexpress ERG.
  • the specification provides an isolated cell or teleost comprising such cells, wherein the activity of ERG is reduced or inhibited to facilitate the assessment vasculogenesis and/or angiogenesis in said teleost compared to a non-modified teleost of the same species.
  • the teleost is modified to under express or inhibit expression of ERG, or to produce a variant ERG polypeptide lacking or exhibiting reduced ERG activity.
  • Teleosts provide a convenient animal model for in vivo bioassays. As shown in the Examples, zebrafish showing mesoderm-restricted overexpression of erg exhibit proliferation of vascular precursors. Accordingly, agents that down modulate proliferation of vascular precursors can readily be identified in zebrafish models. Such agents have therapeutic potential in downregulating vasculogenesis.
  • fli-1 is a useful vascular marker in zebrafish as it is expressed throughout the developing vasculature arising first in the posterior lateral mesoderm at around 12hpf. Transgenic zebrafish stably expressing EGFP from the fli-1 promoter facilitates analysis at a very early stage of development including before onset of circulation.
  • putative agents are tested in zebrafish overexpressing erg and zebrafish are monitored for a change in the level or activity of vascular precursor or the growth of vascular tissue.
  • zebrafish overexpress ERG under the control of a promoter such as an inducible promoter.
  • a promoter such as an inducible promoter.
  • one convenient marker of vascular development is fli-1 and assays are conveniently undertaken in teleosts expressing a marker such as GFP off the fli-1 promoter.
  • the teleost model of erg overexpression is a model for ERG functional activity and agents are tested for their ability to function like erg in development or vasculogenesis.
  • the present invention provides methods for screening and testing agents for their ability to modulate ERG activity, including ERG signalling pathways and/or ERG-associated transcription.
  • ERG regulates the expression of genes encoding vascular cell signalling molecules such as cytokine, chemokine, hormones or their cellular or nuclear receptors or transcription factors.
  • ERG regulates the expression of genes encoding signalling molecules or their receptors or transcription factors that are expressed in the early stages of vascular cell development and differentiation.
  • Exemplary transcriptional targets include without limitation, Tpo, flt3L, SCF, MpI, scl, IL-6, IL-I l, TGF- ⁇ , VEGF and VEGFR and the genetic region of the genes encoding those factors to which ERG polypeptide binds. Modulation of cell or ERG activity may be effected in vitro, in utero, ex vivo or in vivo.
  • the present invention provides agents capable of modulating the activity of ERG polypeptide for use in the treatment of conditions associated with an over supply or an under supply of vascular stem cells and/or progenitor cells.
  • the present invention provides a composition comprising ERG or an agent from which ERG is producible or a variant of either of these which enhances the activity of ERG polypeptide or ERG genetic sequences or ERG transcriptional targets.
  • such compositions or agents are for use in modulating vascular development.
  • modulation is potentiation or upregulation.
  • the present invention also contemplates a composition comprising an agent which down regulates the activity of ERG in a cell, tissue or subject.
  • compositions or agents are for use in modulating vascular cell activity or for treating or preventing clonal hemopathies.
  • enhancement of the activity of ERG in a cell or subject permits the normal or enhanced production i.e., up-regulates the production of vascular tissue from vascular precursors.
  • enhancement of activity of ERG potentiates the development, movement, survival, or proliferation (collectively referred to as "activity") of early angioblasts and their progeny.
  • activity The ability to modulate angioblast and vascular precursor cell numbers and distribution in a subject or in vitro has a wide range of applications in conditions associated with an under supply or activity of cells or an over supply or activity of vascular stem cells.
  • the present invention provides a preparation of vascular cells or vascular precursor cells in a medium comprising an agent that modulates ERG activity in said cells.
  • the present invention further provides methods of screening or diagnosis to determine whether or not a subject has a vascular endothelial cell defect associated with a loss of function of ERG or is susceptible to developing same, the method comprising screening a sample from the subject for a loss of function mutation in ERG or ERG.
  • Subjects may also or additionally be screened for gain of function mutations in ERG or ERG.
  • Subjects may be screened for prostate specific isoforms of ERG.
  • Figure IA to C provides representations of sequence analysis of zebrafish erg.
  • A Percent identity and divergence of full-length protein sequences between various vertebrate ETS family members.
  • B Phylogenic tree describing evolutionary divergence of ETS family homologues and paralogues. Full-length protein sequences were aligned using MUSCLE 5 and phylogeny created using MrBayes. Branch labels are probabilities.
  • C Amino acid sequence alignment of highly conserved ETS DNA-binding domains from a number of vertebrate ETS family transcription factors. This important domain is almost completely conserved between zebrafish Erg and mammalian homologues, while significant identity is also observed with FIi 1 paralogues.
  • Abbreviations: zf: zebrafish/Z) ⁇ m ⁇ rerio; mm mouse/Mus musculus and hu: human/ 'Homo sapiens.
  • Figure 2A to J are photographic representations of WISH analyses showing erg expression during early zebrafish development.
  • A Earliest expression of erg in lateral plate mesoderm at approximately 12 hours post fertilisation (hpf) (dorsal view, filled arrowhead).
  • B Flat-mount preparation shows overlap of early erg expression (blue) with tall expression (red) in vascular (filled arrowheads) but not haematopoietic progenitors (empty arrowhead).
  • D Transverse section showing erg expression in bifurcated cental arterial (filled arrowheads) and lateral venous structures (empty arrowheads).
  • E erg expression at 30 hpf in all developing vascular structures in the brain, trunk and tail of the embryos.
  • Each primer pair was designed to span an intron, providing a genomic DNA-contamination internal negative control.
  • cDNA for each timepoint was synthesised from mRNA collected from WT embryos using a SuperscriptIII RT-PCR kit (Invitrogen) according to manufacturer's instructions, erg primers (5'- GTGGGTTATGACGCTGTCAG-S' (SEQ ID NO: 15) and 5'- CTAACTGCGCTCTCTGCTC-S' (SEQ ID NO: 16)) were designed to amplify from exon
  • bact primers (5'-TGGCATCACACCTTCTAC-3 1 (SEQ ID NO: 17) and 5'- AGACCATCACCAGAGTCC-3' (SEQ ID NO: 18)) were designed to amplify from exon
  • FIG. 3A to T are photographic representations of WISH analyses showing a detailed time-course of erg expression during vasculogenesis.
  • A erg expression first appears at 12 hpf in mesodermal angioblasts. Later, erg positive cells are located in the midline and begin to coalesce into major axial and cranial vessels (B-E).
  • B-E major axial and cranial vessels
  • F By 22 hpf, erg positive cells distinctly define the anterior lateral dorsal aortas, mandibular arches and ventral aorta; lateral dorsal aorta and posterior cardinal vein and posterior caudal artery and caudal vein.
  • G-J Between 24-30 hpf, erg expression also expands to cranial primitive internal carotid artery, primordial midbrain channel, middle cerebral vein, anterior cerebral vein, and caudal and cranial divisions of the internal carotid artery.
  • K erg expression is retained in developing cranial, lateral and posterior vessels while faint expression is observed in developing intersegmental vessels.
  • L erg expression is lost in all major vessels, with expression then restricted to vessels in the developing pharyngeal arch region until 42 hpf (N).
  • Top panel lateral; central panel dorsal; lower panel tail at higher magnification.
  • G-K Top panel lateral, lower panel tail at higher magnification.
  • L-T Top panel: lateral view, lower panel: head at higher magnification.
  • FIG. 4 A to F/f are photographic representations of WISH analyses showing that erg overexpression drives mesodermal angioblast proliferation.
  • D-F/d-f Triple in situ at 14 hpf shows spil and gatal marking anterior myeloid and posterior erythroid progenitors respectively in red andflkl marking angioblast populations in blue. Lateral erg overexpression is traced with LacZ mRNA in turquoise.
  • Figure 5A and B are photographic representations of reporter assays validating the functional binding of ergMO to its target sequence.
  • A Shows EGFP fluorescence in EGFP-only (control) and Target:EGFP (test) groups in the absence of erg ATG-targeting morpholino.
  • B erg ATG-targeting morpholino efficiently knocks down EGFP expression in Target:EGFP but not EGFP-only injected embryos. Uninjected controls are shown for each experiment and wavelength. Rhodamine dextran dye was used a tracer for morpholino injection. Panels show pools of randomly selected embryos. Methodological details are in text. Top panels: bright field (BF); central panels: Rhodamine fluorescence; lower panels: EGFP fluorescence. Bright-field and fluorescent images were taken with 8 ms and 8 s exposure times respectively.
  • Figure 6A and B are representations of results showing that erg acts synergistically with etsrp to regulate vascular development.
  • Panels show GFP-marked vasculature in wild-type and ergMO, etsrpMO and double morphants on the Tg(/7z7:EGFP) line at 48 hpf.
  • erg knock-down with an antisense morpholino oligonucleotide had no effect on vascular development unless introduced on a sensitised etsrp morphant background.
  • Figure 7 is a schematic representation showing a putative role for erg in the transcriptional regulation of vasculogenesis.
  • This highly simplified model represents an analysis of published functional studies of transcriptional regulation of cellular populations in the zebrafish mesoderm such as from Stainier et al, Development, 121: 3141-3150, 1995; Liao et al, Genes Dev, 12: 621-626, 1998; Galloway et al, Dev Cell, 8: 109-116, 2005; Rhodes et al, Dev Cell, 8: 97-108, 2005; Sumanas et al, PLoS Biol, 4: elO, 2006; and Lyons et al, Proc Natl Acad Sci USA, 99: 5454-5459, 2002.
  • Factors in red have been demonstrated as essential by mutant and knockdown experiments, while factors in blue have been implicated by overexpression studies. Factors within boxes are specifically expressed in that lineage. Sharp arrows represent positive regulation, flat arrows represent negative regulation. As disclosed herein erg is positioned downstream of cloche, tall and etsrp, as a positive regulator of angioblast proliferation.
  • Figure 8 is a schematic representation of Erg proteins assayed in the bioassay of Erg activity.
  • Full-length Erg encodes a 427 amino acid protein containing two activation domains (AD), one helix -loop-helix (HLH) putative protein-protein interaction domain and an ETS DNA-binding domain.
  • AD activation domains
  • HSH helix -loop-helix
  • ETS Erg describes a protein in which the ETS DNA-binding domain has been removed, therefore representing a potentially inactive allele of Erg.
  • Figure 9 is a schematic and photographic representation of the bioassay protocol, i) Capped niRNA encoding an Erg allele to be tested is prepared in vitro and observed via gel electrophoresis pre- and post injection (as a control for RNA degradation; faint bands are observed in the bottom panel indicating that degradation has not taken place), ii) mRNA is microinjected into embryos at the 1-cell stage, iii) Readout consists of scoring the phenotypic class at 24 hours post fertilization.
  • FIG 10 is a graphical representation of results from a typical bioassay as described above. Rhodamine injection was used as a negative control. Injection of the ⁇ ETS allele at a high dose (180 pg) resulted in no phenotypic change compared to control animals, while injection of the WT allele at mRNA doses ranging from 4.5-45 pg resulted in a mix of phenotypic classes that shifted towards more severe perturbation of general development and expansion of GFP+ve cells in a dose-dependent manner. These results suggest that ⁇ ETS represents a inactive allele of Erg.
  • 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.
  • the invention is predicated, in part, upon the determination that ERG polypeptide has an important role during vertebrate blood vessel formation, by enhancing angioblast activity, vasculo genesis and angiogenesis.
  • ERG has a role at multiple stages of cardiovascular development. ERG expression is spatiotemporally restricted to angioblasts and developing blood vessels during angioblast migration, vasculogenesis and early angiogenesis. ERG expression is lost in the absence of factors known to regulate angioblast specification, proliferation and vasculogenesis. ERG overexpression specifically drives proliferation of angioblasts in the lateral plate mesoderm. Furthermore, knock-down of ERG in combination with a factor having the activity of Etsrp significantly reduces formation of intersegmental vessels by angiogenesis.
  • ERG agonists are useful in the treatment of conditions where new vascular tissue is desirable.
  • Non-limiting exemplary conditions include tissue trauma, after surgery, heart failure, peripheral arterial disease, burns and skin ulcers.
  • ERG antagonists are useful, for example, where a surfeit of blood vessels promote tumour growth or functional impairment of other tissues such as the brain and nervous system.
  • the term "derived" does not necessarily mean that the cells are directly obtained from a particular source.
  • Reference to a "cell” includes a system of cells such as a particular tissue or organ.
  • modified includes genetically modified but encompasses non- genetic or epigenetic modifications to affect ERG 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 ERG or ERG.
  • 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 ERG or ERG.
  • teleosts are modified using antisense molecules.
  • Reference herein to "modulate” and “modulation” includes completely or partially inhibiting or reducing or down regulating all or part of ERG functional activity and enhancing or up regulating all or part of its functional activity.
  • Functional activity may be modulated by, for example, modulating ERG nucleic acid binding capabilities or transcriptional or translational activity, or its half-life.
  • ERG polypeptide its functional activity may be modulated by, for example, modulating its binding capabilities, its half-life, location in a cell.
  • ERG level or activity may be modulated by modulating ERG expression, transcript stability, or the activity of its regulatory molecules.
  • ERG activity or “functional activity” encompasses any relevant, measurable activity or characteristic of the molecule in proteinaceous or genetic form and includes ERG-specific DNA and protein binding abilities. As disclosed herein, ERG stimulates vasculogenesis and the presently described in vivo assays in teleosts provide a useful method of assessing ERG function.
  • Binding or transcriptional, translational or transactivational activity are preferred ERG activities which are conveniently assessed using standard protocols known in the art as described in Sambrook, Molecular Cloning: A Laboratory Manual, 3 rd Edition, CSHLP, CSH, NY, 2001, Chapters 17 and 18; Ausubel (Ed), Current Protocols in Molecular Biology, 5 th Edition, John Wiley & Sons, Inc, NY, 2002.
  • the ability of ERG to drive transcription may be tested using luminescence reporter assays.
  • ERG ability of ERG 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.
  • Vascular endothelial cell precursor proliferation, migration, differentiation and development can all be monitored using methods known in the art. Other activities assayed include angioblast presence, homing, engraftment, apoptosis and self-renewal.
  • Functional activity in some embodiments is assessed by analysing cells comprising ERG (such as K562, see Rainis et al, 2005 (supra)) for expression of megakaryocyte or leukocytic markers antigens for example, by flow cytometry.
  • ERG ability of ERG to bind DNA via its C-terminal ETS DNA binding domain may also be tested.
  • ERG polypeptide to bind to proteins via the N-terminal pointer domain may also be also tested.
  • ERG polypeptide stimulates a characteristic gene expression profile which serves as a useful marker of ERG activity.
  • the gene expression profile of a cell when ERG polypeptide activity is down-regulated or inhibited serves as a useful marker of lack of ERG 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, second generation sequencing, antibody technology, chromatographic methods such as HPLC or thin layer chromatography or combinations of these. Binding is conveniently detected using antibodies.
  • In vitro or in vivo assays can employ a wide range of markers or indicators of ERG activity using, for example ? the methods exemplified herein.
  • Reference herein to the "activity” or “overactivity” and the like, in relation to 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.
  • cellular development, proliferation, cellular differentiation, cell function such as homing, engraftment, self-renewal, survival differentiation, cell number and cell survival.
  • 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.
  • extension symmetric division where both daughter cells retain stem cell function and which results into expansion of stem cell population
  • maintenance asymmetric division which maintains the stem cell population by producing one daughter stem cell and one daughter cell committed to differentiation
  • committed symmetric division where both daughter cells are committed to differentiate. Accordingly, in relation to vascular precursors reference to "activity" includes reference to extension symmetric division, maintenance asymmetric divisional and/or committed symmetric division.
  • ERG activity of ERG may also be monitored using DNA or protein binding assays, reporter assays or direct or indirect assays of ERG 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 may be used to detect ERG by Western Blotting, cytometric histochemical or ELISA procedures. As discussed herein below, such agents may also distinguish between active and inactive forms of the ERG or between mutant and normal forms of ERG.
  • mutant forms of ERG are forms of ERG (found in a population of subjects) which are associated with aberrant or insufficient vasculogenesis.
  • normal forms of ERG are forms of ERG which are not associated with these conditions. Mutant forms of ERG may also be conveniently be detected using nucleic acid based assays well know in the art and as described herein. Low levels of active polypeptide may be produced as a result of mutations in ERG leading to altered expression levels, altered transcript stability or altered functional activity. Thus, ERG 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 ERG may be monitored using an in vivo teleost bioassay.
  • mutant forms of mammalian ERG, or similarly mutagenised forms of zfERG are injected into the embryo at the 1-cell, 2 to 4 cell, 4 to 8 cell 8-16 cell stage, or 16 to 32 cell stage where the readout for functionality is expansion of vascular progenitors, either by WISH for vascular-specific genes e.g. flkl), or observation of a vascular-specific transgenic embryo (e.g. flil :EGFP or flkl. 1 EGFP) at 12 or 24 hpf.
  • WISH for vascular-specific genes
  • a vascular-specific transgenic embryo e.g. flil :EGFP or flkl. 1 EGFP
  • ERG overexpression may be achieved at later timepoints by transgenesis in which ERG 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 later timepoints following endogenous or exogenous activation of the promoter driving ERG overexpression.
  • 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
  • an expected readout for normal overexpression of ERG may be expansion of flkl/flil +ve cells by WISH or observation of aforementioned transgenic lines, while embryos treated with a drug targeting ERG function may show a reduction in vascular cell expansion.
  • regulatory regions 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.
  • 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 ERG allele or where one or both alleles are modified can be used as model systems to study the effects of ERG in vascular endothelial cell development and function and/or to test for substances which have potential as therapeutic or teratogenic agents when these function are impaired.
  • 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 ERG alleles (including those carrying loxP flanking sequences), usually from a second animal of the same species, as well as insertion of disrupted homologous genes.
  • the endogenous ERG 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 ERG mutations, either somatic or germline.
  • the cell line can be engineered to carry the mutation in the ERG allele, as described above, or by gene modification using zinc finger nucleases (see Meng et al, Nat. Biotechnology, 26(6): 650- 701, 2008; Doyon et al, Nat. Biotech. 26: 702-708, 2008).
  • the phenotype of the cell is determined. Any trait of the cells can be assessed.
  • 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.
  • 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 ERG 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.
  • ERG ERG-UAS system
  • GAL4-UAS system described for example by Fischer et ah, 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.
  • 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.
  • 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.
  • 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.
  • ERG nucleic acid and amino acid sequences of ERG molecules are provided in the sequence listing, further described in Table 1.
  • Human, mouse and zebrafish ERG protein are more than 60% identical (see Figure 1 that shows the percent identity and divergence between the full length amino acid sequences of various vertebrate ETS family members.
  • Figure 1 also shows the high level of conservation between mouse, zebrafish and human ERG ETS binding domains. Accordingly, the terms ERG or ERG in the claims encompass all homologs and isoforms in any animal species including human homologs and isoforms and homologs of veterinary interest.
  • a homolog of ERG or ERG has at least 60% identity to the amino acid sequences of SEQ ID NO: 2, 4, or 6 or any one of SEQ ID NOs: 20, 22, 24, 26, 28, 30, 32, 34, 36 or 38, 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 or their encoding sequences.
  • 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.
  • Cre-LoxP system can be used to provide appropriate conditional ERG 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 U S A., 102(17): 6068-73, 2005) by providing targeted activation (or inactivation).
  • ES cells embryonic stem cells
  • modified cells are injected into the blastocyst or morula or other suitable developmental stage, to generate a chimeric organism.
  • 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.
  • the genetically modified cells or animals may comprise a gene capable of functioning as a marker for detection of modified cells.
  • 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.
  • RNA, cDNA, genomic DNA, synthetic forms and mixed polymers 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.
  • 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.
  • 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.
  • the present invention further contemplates recombinant nucleic acids including a recombinant construct comprising all or part of ERG.
  • the recombinant construct may be capable of replicating autonomously in a host cell. Alternatively, the recombinant construct may become integrated into the chromosomal DNA of the host cell.
  • Such a recombinant polynucleotide comprises a polynucleotide of genomic, cDNA, semisynthetic 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.
  • 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 ERG levels or to down- regulate ERG 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 ERG and, as pharmaceutical compositions for modulating the activity of ERG in a subject in vivo.
  • 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.
  • the present invention provides a genetically modified cell, or non-human animal comprising such cells, wherein a ERG gene is modified and the cell or animal produces a substantially enhanced level or activity of ERG polypeptide, or substantially reduced level or activity of ERG polypeptide compared to a non-modified animal of the same species, or is substantially incapable of producing ERG polypeptides.
  • 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.
  • the genetically modified non-human animal is a teleost.
  • the modified cell or non-human animal is genetically modified and produces a substantially reduced level of ERG or is substantially incapable of producing ERG or produces ERG having substantially reduced or no activity.
  • an ERG gene is modified. Modification may be in one or both alleles and may optionally be within a regulatory region of the gene.
  • the genetic modification resulting in a cell or animal capable of exhibiting a modified level or activity of ERG comprises genetic modification outside the ERG gene to cause expression of genetic or proteinaceous molecules which effectively modulate the activity of ERG or ERG.
  • the modified cell or non-human animal is genetically modified and substantially overproduces ERG having normal or altered activity relative to an unmodified cell or animal of the same species.
  • 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 ERG nucleic acid or ERG polypeptide and exhibiting a substantially modified level or activity of ERG polypeptide.
  • the cell or animal is contacted with the agent and its effect on the activity of ERG or its transcriptional targets determined.
  • the method comprises screening for mutants which exhibit a complementing phenotype and then mapping and identifying the modifying gene.
  • the method comprises screening for agents which enhance the level or activity of ERG in a normal or modified cell.
  • small-molecule libraries are screened for agents which directly or indirectly modulate ERG polypeptide activity.
  • 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 analogues generated by combinatorial chemistry, before proceeding to testing in other animal models.
  • the subject invention provides a use of a cell or non-human animal comprising a modified ERG or ERG and exhibiting a substantially enhanced level or activity of ERG in screening for or testing agents for use in the treatment or prophylaxis of a conditions, states and disorders as described herein.
  • a substantially modified level or activity of ERG 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 vasculogenesis (including angioblast activity) in a subject or cell activity.
  • a reduced level of gene expression of transcription targets or a reporter thereof is detected.
  • 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 ERG level or activity.
  • the reduction may of course be complete loss of ERG activity in a cell or animal.
  • a "modified" level or activity includes enhanced levels of ERG activity relative to pre- treatment levels and may equate to or exceed the level or activity of ERG 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.
  • the present invention further provides a method for identifying agents useful in the treatment or prophylaxis of unwanted conditions associated with too much or too little vasculogenesis such as described herein comprising screening compounds for their ability to modulate the functional activity of ERG polypeptides or modulate ERG activity in inducing vasculogenesis and/or angiogenesis.
  • the modulatory agents of the present invention may be chemical agents such small or large organic or inorganic chemical molecules, peptides, antibodies, polypeptides including dominant negative forms, modified peptides such as constrained peptides, foldamers, peptidomimetics, cyclic peptidomimetics, proteins, lipids, carbohydrates or nucleic acid molecules including antisense or other gene silencing 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 silencing agents such as DNA (gDNA, cDNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (siRNAs), short hai ⁇ in RNAs (shRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs)) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes may be employed.
  • DNA gDNA, cDNA
  • RNA sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (siRNAs), short hai ⁇ in RNAs (shRNAs), micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs)
  • ribozymes ribozymes
  • a variation on antisense and sense molecules involves the use of morpholinos, which 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.
  • therapeutic antibodies have the capacity for intracellular transmission and include antibodies such as shark antibodies, camelids and llama antibodies, scFv antibodies and intrabodies or nanobodies, e.g. scFv intrabodies and V HH intrabodies.
  • antigen binding agents can be made as described by Lui et ah, BMC Biotechnol, 7: 78, 2007; Harmsen & De Haard, Appl. Microbiol. Biotechnol. Nov, 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.
  • Agents in accordance with this aspect of the invention may directly interact with ERG.
  • small molecules, antibodies or peptides, peptidomimetics or analogs and other such molecules may be conveniently employed.
  • genetic mechanisms are used to indirectly modulate the activity of ERG.
  • various strategies are well documented and include mechanisms for pre or post- transcriptional silencing.
  • the expression of antisense molecules, dominant negative, or co- suppression or RNAi or siRNA strategies are particularly contemplated.
  • ERG nucleic acid encoding an ERG polypeptide may be obtained by a process which comprises the steps of screening an appropriate library or extract under stringent hybridisation conditions with a labelled probe having the sequence of a desired nucleic acid and isolating full-length cDNA and genomic clones containing said nucleic acid sequence.
  • stringent hybridisation conditions is where attempted hybridisation is carried out at a temperature of from about 35°C to about 65°C using a salt solution of about 0.9M.
  • 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°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.
  • Hybridisation conditions can also be rendered more stringent by the addition of increasing amounts of formamide, to destabilise the hybrid duplex.
  • particular hybridisation conditions can be readily manipulated, and will generally be chosen as appropriate.
  • 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°C for 90-95% identity and 32 0 C for 70-90% identity.
  • 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).
  • 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 niKNA 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 1 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.
  • ERG polypeptides or fusion proteins comprising ERG 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 and especially Chapters 11, 16 and 17).
  • 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. [00124] Alternatively, they may be identified 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 such agents. Natural products include those from coral, soil, plant, or the ocean or antarctic environments. Small molecule libraries are particularly convenient.
  • 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.
  • biological libraries are 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).
  • 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 Zebrafish Book: Guide For The Laboratory Use Of Zebrafish (3 rd Edition), 1995.
  • ISH in situ hybridisation
  • ISH in situ hybridisation
  • ISH takes advantage of the optical transparency of embryos during the first few days of development, which can be prolonged using l-phenyl-2-thiourea. Labelled antisense RNA probes are used to provide a semiquantitative measure of gene expression through out the entire embryo if required.
  • Vasculo genesis 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.
  • 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
  • Signals can be discriminated and/or analyzed by using pattern recognition software. Changes in the distribution of a protein both spatially and temporally, including a complete absence of a protein, 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., alkaline phosphatase staining (endothelial cells have high levels of endogenous alkaline phosphatase) 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 zebrafish.
  • Various robust methods are available for producing modified zebrafish.
  • the activity of an ERG polypeptide or of other factors in the angioblast vascular development pathway such as without limitation, ETSRP, FLI-I, SCL/TAL-1, FLK-I, ETS-I, ETS-2, GATA-I, TIE-2, FLK-I (VEGFR2), CDH5, and the like is reduced by generating an induced mutation in the endogenous gene or by inhibiting gene expression using nucleic acid interference (RNAi, antisense or genetic silencing).
  • RNAi nucleic acid interference
  • 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, 2008 (supra)).
  • Modified teleosts may be generated by targeting induced local lesions in genomes (TILLING) or from catalogues libraries of insertional mutants described in Wienholds et al., Genome Res 13(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 ERG polypeptides or a transcription target of ERG in a cell include genetic agents (i.e. comprising a nucleic acid molecule) which inhibit production of ERG in a cell at any stage including, for example, post-transcriptional silencing mediated by RNAi (see for example, United States Publication No. 20070042983, 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.
  • Antisense polynucleotide sequences are useful agents in preventing or reducing the expression of ERG.
  • morpholinos may be used as described by Summerton et ah, Antisense and Nucleic Acid Drug Development, 7: 187-195, 1997.
  • Antisense molecules may interfere with any function of a nucleic acid molecule.
  • the functions of DNA to be interfered with can include replication and transcription.
  • 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 ofthe £i?G gene.
  • the present invention provides a transgenic teleost comprising a nucleic acid sequence encoding a reporter protein whose expression is driven by the ERG promoter.
  • transgenic teleosts The production of transgenic teleosts is described in Lawson et al 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.
  • LacZ mRNA may be coadministered as a reporter/tracer for vascular endothelial cells.
  • the agent to be tested is contacted with a system comprising ERG or ERG. Then, the following may be assayed for: the presence of a complex between the agent and ERG or ERG; a change in the interaction between ERG 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).
  • all or part of the ERG gene promoter is operatively linked to a reporter construct and engineered into an expression construct as known to those of skill in the art.
  • a reporter construct 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.
  • an ERG responsive cell line is generated comprising an inducible ERG gene. Potential agents are tested for their ability to up- regulate or down-regulate expression differentiation markers when ERG is activated.
  • an ERG responsive cell line is the human cancer line K562 referred in Ceballos et al., 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).
  • antisense compound is a single-stranded antisense oligonucleotide
  • dsRNA double-stranded RNA
  • oligomeric compound refers to a polymer or oligomer comprising a plurality of monomeric units
  • oligonucleotide refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof.
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • 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.
  • backbone covalent internucleoside
  • 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
  • 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 or compositions in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides).
  • nucleobases i.e. from about 8 to about 80 linked nucleosides.
  • the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.
  • the ERG or ERG homolog polypeptide or agents or compositions of the present invention may be ERG or parts thereof, or ERG or parts thereof or complementary forms or molecules derived or designed from ERG or ERG including variants and fusion proteins comprising ERG or their encoding sequences.
  • Naturally occurring fusions of ERG such as those that occur in prostate and other cancers may also be conveniently tested in the present bioassays. See, for example, SEQ ID NO: 38 and encoding sequences.
  • the present invention provides a composition comprising ERG or a functional variant or ERG or an agent from which either or these is producible which substantially enhances the activity of ERG.
  • said compositions are for use in modulating vasculogenesis or vasculogenesis and angiogenesis.
  • any subject who could benefit from the present methods or compositions is encompassed.
  • subject includes, without limitation, humans and non-human primates, 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.
  • 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 vascular deficiency.
  • the present invention extends to cellular compositions including genetically modified vascular 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 are preferably multipotent or pluripotent.
  • Other cellular compositions comprise vectors such as viral vectors for delivery of nucleic acid constructs as described later herein.
  • the terms “functional form” or “variant”, “functionally equivalent derivative” or “homolog” include molecules which selectively hybridize to ERG 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 ERG polypeptides.
  • ERG nucleotide sequences include those comprising nucleotide sequences set forth in SEQ ID NO: 1 (mouse ERG mRNA), SEQ ID NO: 3 (human ERG-I mRNA (GenBank Accession No.: BC040168)) or SEQ ID NO: 5 (Zebrafish ERG mRNA) or their complements.
  • SEQ ID NO: 1 mimouse ERG mRNA
  • SEQ ID NO: 3 human ERG-I mRNA (GenBank Accession No.: BC040168)
  • SEQ ID NO: 5 Zebrafish ERG mRNA
  • ERG polypeptides include all biologically active naturally occurring forms of ERG as well as biologically active portions thereof and variants and derivatives of these. Fusion proteins comprising all or part of ERG polypeptides are particularly contemplated variants of ERG.
  • Biological activity as determined herein includes modifying vascular progenitor cell activity, the development of vascular tissue (pre-circulatory), modifying early angiogenesis 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 ERG or to one or more functional domains of ERG such that at least one functional activity of ERG 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 ETS domain) or protein binding (such as via the pointer domain).
  • Exemplary ERG amino acid sequences include those comprising sequences set forth in SEQ ID NO: 2 (mouse ERG), SEQ ID NO: 4 (human ERG-2) and SEQ ID NO: 6 (Zebrafish ERG).
  • Reference herein to a "low stringency" includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions.
  • low stringency is at from about 25-30°C to about 42°C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions.
  • Alternative stringency conditions may be applied where necessary, such as "medium stringency", which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
  • 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. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42 0 C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 20°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°C.
  • similarity includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non- identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide sequence comparisons are made at the level of identity and amino acid sequence comparisons are made at the level of similarity. The skilled person will understand that redundancy in the genetic code means that nucleic acid sequences with a high % identity can encode polypeptides of identical sequence.
  • the precent similarity between a particular amino 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 similarities between 60% and 100% are encompassed.
  • the precent 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.
  • a percentage of sequence identity between nucleotide sequences 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.
  • the identical nucleic acid base e.g. A, T, C, G, I
  • 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.
  • derivatives 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 ERG amino acid sequence. Preferably, the derivatives have functional activity or alternatively, modulate ERG functional activity.
  • modulate includes up modulate or up-regulate and down-modulate or down-regulate.
  • ERG As used herein reference to a part, portion or fragment of ERG 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.
  • 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, SEQ ID NO: 3, or SEQ ID NO; 5 or a complementary form thereof.
  • SEQ ID NO: 1 SEQ ID NO: 3
  • SEQ ID NO; 5 or a complementary form thereof.
  • 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 ERG 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. Alternatively, the substitution of like amino acids can be made effectively on the basis of hydrophilicity.
  • hydrophilicity in conferring interactive biological function of a protein is generally understood in the art (U.S. Patent No. 4,554,101).
  • hydrophobic index or hydrophilicity in designing polypeptides is further discussed in U.S. Patent No. 5,691,198.
  • homolog or “homologs” refers herein broadly to functionally and/or structurally related molecules including those from other species.
  • peptide mimetic 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 ah, "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 ERG. 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 O3'--P5' phosphodiester 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 analogues of ERG 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.
  • 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).
  • target molecules may be analyzed by an alanine scan (Wells, Methods Enzymol, 202: 2699-2705, 1991).
  • 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.
  • Analogs of ERG 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.
  • 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 .
  • 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);
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O- acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
  • Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.
  • Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
  • Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
  • Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4- amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids.
  • a list of unnatural amino acid, contemplated herein is shown in Table 4.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids and the introduction of double bonds between C ⁇ and C ⁇ atoms of amino acids.
  • compositions which are prepared according to conventional pharmaceutical 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.
  • compositions comprising an active agent which modulates the activity of ERG or its transcriptional targets for use or when used in modulating vascular endothelial cell activity as defined herein.
  • an active agent which modulates the activity of ERG or its transcriptional targets for use or when used in modulating vascular endothelial cell activity as defined herein.
  • the use of the herein described agent is expressly contemplated in the manufacture of a medicament for the treatment of conditions associated with vascular defects associated with ERG variants.
  • the subject is tested for ERG variants prior to administration.
  • the compounds can be formulated into solid or liquid preparations such as capsules, pills, tablets, lozenges, powders, suspensions or emulsions.
  • 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).
  • 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.
  • the compound may be dissolved in a pharmaceutical carrier and administered as either a solution or a suspension.
  • 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.
  • the compounds 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.
  • 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.
  • these agents 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.
  • 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.
  • the cells of a subject may be tested to determine whether gene or cell therapy with an agent comprising ERG is indicated.
  • the provision of wild type or enhanced ERG function to a cell which carries a mutant or altered form of ERG should in this situation complement the deficiency and result in an improvement in the subject.
  • cells capable of providing normal or enhanced ERG activity (function) may be provided.
  • the ERG allele may be introduced into a cell in a vector such that the gene remains extrachromosomally.
  • artificial chromosomes may be used.
  • 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.
  • 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.
  • plasmid DNA of any size is combined with a polylysine-co ⁇ jugated 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.
  • 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.
  • the construct contains viral sequences sufficient to support packaging of the construct. If the polynucleotide encodes ERG, expression will produce ERG. 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.
  • 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 poly lysine.
  • 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.
  • co-infection with adenovirus can be included to disrupt endosome function.
  • patients who carry an aberrant ERG 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 ERG allele. Preferably only specific cells are targeted.
  • peptides or mimetics or other functional analogues which have ERG activity can be supplied to cells which carry aberrant ERG alleles.
  • Protein can be produced by expression of the cDNA sequence in bacteria, for example, using known expression vectors.
  • 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.
  • supply of molecules with ERG activity should lead to enhanced angioblast proliferation and/or specification, vasculogenesis, angiogenesis and therefore enhanced blood supply to one or more organs or tissues.
  • 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.
  • DGGE denaturing gradient gel electrophoresis
  • SSCP and rSSCP single- stranded conformational polymorphism
  • REF-SSCP REF-SSCP
  • chemical cleavage methods such as CCM, ECM, DHPLC and MALDI-TOF MS and DNA chip technology.
  • the present invention provides methods of diagnosis of conditions associated with modified ERG 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 ERG gene or locus to determine if ERG will be produced or if it will be over-produced or under-produced.
  • the term "aberration" in the ERG gene or locus encompasses all forms of mutations including deletions, insertions, point mutations and substitutions in the coding and non-coding regions of ERG. It also includes changes in methylation patterns of ERG or of an allele of ERG. 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.
  • the diagnostic and prognostic methods of the present invention also include detecting or assessing an aberration in the ERG gene wherein the gene is spliced to form a fusion gene transcript, for example, a TMP 1 RSSUERG gene fusion; see Wang et al, Cancer Res.
  • Useful diagnostic techniques to detect aberrations in the ERG gene include but are not limited to fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot analysis, single-stranded coformational 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 vascular defects can be ascertained by testing any tissue of a human or other mammal for mutations in a ERG 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 ERG 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.
  • SSCP single-stranded conformation polymorphism assay
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • ERG allele tests for confirming the presence or absence of a wild type or mutant ERG 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.
  • amplicon melting analysis Wittwer et al, Clinical Chemistry, 49: 853-860, 2008
  • SSCA single-stranded conformation analysis
  • DGGE denaturing gradient gel electrophoresis
  • Amplification Refractory Mutation System 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.
  • 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.
  • reporter molecule e.g. fluorescent label
  • the technique is described in a range of publications including Hacia et ah, Nature Genetics, 14: 441-447, 1996, Shoemaker et at., Nature Genetics, 14: 450-456, 1996.
  • Alteration of wild type ERG genes can also be detected by screening for alteration of wild type ERG proteins.
  • monoclonal antibodies immunoreactive with ERG can be used to screen a tissue. Lack of cognate antigen would indicate an ERG mutation.
  • Antibodies specific for products of mutant alleles could also be used to detect mutant ERG gene product.
  • 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 ERG) 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).
  • primers used to amplify regions of ERG genetic sequence are routinely derived by the skilled addressee based on known sequences for ERG.
  • EXAMPLE 1 erg is highly conserved between vertebrate lineages
  • the BLAST tool at the Ensembl database was used to search the current zebrafish genome assembly (Z v7).
  • the erg locus is located on chromosome 10 and encodes a predicted 2.8 kb mRNA translating into a 427 amino acid protein with two major functional domains: an N-terminal "pointed domain" implicated in protein-protein interactions; and an C-terminal ETS DNA-binding domain common to all ETS family members. Only one copy of the erg locus was identified in the zebrafish genome; unlike the flil locus (Zhu et al, Dev Biol, 281: 256-269, 2005), the erg gene has not undergone duplication.
  • EXAMPLE 2 Zebrafish as a model amenable to descriptive and functional investigation in vivo
  • vasculogenesis begins at the 6-somite stage with the specification of angioblasts from a putative haemangioblast subset in the lateral plate mesoderm (Liao et al, 1998 (supra)). Between the 10- and 15-somite stages, angioblasts migrate to the midline of the embryo, where they coalesce to form the dorsal aorta and cardinal vein, the first functional embryonic blood vessels. At the 26- somite stage, angiogenesis begins, with secondary vessels sprouting from these primary vessels along the trunk of the embryo (reviewed in (Childs et al, Development, 129: 973- 982, 2002)).
  • ETS factor spil/pu.l drives myeloid specification in the anterior mesoderm (Rhodes et al, 2005 (supra)).
  • Ets-related protein etsrp
  • haemangioblasts towards vascular fate (Sumanas et al., 2006 (supra)).
  • human ets related gene (human ER G) is an ETS family member 96% homologous to FLIl (Maroulakou et al, Oncogene 19: 6432-42, 2000) first identified in 1987 (Reddy et al, Proc Natl Acad Sci U S A, ⁇ W: 6131-6135, 1987).
  • Erg is expressed in mesodermal tissues including precartilaginous, urogenital and endothelial cells, positioning it for functions in cell migration and differentiation, as well as for establishment of endothelial fate in mesenchymal cells (Vlaeminck-Guillem et al , 2000 (supra)). Transient expression of Erg has also been described during murine T-cell lineage specification (Anderson et al, (supra)).
  • HSC hematopoietic stem cell
  • ERG Error-like oligonucleotides/siRNA
  • HAVECs human umbilical vein endothelial cells
  • GeneBloc antisense oligonucleotides/siRNA McLaughlin et al, 2001 , (supra); Huang et al, Blood, 106: 1636-1643, 2005; Birdsey et al, Blood, 111(1): 3498-506, 2008
  • overexpression studies inXenopus Baltzinger et al, Dev Dyn, 216: 420-433, 1999
  • McLaughlin et al., 2001 ⁇ supra described decreased expression of known regulators of angiogenesis, factors important in cellular remodelling and angiogenesis, von Willebrand factor (an adhesive glycoprotein for coagulation of platelets and a proposed factor involved in tumour angiogenesis) (Girma et al, Blood, 70: 605-611, 1987; Zanetta e? al, IntJ Cancer, 85: 281-288, 2000) and RhoA, a small GTP ase involved in cellular adhesion pathways (Nobes et al, J Cell Sci, 108(Pt 1): 225-233, 1995). Furthermore, GenBloc treatment of HUVECs impaired the formation of tubular structures in vitro.
  • ERG mediates angiogenesis in part by transcriptional regulation of VE-cadherin, overexpression of which rescues apoptosis in ERG-deficient HUVECs (Birdsey et al, 2008 (supra)).
  • VE-cadherin overexpression of VE-cadherin
  • XIerg overexpression of XIerg by injection of mRNA led to ectopic endothelial cell accumulation and perturbations of the cardiovascular system (Baltzinger et al 1999 (supra)).
  • Erg was first described in zebrafish as a vascularly-expressed gene by (Weber et al, Blood, 106: 521-530, 2005) based on differential expression in a morpholino microarray analysis, but this report did not present temporo spatial expression patterns or any functional studies. Described herein is the spatiotemporal expression pattern of erg during angioblast specification, vasculogenesis and early angiogenesis.
  • the earliest known factor controlling vascular development is thought to be the acyltransferase lycat, which is located within a deletion in an allele of the cloche mutant, which lacks most hematopoietic and vascular mesodermal derivatives (Stainier et al, 1995 (supra); Xiong et al, Circ Res, 102: 1057-1064, 2008). Downstream of cloche, tall is also required for development of hematopoietic and myeloid lineages, as well as development and formation of the dorsal aorta (Stainier et al, 1995 (supra); Patterson et al, Blood, 105: 3502-3511, 2005).
  • the ETS factor etsrp has been shown to be a key regulator of vasculogenesis downstream of tall and cloche signalling.
  • the expression of erg at the 10- somite (14 hpf) stage was investigated in the lateral plate mesoderm in the absence of each of these factors by WISH, erg expression was absent in cloche ( Figure 21), f ⁇ /i-morphant (Figure 4B) and eterp-morphant embryos (Figure 4C), suggesting that expression of erg at this time-point is dependant on the presence of these factors in the mesoderm.
  • absence of erg expression in cloche and etsrp morphant embryos may reflect the absence of flkl -expressing specified angioblasts in which erg is normally expressed, rather than indicate direct transcriptional regulation of the erg locus.
  • the cardiovascular system is the first fully functional system to develop during zebrafish embryogenesis and is integral for the correct development of many other organs and structures. It is therefore not surprising that such a crucial step in development is regulated by complex transcriptional networks with many integrated safeguards and redundancies.
  • Past functional studies in zebrafish of blood-vessel development have strongly suggested combinatorial or redundant roles for members of the ETS family of transcription factors (Pham et al. , 2007 (supra)). Indeed, of the ETS factors investigated, etsrp alone is essential for the specification of angioblasts during early somitogenesis (Sumanas et al., 2006 (supra)). As shown herein erg is also not alone essential for vasculogenesis.
  • ETS family members are expressed in the lateral plate mesoderm during somitogenesis in the zebrafish, where they have important functions during specification. To elucidate potential functions for ERG in angioblast specification and proliferation, and to further dissect its position in transcriptional and biological pathways, erg was overexpressed from injected mRNA on mutant and morphant backgrounds and the effect on various mesodermal progenitors at 14 hpf was assessed.
  • FIG. 8 provides a schematic representation of an agent suitable for testing in the subject model of erg functional activity.
  • Full-length Erg encodes a 427 amino acid protein containing two activation domains (AD), one helix-loop-helix (HLH) putative protein-protein interaction domain and an ETS DNA-binding domain.
  • AD activation domains
  • HSH helix-loop-helix
  • ETS Erg describes a protein in which the ETS DNA-binding domain has been removed, therefore representing a potentially inactive allele of Erg.
  • FIG. 9 A schematic and photographic representation of the biassay protocol is provided in Figure 9.
  • Capped mRNA encoding an Erg allele to be tested is prepared in vitro and observed via gel electrophoresis pre- and post injection (see Figure 9 (i). As a control for RNA degradation; faint bands are observed in the bottom panel indicating that degradation has not taken place).
  • mRNA is microinjected into embryos at the 1-cell stage (see Figure 9 (ii). The readout consists of scoring the phenotypic class at 24 hours post fertilization (see Figure 9 (iii).
  • over-expression of an active allele of Erg is expected to result in increasingly dismorphic embryos (classes I-IV) with an expanded population of GFP-positive cells in the Tg(/?z7 ⁇ :EGFP) strain in a dose-dependent manner.
  • ETS factors act in complex with etsrp to drive various stages of haemangioblast specification, vasculogenesis and angiogenesis.
  • EXAMPLE 5 erg acts synergistically with etsrp to regulate vascular development
  • MOs antisense morpholino antisense oligonucleotides
  • Antisense MOs bind to sense mRNA targets and inhibit translation creating a transient phenocopy of a gene knockout.
  • an in vivo reporter assay was employed.
  • the erg 1.2 kb coding sequence was amplified from an adult kidney cDNA library using the Kozak optimised primers: (A) 5'- GCGGAATTCCATGACGGCGTCTGCAGCCGC-3' (SEQ ID NO: 7) and (B) 5'- GCGCCTCGAGGTTCTTCTAGTAGTATGAGC-3' (SEQ ID NO: 8).
  • the 528 bp construct used for in situ hybridisation was amplified using (A) and (C) 5'- GCGCCTCGAGGTTGGGAAAGATGAAGTTGGC-3' (SEQ ID NO: 9).
  • Antisense and sense riboprobes for erg were generated from a 528 bp fragment targeting the 5' end of erg mRNA, which lacks the highly conserved ETS domain.
  • the template was linearised with EcoKL and Stul for transcription with T3 and SP6 polymerases respectively.
  • In situ hybridisation experiments were performed as described previously (Lieschke et al, Dev Biol, 246: 274-295, 2002; Oates et al, Blood, 94: 2622-2636, 1999). Embryos presented in figures are representative of > 10 examples unless otherwise stated. Erg overexpression constructs
  • erg morpholino antisense oligonucleotide ergMO (5' CAGACGCCGTCATCTGCACGCTCAG -3' (SEQ ID NO: 12)) was designed to target the ATG start-codon, all other morpholinos described are previously published: Control (Hogan et al, Dev Biol, 276: 508-522, 2004; Rhodes et al, 2005 ⁇ supra); etsrp (Sumanas et al., 2006 (supra)), and tall (Dooley et al, 2005 (supra)).
  • the ergMO validation construct was generated by cloning oligonucleotides (5' TTGCAGGATCCTGAGCGTGCAGATGACGGCGTCTGCAATCGATTCG -3' (SEQ ID NO: 13)) and (5'-
  • etsrp morpholino was injected at 2 ng/ ⁇ l in epistasis experiments and at 0.125 ng/ ⁇ l as a sensitising dose in synergy experiments.
  • the tall morpholino was injected at 4.19 ng/ ⁇ l
  • erg morpholino was injected at 2.84 ng/ ⁇ l
  • control morpholino at 2.085 ng/ ⁇ l.
  • Overexpression of erg traced by LacZ expression was performed using injectates containing 50 ng/ ⁇ l of each mRNA. Average injection bolus for 1-2 cell stage injections was 1.8 nl, while 8-16 cell injections were 0.26 nl.
  • Wild type and variant ERG cDNA are cloned using standard procedure (Ausubel). DNA binding is determined using electrophoretic mobility shift assays. The effect of any mutation on function is determined using reporter gene assays and biological assays such as the presently described zebrafish overexpression bioassay.
  • the bioassay assesses the functional activity of an agent selected from the group consisting of ERG or a variant thereof or of an agonist or antagonist of ERG activity or of the activity of an ERG transcriptional target in vascular endothelial tissue in vivo.
  • the method comprises: (i) contacting the agent and a model system comprising a teleost (zebrafish) and (ii) determining the effect of the agent on vasculogenesis in the teleost relative to controls.
  • the teleost is contacted with an agent to overexpress ERG to induce vascular precursor activity (migration, proliferation, differentiation or development) and/or vasculogenesis and further contacted with an Erg antagonist or a putative Erg antagonist.
  • the teleost may be a wild type zebrafish or a modified form, modified perhaps to sensitise the model system for testing one or more agents.
  • a modified teleost overexpresses or underexpresses one or more molecules expressed during vasculogenesis such as ETSRP, ETSRP, FLI-I, SCL/TAL-1, FLK-I, ETS-I, ETS-2, GATA-I, FLK-I (VEGFR2) and CDH5, and the like.
  • the teoleost is screened for expression of one or more markers of vasculogenesis such as expression of FLK-I, FLI-I, SCL, and/or ERG or of detectable reporters of such expression.
  • zebrafish embryos are contacted at the approximately 8 to 16 cell stage.
  • the target genes of ERG that play a role in vasculogenesis and angiogenesis are identified using techniques such as chromatin immunoprecipitation and microarrays.
  • the identification of nucleic acid sequences bound by ERG is conveniently assessed using genome-wide location analyses such as ChIP-on Gene analysis such as described by Horak et al, Methods EnzymoL, 350: 469-483, 2002.
  • Erg regulates the expression of signaling molecules or their receptors such as a growth factor, cytokine, hormone or chemokine, or their receptor.
  • the signalling molecules or their cellular or nuclear receptors are expressed early in haemangioblast or angioblast specification.
  • the sequence of the bound nucleic acids can be determined and how often they are represented in the cell lysate. This indicates the gene promoters to which ERG binds in a given cell, and therefore, its transcriptional targets.
  • ENU mutagenesis modifier screen are performed i.e. a screen for mutations that can enhance or suppress the phenotypes caused by Erg- mutation in Erg.
  • This will identify two classes of genes: 1) those that act within the ERG pathway, and 2) genes that act outside the ERG pathway, but which when mutated, produce biologic effects that can enhance or suppress that induced by mutations in ERG.
  • the present invention therefore also contemplated modified animals comprising two or more different mutations the ERG signalling pathway or its transcriptional targets.
  • class 2) genes which when mutated suppresses the ERG phenotype are further targets for the development of agents useful in the present invention.
  • Non-conventional Code Non-conventional Code amino acid amino acid ⁇ -aminobutyric acid Abu L-N-methylalanine Nmala ⁇ -amino- ⁇ -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-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-Nmethylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

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Abstract

L’invention concerne le facteur de transcription ERG et identifie un rôle de l'ERG dans le développement et les tissus vasculaires des vertébrés. L'invention concerne des dosages biologiques de l'activité fonctionnelle de l'ERG basés sur la production de l'ERG dans des téléostes modifiés. L'activité de variants de l'ERG peut être testée par des dosages chez le sujet. D'autres dosages biologiques fournissent des procédés pour identifier ou doser la capacité des agents à moduler l'activité des précurseurs vasculaires des vertébrés ou la vasculogenèse. L'invention concerne de plus des procédés de modulation du développement vasculaire comprenant la mise en contact d'un sujet, d'une cellule ou de tissus avec un agoniste ou un antagoniste de l'ERG ou d’un homologue de l’ERG. L'invention concerne également des téléostes modifiés, des marqueurs de la vasculogenèse et des cellules modifiées.
PCT/AU2009/001057 2008-08-19 2009-08-19 Modulation du développement vasculaire par le facteur de transcription erg WO2010019995A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019171191A1 (fr) * 2018-03-05 2019-09-12 Dr. Reddy's Institute Of Life Sciences Modèles de poisson zèbre embryonnaires utilisant un knock-down médié par dnazymes

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BIRDSEY, G. M. ET AL.: "Transcription factor Erg regulates angiogenesis and endothelial apoptosis through VE-cadherin", HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY, vol. 111, no. 7, 1 April 2008 (2008-04-01), pages 3498 - 3506 *
ELLETT, F. ET AL.: "The role of the ETS factor erg in zebrafish vasculogenesis", MECHANISMS OF DEVELOPMENT, vol. 126, 2009, pages 220 - 229 *
LIU, F. ET AL.: "Genome-wide analysis of the zebrafish ETS family identifies three genes required for hemangioblast differentiation or angiogenesis", CIRCULATION RESEARCH, vol. 103, 2 October 2008 (2008-10-02), pages 1147 - 1154 *
REMY, P. ET AL.: "The Ets-transcription factor family in embryonic development: lessons from the amphibian and bird", ONCOGENE, vol. 19, 2000, pages 6417 - 6431 *
SATO, Y.: "Role of ETS family transcription factors in vascular development and angiogenesis", CELL STRUCTURE AND FUNCTION, vol. 26, 2001, pages 19 - 24 *
SEIFERT, T. ET AL.: "Vasculogeneic maturation of E14 embryonic stem cells with evidence of early vascular endothelial growth factor independency", DIFFERENTIATION, vol. 76, 2008, pages 857 - 867 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019171191A1 (fr) * 2018-03-05 2019-09-12 Dr. Reddy's Institute Of Life Sciences Modèles de poisson zèbre embryonnaires utilisant un knock-down médié par dnazymes

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