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WO2008025069A1 - Procédés de modulation de l'activité cellulaire et compositions à cet effet - Google Patents

Procédés de modulation de l'activité cellulaire et compositions à cet effet Download PDF

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WO2008025069A1
WO2008025069A1 PCT/AU2007/001243 AU2007001243W WO2008025069A1 WO 2008025069 A1 WO2008025069 A1 WO 2008025069A1 AU 2007001243 W AU2007001243 W AU 2007001243W WO 2008025069 A1 WO2008025069 A1 WO 2008025069A1
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cell
suzl2
agent
activity
cells
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Douglas James Hilton
Ian Majewski
Warren Scott Alexander
Marnie Elisabeth Blewitt
Carolyn Anne De Graaf
Bahlo MELANIE
McManus EDWARD
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Walter and Eliza Hall Institute of Medical Research
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Walter and Eliza Hall Institute of Medical Research
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Definitions

  • the present specification relates to the regulation of endogenous gene expression and the identification of epigenetic modulators thereof.
  • the present invention provides methods for enhancing the number and/or activity of hematopoietic cells and/or their progenitor cells such as hematopoietic stem cells.
  • the invention pertains to the treatment or prevention of conditions associated with insufficient or insufficiently active hematopoietic cells, hematopoietic stem cells, or myeloid- or lymphoid-restricted progenitor cells.
  • HSC Hematopoietic stem cells
  • multipotency which is the developmental capacity to form all types of blood cell
  • self renewal which is the capacity to generate daughter HSCs, and relative quiescence.
  • HSCs have three alternative fates: self-renewal, differentiation or programmed cell death.
  • both daughter cells adopt the same fate, while in an "asymmetric” cell division they adopt different fates.
  • HSCs undergo “commitment,” which is a stable change in internal state.
  • “Lineage commitment” results in an internal change that restricts the number of lineages into which a cell's progeny can differentiate.
  • the process of "differentiation” involves cells becoming structurally and functionally distinct.
  • HSC are important cellular precursors and they are recognised by the medical and allied health fields as a high priority area for research and development of new treatment and prophylactic strategies.
  • Mature blood cells have a diverse morphology and function. They are generally unable to proliferate and their numbers are replenished from a small population of HSC. Hematopoiesis proceeds through a series of lineage commitment steps, in which HSC progeny become progressively more restricted in their differentiation and proliferative potential. This model of hematopoiesis arose in the late 1960s, when colony forming unit (CFU) assays led to the identification of progenitor cells with differing developmental, proliferative and self-renewal potential (Bradley et al, Nature, 2/4(87):511, 1967).
  • CFU colony forming unit
  • FACS fluorescence activated cell sorters
  • Positive regulators of HSC include IL-6, IL-I l, Flt3L, stem cell factor (SCF) and thrombopoietin (Tpo). These cytokines act synergistically in vitro to promote proliferation of HSC. However, the role of cytokines in directing lineage commitment, if any, has not been clarified. Members of the Homeobox (Hox) gene family have been implicated in the control of HSC expansion in vitro (Amsellem et al, Nat, Med., 9(11):1423-1427, 2003). The polycomb group gene Bmi-1 has been shown to be essential for sustained hematopoiesis. However, the role of these genes, if any, in lineage commitment is uncertain.
  • Hox Homeobox
  • PU.1 is an Ets family member that plays a critical role in both myeloid and lymphoid differentiation (McKercher et al, EMBO. J., /5(20):5647-5658, 1996; Scott et al, Science, 2 ⁇ J5(5178):1573-1577, 1994).
  • GATA-I is required for erythroid and megakaryocyte development (Pevny et al, Nature, J4P(6306):257-260, 1991).
  • GATA-I GATA-I regulates lineage choice by common myeloid progenitors (CMP) (Zhu et al, Oncogene, 2/(21):3295-3313, 2002).
  • CMP common myeloid progenitors
  • PU.1 prevents megakaryocyte and erythroid differentiation by antagonising GATA-I and instead facilitates differentiation along myeloid pathways.
  • Platelets are small, anuclear fragments of megakaryocytes that circulate in the blood and make essential contributions to functions such as blood clotting and wound healing. Like all lineages of blood cells, regulatory mechanisms in the body ensure that precise numbers of platelets are generated at steady-state to replace those that are functionally expended or removed from the circulation, as well as allowing rapid responses to emergency requirements such as haemorrhage. Platelets are shed by megakaryocytes: large, polyploid cells in hematopoietic tissues produced by specific progenitor cells. In normal individuals, precise control of proliferation, differentiation, survival and clearance of these cells ensures maintenance of homeostasis, and reduces the likelihood of haemorrhage should platelet counts fall or thrombosis resulting from excess platelet production.
  • Thrombopoietin plays a key role in platelet homeostasis, by regulating the level of platelet production to maintain optimal circulating levels. If this delicate balance is perturbed, thrombocytopenia, or low platelet count, can ensue.
  • Thrombocytopenia is a common problem in the clinic, particularly in hematological and oncological practice. It can occur congenitally, with a number of inherited disorders having been defined (Drachman, Blood, 703:390-398, 2004), but the majority of thrombocytopenias seen in the clinic are the result of other causes. It can be a major problem for patients undergoing cancer chemotherapy.
  • thrombocytopenia is also frequently encountered in myelodysplastic syndromes (MDS), idiopathic thrombocytopenia purpura (ITP) and chronic liver disease, and is associated with viral infections, particularly AIDS (Kuter et al, Blood, 700:3457-3469, 2002). In these more chronic contexts, thrombocytopenia may result from defective platelet production or elevated platelet destruction, often as the result of autoimmune reactions.
  • Deficiencies in platelet levels or function can lead to haemorrhagic episodes and this condition can be the result of congenital or acquired syndromes such as von Willebrand disease, Bernard-Soulier syndrome, Glanzmann's thrombasthenia, asprin-like defects, myeloproliferative disorders, liver disease and uremia.
  • Treatment for low platelet numbers includes platelet transfusion and, potentially, administration of thrombopoietin (TPO).
  • TPO thrombopoietin
  • Platelet-mediated thrombosis is a major mechanism leading to vascular diseases such as cardiovascular disease, cerebrovascular disease and peripheral vascular disease. Control of platelet levels or activity is an essential component of anti-thrombosis treatments. Pro- thrombotic states are seen in subjects with conditions such as myeloproliferative disorders, chronic pulmonary obstructive disease and essential thrombocytosis.
  • polycomb protein includes a single polycomb protein, as well as two or more polycomb proteins; and so forth.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>l (SEQ ID NO:1), ⁇ 400>2 (SEQ ID NO:2), etc.
  • SEQ ID NO:1 sequence identifiers ⁇ 400>l
  • SEQ ID NO:2 sequence identifiers
  • Suzl2 is an expression product of Suzl2.
  • the term “Suzl2” or “Suzl2” or “Ezh2” or “Ezh2” or “Eed” or “Eed” is used to encompass all functionally analogous homologs, including orthologs and paralogs, isoforms and variants in any species.
  • a genetic screen for mutations that suppress thrombocytopenia in mice has led to the identification of a polycomb protein Suzl2 or the Suzl2 gene as a target for pharmacological or physiological agents useful in modulating the number and/or activity of hematopoietic cells and/or their progenitor cells.
  • Suzl2 is functionally compromised, the number and/or activity of hematopoietic cells or their progenitor cells is enhanced.
  • Plt8 the isolation and characterisation of a mutation in Suzl2, termed Plt8, identified in a mutagenesis screen performed on sensitized mice that lack the thrombopoietin receptor (c-mpf ' ).
  • the Suppressor of Zeste 12 (Su(Z)] 2) locus was first identified in a genetic screen performed in Drosophila melanogaster to discover factors that repress gene transcription. Flies that lack functional Su(Z) 12 show loss of segment identity, consistent with miss- expression of homeobox (Hox) genes during development (Birve et al, Development, /25:3371-3379, 2001).
  • Hox homeobox
  • the spatial and temporal control of Hox gene expression is mediated by Polycomb group (PcG) proteins which act as negative regulators and Trithorax group proteins that promote transcription (Lund et al, Curr. Opin. Cell. Biol., 75:239-246, 2004).
  • PRC2 The components of PRC2 are broadly conserved between Drosophila and vertebrates, and include Suzl2, the methyl-transferase Enhancer of Zeste 2 (Ezh2) and various forms of the embryonic ectoderm development protein (Eed) (Cao et al, Curr. Opin. Genet. Dev., 74:155-164, 2004).
  • Reference herein to a polycomb repressive complex polypeptide includes reference to a polycomb protein (PcG).
  • the human homolog of Su(Z) 12 was identified by two independent groups; first, as a gene disrupted in a translocation common in endometrial stromal tumours (Koontz et al, Proc. Natl. Acad. Sci. U.S.A., 95:6348-6353, 2001), and second, as a target gene of the E2F family of transcription factors (Weinmann et al, Methods, 26:31 '-47, 2002). Both studies implicate deregulation of PRC2 function in tumorigenesis. Over-expression of both Suzl2 and Ezh2 has been reported in breast, colon and bladder malignancy (Collett et al, Clin. Cancer Res.
  • Murine models of PRC2-def ⁇ ciency have demonstrated an absolute requirement for all three components (Suzl2, Ezh2 and Eed) for proper development during embryogenesis (O'Carroll et al, MoI. Cell. Biol., 27:4330-4336, 2001; Pasini et al, Embo. J., 23:4061- 4071, 2004; Shumacher et al, Nature, 353:250-253, 1996). Further investigation of PRC2 function in the adult mouse has therefore been restricted to the use of conditional targeted alleles, which have been generated for Ezh2 (Su et al, Nat. Immunol., 4: 124-131, 2003), and viable hypomorphic alleles that include Eed 1989 (Shumacher et al, 1996 ⁇ supra)).
  • Histone proteins carry extensive post- translational modifications that influence the packaging of DNA and the accessibility of DNA sequence to transcription factors (Jenuwein et al, Science, 2001 (supra)). Acetylation of histone proteins is generally associated with regions that are being actively transcribed. Acetylation acts to neutralize the positive charge of lysine residues, thereby reducing the affinity between histone proteins and the phosphate residues in the DNA backbone that carry a negative charge.
  • HDACs Histone Acetyl- Transferases
  • HDACs Histone De-Acetylases
  • HDACi HDAC-inhibitors
  • HDACi Inhibition of HDACs has been shown to have clinical relevance in the treatment of various forms of leukaemia and also in solid tumours.
  • the administration of HDACi is thought to affect a small subset of genes, rather than causing wholesale changes to gene expression, which may be due to the inability of HDACi to counteract higher-order silencing regulated by additional processes such as DNA-methylation (Peart et al., Proc. Natl. Acad. Sci. U.S.A., 702:3697-3702, 2005).
  • Suzl2 is a polycomb protein (PcG) that forms an important part of polycomb repressive complexes (PRCs) that repress transcription by modifying chromatin.
  • PcG polycomb protein
  • PRCs polycomb repressive complexes
  • the present invention relates to the discovery that agents that de-repress or enhance gene expression and particularly transcription of genes in HSC and/or their descendants are useful for enhancing the number of HSC and hematopoietic lineage cells derived therefrom.
  • the number of myeloid and/or myeloid progenitor cells is enhanced.
  • the number of lymphoid and/or lymphoid progenitor cells is enhanced.
  • the number of B-cell progenitor cells and/or B-cells is enhanced.
  • mice As shown in the present Tables and Examples, levels of myeloid and lymphoid cells and their precursors are elevated in Suzl2 deficient PU8/+ mice. This observation was made primarily in mice that were c-mpV 1' which exhibit thrombocytopenia and which therefore provide a sensitised system for detecting agents which modulate platelet number.
  • the number of myeloid and/or myeloid progenitor cells is enhanced.
  • the number of lymphoid and/or lymphoid progenitor cells is enhanced.
  • the number of B-cell progenitor cells and/or B-cells is enhanced.
  • the specification describes a method of identifying agents that modulate the level or activity of Suzl2 or a complex comprising Suzl2 in vivo wherein the method comprises administering the agent to a genetically modified animal model of thrombocytopenia and monitoring the number and/or activity of platelets in the animal wherein a change in the number and/or activity of platelets in the presence of the agent indicates that the agent is effective in vivo.
  • the animal is c-mp ⁇ ' .
  • a method of testing or monitoring the effect of an epigenetic modifier agent in a subject comprising administering the agent and monitoring the number and/or activity of platelets in the subject wherein a change in the number and/or activity of platelets as a result of said administration is a measure of the effect of the agent on the subject.
  • the subject is a human or mammalian subject.
  • the epigenetic modifier modulates the level or activity of a polycomb polypeptide or gene.
  • the agent is an inhibitor of the level or activity of Suzl2 or Suzl2.
  • the agent is a demethylating agent, a histone de-acetylase inhibitor or a histone acetyl-transferase mimic.
  • the Suzl2 mutation or modification provides de-repressed transcription through reducing chromatin methylation.
  • Demethylating agents are also expected to facilitate de-repression of transcription.
  • acetylation of chromatin for example by histone acetyl transferases mimics, or by inhibition of histone de-acetylases by histone de-acetylase inhibitors is expected to de-repress transcription.
  • down regulation of the level or activity of one of more PRCs or other members of the network of interacting molecules to which Suzl2 belongs is useful in modulating, and particularly enhancing, the number or activity of hematopoietic cells and/or their progenitor cells.
  • the agents described herein modulate the level or activity of PRC2 target genes or their expression products.
  • the present invention provides, therefore, in some embodiments, methods of modulating the number and/or activity of hematopoietic cells and/or their progenitor cells.
  • the methods comprise down regulating the level or activity of one or more PRC polypeptides, PcG or other members of the network of interacting molecules to which Suzl2 belongs.
  • the level or activity of a target of a PRC is modulated.
  • the PRC complex is PRCl.
  • the PRC complex is PRC2/3.
  • the number of myeloid progenitor cells is enhanced.
  • the number of lymphoid progenitor cells is enhanced.
  • the number of leukocytes is enhanced.
  • the agent down regulates the level or activity of Suzl2 polypeptide or the Suzl2 gene.
  • the Plt8 mutation is a dominant mutation that shows its phenotypic effect in heterozygous form. Accordingly, Suzl2 or Suzl2 is a particularly attractive target for the development of pharmaceutical compositions that inhibit Suzl2 function or activity. Specifically, such agents are useful in the manufacture of medicaments that effectively down modulate gene expression within hematopoietic progenitor cells.
  • PcGs effect transcriptional repression by modifying histone proteins.
  • reduced Suzl2 levels or activity leads to de-repression of transcription in hematopoietic cells or their precursors and enhanced activity and/or proliferation of these cells. This is unexpected in view of the teachings of the prior art which indicate that down regulation of Suzl2 is useful in the treatment of cancer by inhibition of proliferation.
  • Reference to "enhancing the activity of hematopoietic cells and/or their precursors" encompasses enhancing lineage commitment and/or differentiation into a particular blood cell type.
  • Reference to the "activity of PRC or PcG proteins” means the functional activity of one or more PRC proteins or PcG in modulating gene expression via a cascade of reactions encompassing one or more modes of action such as, without limitation: methylation, demethylation, acetylation, deacetylation, ubiquitylation, phosphorylation, dephosphorylation, methyltransferase activity, protein-, nucleic acid- and nucleosome- binding. Further, the activity of PRC proteins or PcG proteins may be reduced by modulating the level or activity of nucleic acid molecules encoding one or more PRC proteins using strategies known in the art and/or described further herein.
  • the methods comprise administering to a mammalian subject, or contacting cells therefrom, with an agent that down regulates the level or activity of one or more polycomb group proteins (PcG) or polycomb repressive complexes (PRC) or other members of the network of interacting molecules to which Suzl2 belongs, or their encoding nucleic acid molecules.
  • PcG and PRC polypeptides include Suzl2, Ezh2 and Eed and functional homologs thereof.
  • the present invention is to be understood to encompass methods of modulating the level or activity of hematopoietic progenitor cells comprising contacting cells with an effective amount of an agent that up-regulates the level or activity of a gene to which PRCl or PRC2/3 binds.
  • the gene activity is transcriptional or translational activity.
  • the agent is provided together with an agent that down regulates the level or activity of PRCl and/or PRC2/3.
  • small molecules are determined that interact with the expression product/s of genes to which PRC polypeptides bind.
  • agents including cytokines and pharmacological agents such as antisense molecules or small peptide or non-peptide inhibitors may be envisaged by the skilled artisan that are capable of down regulating the level or activity of PRC or up-regulating the level or activity of PRC target genes.
  • the present invention provides for the use of PRC inhibitors in the treatment or prevention of conditions associated with thrombocytopenia.
  • the invention provides for the use of these agents in the preparation of a medicament for the treatment or prophylaxis of thrombocytopenia.
  • the condition is associated with leukopenia or pancytopenia.
  • the methods are used to up-regulate the number or activity of progenitor cells. These methods and medicaments may be applied, for example, to enhance HSC function prior to bone marrow transplantation.
  • the agent enhances gene expression by modulating the level or activity of components in the PRC pathway, which culminates in chromatin modification and transcriptional repression.
  • the agents are conveniently in a composition comprising the agent and one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • the agents may also be used in conjunction with further modulators of the number or activity of hematopoietic stem cells. Consequently, the present invention provides compositions or two- or multi- part pharmaceutical compositions comprising in one embodiment at least one inhibitor of transcriptional repression and one modulator of a transcription factor.
  • the transcription factor is Myb.
  • the agent is an inhibitor that reduces the level or activity of Myb transcription factor.
  • antagonists and agonists of the target molecules identified in accordance with the present invention.
  • antagonists and agonists may comprise all or part of the target molecules themselves, in genetic or proteinaceous form, or their complementary sequences, chemical analogs, mimetics, sense or antisense molecules including inhibitory RNA-type agents, antibodies, or other molecules in the genetic network to which the target molecules identified by the instant methods belong or their derivatives.
  • the antagonists or agonists may be synthetic chemicals or natural products identified by screens known in the art. Once a target molecule identified as described herein, a wide range of screening strategies known in the art are available for the identification, production, design and development of antagonists or agonists.
  • the rational design of molecules which interact with an active or binding site of a proteinaceous target molecule may be achieved using the solution or crystal structures of the target and/or target-ligand complexes. Spectroscopic and computer modelling techniques are generally used to determine a solution structure and subsequently the three dimensional structure can be displayed and manipulated using computer enhanced algorithms for the design of agonists or antagonists.
  • endogenous PRC binding molecules are identified and employed in the present invention.
  • Biologically active portions of the subject polypeptides are also characterised by having one or more binding functions/sites of a reference full length molecule and one or more functional activities thereof or by not having one or more functional/binding activities of the full length reference molecule.
  • a single basis pair deletion in the splice acceptor site of the sixteenth exon of Suzl2 of the Plt8/+ mutant mouse profoundly alters its ability to be active in vivo.
  • the present invention provides methods of screening or testing for agents useful in modulating the number or activity of hematopoietic cells and/or their progenitor cells.
  • Modified non-human animals and isolated cells comprising a mutation or modification one or more polycomb protein family genes are also provided.
  • the invention also provides methods of screening or testing subjects for mutations in the Suzl2 gene or one or more genes encoding PRC molecules or their associated regulatory molecules indicative of a particular genetic basis for defects in hematopoiesis such as, without limitation, thrombocytosis, myelofibrosis, thrombocytopenia, leukopenia, progenitor or stem cell defects in the subject.
  • Any agent that affects the targets identified in the present invention may be employed to modulate the number or activity of hematopoietic cells and/or their progenitors.
  • agents and compositions of the present invention include, for example, small or large chemical molecules, peptides, polypeptides including antibodies, modified peptides such as constrained peptides, foldamers, peptidomimetics, cyclic peptidomimetics, proteins, lipids, carbohydrates or nucleic acid molecules including antisense or other gene silencing molecules.
  • Agents may comprise naturally occurring molecules, variants (including analogs) thereof as defined herein or non-naturally occurring molecules or variants thereof.
  • Figure 1 is a graphical representation showing the isolation of two dominant mutations that suppress thrombocytopenia. Platelet counts of Gi progeny are shown for two ENU- treated males 2019 and 2026, and for control c-mpl 'A mice that were not treated with ENU. Offspring with platelet counts above 300 x lOVml (marked by arrows) were thought to carry a mutation that suppressed thrombocytopenia and were selected for progeny testing.
  • Figure 2 is a graphical representation of data showing the results of F 2 -mapping.
  • the PU8 mutation is located on mouse chromosome 11.
  • A Mice that carried C57BL/6 DNA on chromosome 11 (designated 'PU8/+ ', grey bar) were found to have a higher mean platelet count than a control F 2 population, or mice homozygous 129/Sv ('+/+', black bar).
  • B mice homozygous 129/Sv
  • Figure 3 is a graphical representation of the fine mapping strategy undertaken to localize the PU8 mutation and to further reduce the candidate interval. Specifically, F 2 mice were genotyped with additional polymorphic microsatellite markers on chromosome 11. In all 531 F 2 mice were used in this study. Progeny testing confirmed that mice which carry C57BL/6 at this point in the genome carry a mutation which results in high platelet counts. Furthermore, no mice were found to be homozygous C57BL/6 over the interval between CAR28 and CAR48, suggesting the presence of a mutation that is homozygous lethal.
  • Figure 4 is a representation of data showing that the PU8 mutation is a single base pair deletion in Suzl2.
  • a schematic representation of the Suzl2 locus is shown; an arrow marks the position of the mutation.
  • the mutation is a single base pair deletion upstream of the last coding exon of Suzl '2, which disrupts the splice acceptor site of exon 16.
  • the loss of a single guanine nucleotide (g) is evident when sequence was analysed from mice that were heterozygous for the Plt8 mutation.
  • Figure 5 is a representation of data showing that the Plt8 mutation alters splicing ofSuzl2.
  • (A) is a schematic representation of the Suzl2 locus showing intron and exon structure.
  • Figure 7 is a photographic representation (A) showing protein expression and a graphical representation (B) showing gene expression in GlME hematopoietic cells (Stachura et al Blood, 107 (l):87-97, 2006).
  • GlME cells were infected with retroviral constructs that direct expression of short hairpin RNAs (shRNAs) that have been designed to reduce expression of PRC components.
  • shRNAs short hairpin RNAs
  • the megakaryocy e cell line GlME was used as a model system to study changes in gene expression that are associated with reduced PRC2 function in hematopoietic cells.
  • GlME cells were infected with various retroviral constructs that direct expression of short hairpin RNAs (shRNAs) that have been designed to reduce expression of a target gene (Suzl2, Ezh2 or Eed).
  • shRNAs short hairpin RNAs
  • NONS scrambled sequence
  • LMP empty vector
  • Cells were maintained in puromycin, a drug that will selectively kill cells that have not been infected with the retrovirus.
  • a western blot (A) was performed to monitor protein expression in transfected GlME cells, specific primary antibodies were used to measure the level of Suzl2, Ezh2, ERK 1/2, Histone 3 Lysine 27 tri-methylation (H3-K27-3Me) and total Histone 3 (H3-total).
  • Figure 8 is a graphical representation showing the results of competitive transplantation studies performed to test the ability of Suzl2 Plt8/+ stem cells to repopulate the hematopoietic compartment of lethally irradiated recipients.
  • Irradiated recipient mice (Ly5.1 + ) were transplanted with an equal number of bone marrow cells from a test marrow (Ly5.2 + ) and competitor marrow (Ly5.1 + ). In total 2 xlO 6 cells were injected into each recipient. In each case the competitor marrow shared the same MpI genotype as the test marrow. Data shown represent the ratio of Ly5.2/Ly5.1 in total leukocytes, B cells, T cells and myeloid cells (GrlMacl).
  • the ratio should equal 1.
  • the mutant cells show a greater contribution (ratios above 1), and the difference between MpI ' ' " Suzl2 plt8/+ and Mpl ⁇ ;' Suzl2 +/+ marrow is significant in both total cells and in the B-cell lineage. The same trend is evident in MpI+/+ samples.
  • Each column is representative of 3-4 individual test marrows, that had been transplanted into ⁇ 5 recipients each. * denotes significance (pO.Ol).
  • Figure 9 is a graphical representation of sequence data. DNA was extracted from PLT8 mice with elevated platelet counts for sequence analysis. A single base pair deletion was identified in heterozygous mice (Suzl2 Plt8/+ ) and in homozygous tissue obtained from embryos (Suzl2 p " 8/Plt8 ) (large arrow). The deletion disrupts the splice acceptor site upstream of exon 16.
  • Figure 10 is a photographic representation of Western blotting data showing protein expression levels in lysates prepared from sex-matched mouse embryos (E12.5). Suzl2 and Ezh2 protein levels were reduced in Suzl ⁇ h8/Jr embryos. Suzl2 protein levels were equivalent in Suzl2 P t8 + embryos and embryos heterozygous for the genetrap allele (Suzl2 502gt/+ ). Equivalent amounts of protein were run in each lane, Histone H3 was used to verify equal loading. Western blot signal intensity was quantified using a densitometer; results represent the average of two independent experiments (at right).
  • Figure 11 is a graphical representation showing enhanced CFU-S frequency in bone marrow derived from Suzl2 Pll8/+ mice compared to wildtype littermates. Irradiated recipients received 1.5 xlO 5 nucleated bone marrow cells from c-mpV 1' donors or 7.5 x 10 4 cells from c-mpl +/+ donors. Data represent the mean of 4-6 mice of each genotype and error bars show the standard error of the mean. Statistical significance was assessed using an unpaired t-test.
  • Figure 12 is a graphical representation of data showing that Suzl2 deficiency enhances progenitory activity.
  • Irradiated recipients (Ly 5.1 + ) were transplanted with an equal number of bone marrow cells from a test animal (Ly5.2 + ) and a wildtype competitor
  • Ly5.1 + , Ly5.2 + and various lineage markers e.g. B220, Macl and CD4 to measure the contribution of the test marrow to hematopoiesis. Equal contribution from test and competitor would result in 50% of cells being positive for the Ly5.2 + marker. Suzl2 p " 8/+ cells made a greater contribution than wildtype on both a c-Mpl +/+ and a c-Mpl' ' background. (B) Secondary recipients were analysed three months after transplantation.
  • Each column is the average of 3-4 test marrows that have been transplanted into 5 recipients.
  • An asterisk denotes statistical significance (p ⁇ 0.004) corrected for multiple testing.
  • Figure 13 is a photographic representation showing inhibition of Suzl2 expression by shRNA-mediated silencing in vivo.
  • Bone marrow extracted from 5-FU treated mice was infected with either the LMS-Nons or the LMS-Suzl2 virus and transplanted into recipient mice.
  • Thymocytes were isolated 12 weeks after transplantation and fractionated based upon expression of GFP (+ or -); low or intermediate populations were detected in some mice (low).
  • Protein lysates were prepared from sorted cells and Western blotting was performed to detect expression of Suzl2, Ezh2 or histone H3. Non-specific bands have been marked (*) and an arrow used to denote residual Suzl2 signal that persisted after the membrane was stripped.
  • Figure 14 is a graphical representation of data showing that inhibition of Suzl2 by shRNA-mediated silencing elevates HSC contribution to hematopoiesis.
  • A Bone marrow extracted from 5-FU treated mice was infected with either the LMS-Nons or the LMS- Suzl2 virus and transplanted into recipient mice. Three independent infections were performed and in each case infected cells were transplanted into five recipient animals. A selection of primary recipients (9-11) were used as donors for secondary transplants, in each case cells were transplanted into 3-5 recipient mice. The frequency of cells that carried the virus (GFP+) was monitored prior to transplantation (input) and 8-12 weeks after transplantation in primary or secondary recipients.
  • GFP+ The frequency of cells that carried the virus
  • Table 1 provides a summary of sequence identifiers.
  • Table 2 provides an amino acid sub-classification.
  • Table 3 provides a list of exemplary and preferred amino substitutions.
  • Table 4 provides a list of non-convention amino acids.
  • GM granulocyte-macrophage colonies
  • G granulocyte colonies
  • M macrophage colonies
  • Eo eosinophil colonies
  • Meg megakaryocyte colonies.
  • Table 11 provides the megakaryocyte progenitor number in c-mpl ⁇ " mice with mutations in both Suzl2 and c-Myb. Data represent the mean and standard deviation of megakaryocyte colony number in cultures of bone marrow, 2.5 xlO 5 cells were plated in each dish. Cultures were prepared from three c-mpT 1' mice of each genotype (except for c- myb plt4/+ Suzl2 +/+ for which two mice were cultured) and were stimulated with SCF/IL- 3/Epo or with IL-3 alone.
  • Table 12 provides the genes that are up-regulated in GlME cells that express shRNA- Sul2.
  • To identify genes that are regulated by Suzl2 a global analysis of gene expression was performed with GlME cells that expressed shRNA-Suzl2. A large number of genes showed altered expression in Suzl2 knockdown cells when compared to the non-specific control (shRNA-Nons) (194 genes with an adjusted p-value below 0.05). Genes elevated in expression with a fold change >1.8 are listed above.
  • Table 13 provides the genes that are down-regulated in GlME cells that express shRNA- Sul2. To identify genes that are regulated by Suzl2 a global analysis of gene expression was performed with GlME cells that expressed shRNA-Suzl2.
  • Suzl2 knockdown cells A small number of genes showed reduced expression in Suzl2 knockdown cells when compared to the non-specific control (shRNA-Nons) (14 genes with an adjusted p-value below 0.05). As expected, Suzl2 was identified as one of the transcripts under-represented in the Suzl2 knockdown cells.
  • Table 14 provides the confirmation of gene expression changes in GlME cells that express shRNA-Sul2. Quantitative real-time PCR (QPCR) was performed on cDNA samples prepared from GlME cells infected with either the LMP-Suzl2 or the LMP-Nons retrovirus. Gene specific primers and probe sets were acquired from Applied Biosystems.
  • Hprtl The expression level of Hprtl was used to normalise for sample abundance, and the relative quantification ( ⁇ C t ) method was used to compare gene expression between LMS- Suzl2 and the LMS-Nons control.
  • Polycomb proteins and complexes comprising them have a role in modifying chromatin to repress gene expression. Disruption of PRC function has been associated with tumorgenesis. Similarly, inhibition of protein deacetylases, which deacetylate histones and represses transcription, is being used as a method for inhibiting cancer cell proliferation.
  • the present invention is predicated, in part, on the discovery that the number and/or activity of hematopoietic progenitor cells and/or their descendants can be enhanced using physiological or pharmacological agents that de-repress (or enhance) gene expression in these cells.
  • PRC polypeptide or "PRC protein” is a polypeptide or protein that binds to and forms part of a PRC.
  • the term also encompasses the expression products (polypeptide) of PRC-target genes. That is, those polypeptides whose level or activity is specifically regulated by the activity of PRC polypeptide in hematopoietic cells, such as GIME cells.
  • Exemplary PRC members include the PcG proteins Suzl2, Ezh2 and Eed. These proteins are mammalian homologs of Drosophilia proteins Su(Z) 12, E(Z) and Esc, respectively
  • compound used to refer to a chemical compound that induces a desired pharmacological and/or physiological effect.
  • the terms also encompass pharmaceutically acceptable and pharmacologically active ingredients of those active agents specifically mentioned herein including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs and the like.
  • agent When the terms “compound”, “active agent”, “pharmacologically active agent”, “medicament”, “active” and “drug” are used, then it is to be understood that this includes the active agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, enantiomers, metabolites, analogs, etc.
  • agent is not to be construed as a chemical compound only but extends to peptides, polypeptides and proteins as well as genetic molecules such as RNA, DNA and chemical analogs thereof.
  • modulator is an example of an "agent, pharmacologically active agent, medicament, active and drug which modulates the number or activity of hematopoietic cells and/or their progenitors.
  • prodrug includes variants that are converted in vivo into the agents of the invention.
  • prodrug also encompasses the use of fusion or attached proteins or peptides comprising cell-permeant proteins or peptides. These agents enhance transport or agents across cellular membranes and include membrane permeable sequence, the tat peptide and antennapedia (penetratin).
  • an "effective amount” means an amount necessary to at least partially attain the desired response.
  • An effective amount for a human subject lies in the range of about 0.1 ng/kg body weight/dose to lg/kg body weight/dose. In some embodiments, the range is about l ⁇ to Ig, about lmg to Ig, lmg to 500mg, lmg to 250mg, lmg to 50mg, or l ⁇ to lmg/kg body weight/dose.
  • Dosage regimes are adjusted to suit the exigencies of the situation and may be adjusted to produce the optimum therapeutic dose. For example, several doses may be provided daily, weekly, monthly or other appropriate time intervals.
  • subject agents may be used neat however, typically, subject agents are formulated as pharmaceutical compositions at a concentration of about 0.1mg/m to 100mg/ml, such as 1 to 10mg/ml.
  • Formulations comprising lOmg of active ingredient or more broadly O.lmg to 200mg per tablet are suitable representative dosage forms.
  • gene is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Reference herein to a “gene” is also taken to include:- (i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3'- untranslated sequences of the gene.
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3'- untranslated sequences of the gene.
  • progenitor cell or “precursors” and the like encompass undifferentiated hematopoietic stem cell and any one or more of the blood cell types which arise from HSC.
  • the term refers to multipotent cells as well as the various different forms of myeloid- or lymphoid-restricted cells that ultimately give rise to fully differentiated mature blood cells.
  • HSC In adults, HSC reside in the bone marrow, peripheral blood, lung, liver, spleen and other organs. HSC are the first in a hierarchy of progenitor cells. They are capable of long-term self renewal (long term (LT)-HSCs).
  • LT-HSCs differentiate into short-term multipotent HSCs, (ST-HSCs) that retain the ability to produce all blood types but only proliferate for a relatively short time.
  • lymphoid progenitors arise that ultimately produce immune cells
  • myeloid progenitors arise that ultimately produce mainly red blood cells and platelets and some innate immune cells.
  • These progenitor cells have various abilities to proliferate and differentiate and from these cells ultimately arise terminally differentiated cells.
  • reference to HSC and hematopoietic progenitors include all the above mentioned progenitor cells and reference to hematopoietic or blood cells include any of their terminally differentiated descendants.
  • HSC hematopoietic stem cell
  • CLP common lymphoid precursor
  • CMP common myeloid precursor
  • GMP granulocyte-macrophage precursor
  • MEP megakaryocyte-erythroid precursor
  • CFU-GM colony forming unit- granulocytic/macrophage
  • CFU-G colony forming unit-granulocytic
  • CFU-M colony forming unit-macrophage
  • CFU-Mk colony forming unit-megakaryocytic
  • BFU-e Burst- forming unit erythroid
  • CFU-E colony forming unit-erythroid cells.
  • references to "modulating”, “modulated” or “modulator” and the like includes down modulating, inhibiting antagonising, decreasing or reducing and up modulating, increasing, potentiating, agonising, prolonging, stimulating or enhancing as well as agents that have this effect. Any subject who could benefit from the present methods or compositions is encompassed.
  • the term "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.
  • the present invention provides a method of modulating the number and/or activity of hematopoietic cells or their progenitors in a subject, the method comprising administering to the subject an effective amount of an agent that inhibits the activity of histone deacetylases.
  • histone deacetylase inhibitors are used in conjunction with further additional agents described herein such as, for example, demethylating agents.
  • Protein deacetylase inhibitors may be selected from the group comprising; short chain fatty acids such as butyric acid, valproic acid, and sodium acid phenybutyrate; hydroxamic acids such as SAHA, oxamflatin and TSA; cyclic tetrapeptides such as depipeptide and apicidin; benzamides such as MS-275; ketones such as trifluoromethyl kentone and miscellaneous agents such as depudecin.
  • short chain fatty acids such as butyric acid, valproic acid, and sodium acid phenybutyrate
  • hydroxamic acids such as SAHA, oxamflatin and TSA
  • cyclic tetrapeptides such as depipeptide and apicidin
  • benzamides such as MS-275
  • ketones such as trifluoromethyl kentone and miscellaneous agents such as depudecin.
  • subject includes, without limitation, humans and non-human primates, animals, livestock animals, companion animals, laboratory test animals, captive wild animals, reptiles and amphibians, fish, birds etc.
  • 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.
  • Reference to modulating the "activity" of a target includes reference to the level or number of molecules/cells or the concentration of the target or the functional activity of the target or cell.
  • the activity of a polypeptide may be enhanced by increasing the level of transcription or translation of an encoding DNA or RNA.
  • the activity of a polypeptide may also be decreased by reducing the level of transcription or translation such as by inhibiting promoter or enhancer activity or by the use of antisense/iRNA strategies now routine in the art.
  • the level of one or more PRC polypeptides in hematopoietic cells may be modulated by administering agents from which the polypeptide or its regulators are producible, such as a genetic construct encoding a functional form of the polypeptide.
  • the genetic construct encodes a regulator of expression of the target polypeptide such as an antisense molecule, iRNA, shRNA promoter or repressor or enhancer.
  • a regulator of expression of the target polypeptide such as an antisense molecule, iRNA, shRNA promoter or repressor or enhancer.
  • 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 a gene encoding a PRC polypeptide or a functional variant thereof.
  • 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, semi-synthetic or synthetic origin which, by virtue of its origin or manipulation: (i) is not associated with all or a portion of a polynucleotide with which it is associated in nature; (ii) is linked to a polynucleotide other than that to which it is linked in nature; or (iii) does not occur in nature.
  • 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 PRC levels or to down-regulate the level of one or more PRC polypeptides such as via antisense means or RNAi-mediated gene silencing.
  • Such constructs are also useful in generating animal models and cells carrying modified alleles of genes encoding PRC polypeptides. Such animals and cells and compositions comprising them are discussed briefly towards the end of the description.
  • Other recombinant constructs include sequences comprising PRC-target gene sequence i.e. comprising all or part of a gene encoding the expression products of a PRC-target gene. In some embodiments such targets encode transcriptional repressors or enhancers.
  • antisense polynucleotide sequences are useful agents in preventing or reducing the expression of RNAs.
  • morpholines may be used as described by Summerton et al. (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. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise.
  • RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA.
  • One preferred result of such interference with target nucleic acid function is modulation of the expression of a PRC gene or a PRC -target gene.
  • antisense compound is a single-stranded antisense oligonucleotide
  • double-stranded structures such as double-stranded short hairpin RNA (dsRNA) molecules such as stem-loop RNAs and microRNA-30 based shRNAs
  • dsRNA double-stranded short hairpin RNA
  • shRNA shRNA
  • 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. 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.
  • modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.
  • nuclease-resistant phosphorothioates that hybridise to nucleotides within the open reading frame of a PcG or a PRC mRNA will induce RNAseH-mediated degradation.
  • antisense RNA selected to inhibit one or more of Suzl2, Ezh2 and Eed effectively down regulates the production of the encoded polypeptide in hematopoietic cells.
  • the genetic agents or compositions in accordance with this aspect of the 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 agents of the present invention in some embodiments comprise Suzl2 or a functional fragment or functional variant thereof, or in genetic form a Suzl2 gene or a functional part or functional variant thereof or complementary forms of these. In other embodiments, the agents comprise Ezh2 or Eed or function fragments or functional variants thereof or complementary forms thereof.
  • the present invention provides a composition comprising Suzl2 or Suzl2 (ie the molecule in genetic or proteinaceous form) or a functional variant thereof which substantially reduces the activity of Suz 12 or Suzl2 for use in enhancing the number and/or activity of hematopoietic cells and/or their precursors.
  • Compositions may be designed for ex vivo or in vivo applications.
  • the compositions comprise Ezh2 or Eed or Ezh2 or Eed or a functional variant of either of these, which substantially reduces the activity of Ezh2 or Eed polypeptides or Ezh2 or Eed genes for use in enhancing the number and/or activity of hematopoietic cells and/or their precursors.
  • the modulatory agents of the present invention may be chemical agents such as a synthetic or recombinant molecules, polypeptides, peptides, modified peptides or proteins, lipids, glycoproteins or other naturally or non-naturally occurring molecules, variants, derivatives or analogs thereof.
  • genetic agents such as DNA (gDNA, cDNA, PNA), RNA (sense RNAs, antisense RNAs, mRNAs, tRNAs, rRNAs, small interfering RNAs (siRNAs), ShRNAs, micro RNAs (miRNAs), small nucleolar RNAs (SnoRNAs, small nuclear (SnRNAs)) ribozymes, aptamers, DNAzymes or other ribonuclease-type complexes may be employed. Agents in accordance with this aspect of the invention may directly interact with Suzl2.
  • antibodies or antigen binding fragments, peptides, modified peptides, oligosaccharides, foldamers, peptidomimetics or analogs, synthetic or naturally occurring small or intermediate molecules and other such molecules may be conveniently employed.
  • genetic mechanisms are used to indirectly modulate the activity of hematopoietic progenitor cells. Genetic mechanisms include gene silencing approaches as well as gene expression approaches to endogenously produce the present agents such as peptides, polypeptides and nucleic acid molecules.
  • RNA and DNA aptamers are also contemplated as exogenous agents.
  • RNA and DNA aptamers can substitute for monoclonal antibodies in various applications (Jayasena, Clin. Chem., 45(9):1628-1650, 1999; Morris et al, Proc. Natl. Acad. ScL, USA, P5(6):2902-2907, 1998).
  • Aptamers are nucleic acid molecules having specific binding affinity to non-nucleic acid or nucleic acid molecules through interactions other than classic Watson-Crick base pairing. Aptamers are described, for example, in U.S. Pat. Nos. 5,475,096; 5,270,163; 5,589,332; 5,589,332; and 5,741,679.
  • agents which modulate the level or activity of Suzl2 genes or Suzl2 polypeptides may be derived from Suzl2 or Suzl2 or be variants of Suzl2. Alternatively, they may be identified in in vitro or in vivo screens. Natural products, combinatorial synthetic organic or inorganic compounds, peptide/polypeptide/protein, nucleic acid molecules and libraries or phage or other display technology comprising these are all available to screen or test for suitable agents. Natural products include those from coral, soil, plant, or the ocean or antarctic environments. Various domains of PRC family members may be specifically targeted or screened, such as the VEFS box required for interaction between at least Suzl2 and Ezh2, or a zinc-finger binding motif.
  • the agent to be tested is contacted with a system comprising a PcG or PRC protein genetic sequence. Then, the following may be assayed for: the presence of a complex between the agent and the target, a change in the activity of the target, or a change in the level of 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.
  • the present agents inhibit enzymes required for PRC or Suzl2 function or activity. As shown by Tan et al (2007) chemical inhibition of the methyl donor required for PRC2 function or activity is an effective method of reducing PRC2 function. In some embodiment, the agents inhibit s-adenosylhomocysteine hydrolase. In other embodiments, the agent is 3-Deazaneplanocin A (DZNep, NSC 617989).
  • Bound polypeptide is detected by various methods.
  • a similar method involving peptide synthesis on beads, which forms a peptide library in which each bead is an individual library member, is described in U.S. Patent No. 4,631,211 and a related method is described in International Publication No. WO 92/00091.
  • a significant improvement of the bead-based methods involves tagging each bead with a unique identifier tag, such as an oligonucleotide or electrophoretic tag, so as to facilitate identification of the amino acid sequence of each library member. These improved bead-based methods are described in International Publication No. WO 93/06121.
  • Another chemical synthesis screening method involves the synthesis of arrays of peptides (or peptidomimetics) on a surface wherein each unique peptide sequence is at a discrete, predefined location in the array.
  • the identity of each library member is determined by its spatial location in the array.
  • the locations in the array where binding interactions between a predetermined molecule and reactive library members occur is determined, thereby identifying the sequences of the reactive library members on the basis of spatial location.
  • display systems which enable a nucleic acid to be linked to the polypeptide it expresses.
  • Selection protocols for isolating desired members of large libraries are known in the art, as typified by phage display techniques.
  • Such systems in which diverse peptide sequences are displayed on the surface of filamentous bacteriophage, are useful for creating libraries of antibody fragments (and the nucleotide sequences that encoding them) for the in vitro selection and amplification of specific antibody fragments that bind a target antigen.
  • the nucleotide sequences encoding the V H and V L regions are linked to gene fragments which encode leader signals that direct them to the periplasmic space of E.
  • coli and the resultant antibody fragments are displayed on the surface of the bacteriophage, typically as fusions to bacteriophage coat proteins (e.g., pill or pVIII).
  • antibody fragments are displayed externally on lambda phage capsids (phage bodies).
  • An advantage of phage-based display systems is that selected library members can be amplified simply by growing the phage containing the selected library member in bacterial cells.
  • nucleotide sequence that encode the polypeptide library member is contained on a phage or phagemid vector, sequencing, expression and subsequent genetic manipulation is relatively straightforward.
  • Corresponding technologies are applied to combinatorial libraries of small organic molecules.
  • Antibodies including anti-idiotypic antibodies, chaemeric antibodies and humanised antibodies and antigen binding fragments thereof are useful agents for down regulating specific targets in a cell.
  • Antibodies that down regulate histone methylation or acetylation are contempled.
  • Antibodies that down regulate the level or activity Suzl2 or a PRC complex comprising Suzl2 are also contemplated in some embodiments.
  • PRC function may be down regulated by interfering with PRC-histone interactions, such as the ability of PRC components to methylate histone proteins.
  • variant histone proteins that lack K(lysine)27 will bind to PRC2 but are not able to be methylated by PRC- mediated reactions and will therefore competivively inhibit PRC function.
  • agents such as antibodies that interfere with PRC2-histone binding as specific epitopes provide specific inhibition of PRC function.
  • antibodies and other agents are particularly preferred which are capable of traversing biological membranes to gain access to intracellular and intravesicular portions of the cell.
  • antibody is used in the broadest sense and specifically covers single monoclonal antibodies and antibody compositions with polyepitopic specificity.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
  • each monoclonal antibody is directed against a single determinant on the antigen.
  • the monoclonal antibodies herein include hybrid and recombinant antibodies produced by splicing a variable (including hypervariable) domain of an antibody, such as an anti-Suzl2 or anti-PRC2 antibody with a constant domain (e.g.
  • “humanized” antibodies or a light chain with a heavy chain, or a chain from one species with a chain from another species, or fusions with heterologous proteins, regardless of species of origin or immunoglobulin class or subclass designation, as well as antibody fragments (e.g., Fab, F(ab').sub.2, and Fv), so long as they exhibit the desired biological activity. See, e.g. U.S. Pat. No. 4,816,567 and Mage and Lamoyi, in Monoclonal Antibody Production Techniques and Applications, pp. 79-97 (Marcel Dekker, Inc.: New York, 1987).
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler and Milstein, Nature, 256:495 (1975), or may be made by recombinant DNA methods. U.S. Pat. No. 4,816,567.
  • the “monoclonal antibodies” may also be isolated from phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (1990), for example.
  • Humanized forms of non-human (e.g. murine) antibodies are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
  • humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity.
  • CDR complementary determining region
  • Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • the humanized antibody may comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • the three-dimensional structure of Suzl2 or a PRC polypeptide or a PRC binding molecule or an expression product of a PRC-target gene facilitates the design of binding agents that de-repress transcription to enhance the number or activity of hematopoietic cells and/or their progenitor cells.
  • the skilled person can screen three-dimensional structure databases of compounds to identify those compounds having functional groups that will fit into one or more of the binding sites. Combinational chemical libraries can be generated around such structures to identify those with high affinity binding to PRC binding sites. Agents identified from screening compound databases or libraries are then fitted to three-dimensional representations of PRC binding sites in fitting operations, for example, using docking software programs.
  • a potential modulator may be evaluated "in silico" for its ability to bind to a PRC active site prior to its actual synthesis and testing.
  • the quality of the fit of such entities to binding sites may be assessed by, for example, shape complementarity by estimating the energy of the interaction. (Meng et al., J. Comp. Chem., 75:505-524, 1992).
  • the design of chemical entities that associate with components of PRC generally involves consideration of two factors.
  • the compound must be capable of physically and structurally associating with PRC members.
  • Non-covalent molecular interactions important in the association of PRC members with their interacting partners include hydrogen bonding, van der Waal's and hydrophobic interactions.
  • the compound must be able to assume a conformation that allows it to associate with a PRC polypeptide. Although certain portions of the compound will not directly participate in this association with PRC, those portions may still influence the overall conformation of the molecule.
  • Such conformation requirements include the overall three-dimensional structure and orientation of the chemical entity or compound in relation to all or a portion of the active site, or the spacing between functional groups of a compound comprising several chemical entities that directly interact with a PRC member. Similar considerations apply to design of agents that interact with the expression products of PRC-target genes.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e. the replacement group will have approximately the same, size, shape, hydrophobicity and charge as the original group. It should of course, be understood that components known in the art to alter conformation should be avoided.
  • Putative binding agents may be computationally evaluated and designed by means of a series of steps in which chemical entities or fragments are screened and selected for their ability to associate with the one or more binding sites. Selected fragments or chemical entities may then be positioned in a variety of orientations, or "docked,” to target binding sites. Docking may be accomplished using software, such as QUANTA and SYBYL, followed by energy minimization and molecular dynamics with standard molecular mechanics force fields, such as CHARMM or AMBER. Specialised computer programs may be of use for selecting interesting fragments or chemical entities.
  • Useful programs to aid the skilled addressee in connecting chemical entities or fragments include CAVEAT (University of California, USA), 3D database systems and HOOK (Molecular Simulations, USA)
  • De-novo ligand design methods include those described in LUDI (Molecular Simulations, USA), LEGEND (Molecular Simulations, USA), LeapFrog (Tripos Inc.,) SPROUT (University of Leeds, UK) and the like.
  • Standard homology modelling techniques may be employed in order to determine the unknown three-dimensional structure or molecular complex.
  • Homology modelling involves constructing a model of an unknown structure using structural coordinates of one or more related protein molecules, molecular complexes or parts thereof. Homology modelling may be conducted by fitting common or homologous portions of the protein whose three-dimensional structure is to be solved to the three-dimensional structure of homologous structural elements in the known molecule. Homology may be determined using amino acid sequence identity, homologous secondary structure elements and/ or homologous tertiary folds. Homology modelling can include rebuilding part or all of a three-dimensional structure with replacement of amino acid residues (or other components) by those of the related structure to be solved.
  • binding agents are designed with a deformation energy of binding of not greater than about 10 kcal/mole, more preferably not greater than 7kcal/mole.
  • Computer software is available to evaluate compound deformation energy and ectrostatic interactions. For example, Gaussian 98, AMBER, QUANTA, CHARMM, INSIGHT II, DISCOVER, AMSOL and DelPhi.
  • Libraries of small organic molecules can be generated and screened preferably using high- throughput technologies known to those of skill in this area. See for example US Patent No. 5,763,263 and US Application No. 20060167237. Combinatorial synthesis provides a very useful approach wherein a great many related compounds are synthesised having different substitutions of a common or subset of parent structures. Such compounds are usually non-oligomeric and may be similar in terms of their basic structure and function,- varying in for example chain length, ring size or number or pattern of substitutions. Virtual libraries may also, as mentioned above, be constructed and compounds tested in silico (see for example, US Application No. 20060040322) or in vitro or in vivo assays known in the art.
  • agents are derived from genetic sequences encoding PRC, PcG or PRC- target gene products or their complementary forms.
  • the terms functional form or variant, functionally equivalent derivative or homolog include molecules that selectively hybridize to PRC genes or PRC -target genes or a complementary form thereof over all or part of the genetic molecule under conditions of medium or high stringency at a defined temperature or range of conditions, or which have about 60% to 80% sequence identity to the nucleotide sequence defining PRC genes or PRC-target genes.
  • Illustrative PRC nucleotide sequences include those comprising nucleotide sequences set forth in SEQ ID NO: 1 or 3 (mouse or human Suzl2 mRNA).
  • SEQ ID NO: 1 or 3 ouse or human Suzl2 mRNA.
  • the term “Suzl2 gene” expressly encompass all forms of the gene including regulatory regions such as those required for expression of the coding sequence and genomic forms or specific fragments including probes and primers, antisense molecules and constructs comprising same or parts thereof as well as cDNA or RNA and parts thereof.
  • PRC nucleotide sequences include those comprising nucleotide sequences set forth in SEQ ID NO: 5 or 7 (mouse or human Ezh2 mRNA).
  • Ezh2 gene expressly encompass all forms of the gene including regulatory regions such as those required for expression of the coding sequence and genomic forms or specific fragments including probes and primers, antisense molecules and constructs comprising same or parts thereof as well as cDNA or RNA and parts thereof.
  • PRC nucleotide sequences include those comprising nucleotide sequences set forth in SEQ ID NO: 9 or 11 (mouse or human Eed mRNA).
  • Eed gene expressly encompass all forms of the gene including regulatory regions such as those required for expression of the coding sequence and genomic forms or specific fragments including probes and primers, antisense molecules and constructs comprising same or parts thereof as well as cDNA or RNA and parts thereof.
  • T n of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner et al, Eur. J. Biochem., 46:%2>, 1974).
  • Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6 x SSC buffer, 0.1% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 0.1% w/v SDS at a temperature in the range 2O 0 C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.
  • the nucleic acid molecule encoding a PRC polypeptide comprise a sequence of nucleotides as set forth in SEQ ID NOs: 1, 3, 5, or 7 or which hybridises thereto or to a complementary form thereof under medium or high stringency hybridisation conditions.
  • the hybridisation region is about 12 to about 80 nucleobases or greater in length.
  • the precent identity between a particular nucleotide sequence and a reference sequence is about 30%, or 65% or about 70% or about 80% or about 85% or more preferably about 90% similarity or greater as about 95%, 96%, 97%, 98%, 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.
  • the present invention contemplates the use of a full-length PRC polypeptide or variants comprising biologically active portions of those polypeptides.
  • variants are inhibitors that bind to other PRC members of their targets and inhibit PRC function.
  • a biologically active portion comprises one or more binding domains or motifs or structures.
  • a biologically active portion of a full-length polypeptide can be a polypeptide which is, for example, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 300, or more amino acid residues in length.
  • the PRC polypeptides of the present invention include all biologically active or functional naturally occurring forms of PRC as well as variants comprising biologically active portions thereof and derivatives of these.
  • a PRC polypeptide or variants thereof including agonists or antagonists may be delivered to hematopoietic cells in proteinaceous forms as part of a delivery construct designed to allow appropriate intracellular targeting.
  • polypeptides comprising a sequence of amino acids having about 60% sequence identity to a PRC polypeptide or proteinaceous product of a PRC target gene.
  • Illustrative Suzl2 polypeptides comprise a sequence of amino acids substantially as set out in SEQ ID NO: 2 or 4 or are encoded by a sequence of nucleotides as set out in SEQ ID NOs: 1 or 3.
  • Illustrative PRC polypeptides comprise all or part of amino acid sequences set forth in SEQ ID NO: 6, 8, 10 or 12, or are encoded by a contiguous sequence of nucleotides as set out in SEQ ID NO: 5, 7, 9 or 11.
  • amino acid refers to compounds having an amino group and a carboxylic acid group.
  • An amino acid may be a naturally occurring amino acid or non- naturally occurring amino acid and may be a proteogenic amino acid or a non-proteogenic amino acid.
  • the amino acids incorporated into the amino acid sequences of the present invention may be L-amino acids, D-amino acids, ⁇ -amino acid, ⁇ -amino acids, sugar amino acids and/or mixtures thereof.
  • variants include proteins derived from the native protein by deletion (so-called truncation) or addition of one or more amino acids to the N-terminal and/or C-terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein.
  • variant proteins encompassed by the present invention are biologically active, that is, they continue to possess at least one biological activity or binding domain of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native PRC polypeptide will have at least 40%, 50%, 60%, 70%, generally at least 75%, 80%, 85%, preferably about 90% to 95% or more, and more preferably about 98% or more sequence similarity with the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein using default parameters.
  • a biologically active variant of a PRC polypeptide may differ from that polypeptide or parts thereof generally by as much as 100, 50 or 20 amino acid residues or suitably by as few as 1-15 amino acid residues, as few as 1-10, such as 6-10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • a PRC polypeptide/peptide may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, amino acid sequence variants of a PRC polypeptide can be prepared by mutations in the DNA. Methods for mutagenesis and nucleotide sequence alterations are well known in the art. See, for example, Kunkel (Proc. Natl. Acad. Sci. USA, 52:488-492, 1985), Kunkel et al, (Methods in Enzymol., 154:367- 382, 1987), U.S. Pat. No.
  • REM Recursive ensemble mutagenesis
  • Variant PRC polypeptides may contain conservative amino acid substitutions at various locations along their sequence, as compared to a reference amino acid sequence.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, which can be generally sub- classified as follows: Acidic: The residue has a negative charge due to loss of H ion at physiological pH and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH. Amino acids having an acidic side chain include glutamic acid and aspartic acid.
  • the residue has a positive charge due to association with H ion at physiological pH or within one or two pH units thereof (e.g., histidine) and the residue is attracted by aqueous solution so as to seek the surface positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium at physiological pH.
  • Amino acids having a basic side chain include arginine, lysine and histidine.
  • Charged The residues are charged at physiological pH and, therefore, include amino acids having acidic or basic side chains (i.e., glutamic acid, aspartic acid, arginine, lysine and histidine).
  • Hydrophobic The residues are not charged at physiological pH and the residue is repelled by aqueous solution so as to seek the inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a hydrophobic side chain include tyrosine, valine, isoleucine, leucine, methionine, phenylalanine and tryptophan.
  • Neutral/polar The residues are not charged at physiological pH, but the residue is not sufficiently repelled by aqueous solutions so that it would seek inner positions in the conformation of a peptide in which it is contained when the peptide is in aqueous medium.
  • Amino acids having a neutral/polar side chain include asparagine, glutamine, cysteine, histidine, serine and threonine.
  • proline This description also characterises certain amino acids as “small” since their side chains are not sufficiently large, even if polar groups are lacking, to confer hydrophobicity.
  • "small” amino acids are those with four carbons or less when at least one polar group is on the side chain and three carbons or less when not.
  • Amino acids having a small side chain include glycine, serine, alanine and threonine.
  • the gene-encoded secondary amino acid proline is a special case due to its known effects on the secondary conformation of peptide chains.
  • the structure of proline differs from all the other naturally-occurring amino acids in that its side chain is bonded to the nitrogen of the ⁇ - amino group, as well as the ⁇ -carbon.
  • amino acid similarity matrices include proline in the same group as glycine, serine, alanine and threonine. Accordingly, for the purposes of the present invention, proline is classified as a "small" amino acid.
  • Amino acid residues can be further sub-classified as cyclic or noncyclic, and aromatic or nonaromatic, self-explanatory classifications with respect to the side-chain substituent groups of the residues, and as small or large.
  • the residue is considered small if it contains a total of four carbon atoms or less, inclusive of the carboxyl carbon, provided an additional polar substituent is present; three or less if not.
  • Small residues are, of course, always nonaromatic.
  • amino acid residues may fall in two or more classes. For the naturally-occurring protein amino acids, sub-classification according to this scheme is presented in the Table 2.
  • Conservative amino acid substitution also includes groupings based on side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine.
  • amino acid substitutions falling within the scope of the invention are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. After the substitutions are introduced, the variants are screened for biological activity.
  • similar amino acids for making conservative substitutions can be grouped into three categories based on the identity of the side chains.
  • the first group includes glutamic acid, aspartic acid, arginine, lysine, histidine, which all have charged side chains;
  • the second group includes glycine, serine, threonine, cysteine, tyrosine, glutamine, asparagine;
  • the third group includes leucine, isoleucine, valine, alanine, proline, phenylalanine, tryptophan, methionine, as described in Zubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).
  • a predicted non-essential amino acid residue in a PRC polypeptide is typically replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of the polynucleotide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for an activity of the parent polypeptide to identify mutants which retain that activity.
  • the encoded peptide can be expressed recombinantly and the activity of the peptide can be determined.
  • the present invention also contemplates variants of the naturally-occurring PRC polypeptide sequences or their biologically-active fragments, wherein the variants are distinguished from the naturally-occurring sequence by the addition, deletion, or substitution of one or more amino acid residues.
  • variants will display at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % identity to a reference PRC polypeptide sequence as, for example, set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12.
  • sequences differing from the native or parent sequences by the addition, deletion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 or more amino acids but which retain certain properties of the reference PRC polypeptide are contemplated.
  • the present variant PRC polypeptides also include polypeptides that are encoded by polynucleotides that hybridize under stringency conditions as defined herein, especially high stringency conditions, to PRC polynucleotide sequences, or the non-coding strand thereof.
  • variant polypeptides differ from an PRC polypeptide sequence by at least one but by less than 50, 40, 30, 20, 15, 10, 8, 6, 5, 4, 3 or 2 amino acid residue(s).
  • variant polypeptides differ from the corresponding sequence in any one of SEQ ID NOs: 2, 4, 6, 8, 10 or 12 by at least 1% but less than 20%, 15%, 10% or 5% of the residues. If this comparison requires alignment the sequences should be aligned for maximum similarity. (“Looped" out sequences from deletions or insertions, or mismatches, are considered differences.) The differences are, suitably, differences or changes at a nonessential residue or a conservative substitution. A sequence alignment for PRC proteins from a range of mammalian species is used to demonstrate conserved residues.
  • a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of an embodiment polypeptide without abolishing or substantially altering one or more of its activities.
  • the alteration does not substantially alter one of these activities, for example, the activity is at least 20%, 40%, 60%, 70% or 80% of wild-type.
  • An "essential" amino acid residue is a residue that, when altered from the wild-type sequence of an polypeptide agent of the invention, results in abolition of an activity of the parent molecule such that less than 20% of the wild-type activity is present.
  • a variant polypeptide includes an amino acid sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or more similarity to a corresponding sequence of a PRC polypeptide as, for example, set forth in SEQ ID NOs: 2, 4, 6, 8, 10 or 12, and has at least one activity of that PRC polypeptide.
  • Polypeptide agents may be prepared by any suitable procedure known to those of skill in the art.
  • the polypeptides may be prepared by a procedure including the steps of: (a) preparing a chimeric construct comprising a nucleotide sequence that encodes at least a portion of a PRC polypeptide or a functional variant thereof and that is operably linked to one or more regulatory elements; (b) introducing the chimeric construct into a host cell; (c) culturing the host cell to express the PRC polypeptide or variant thereof; and (d) isolating the PRC polypeptide or variant of either of these polypeptides from the host cell.
  • the nucleotide sequence encodes at least a portion of the sequence set forth in SEQ ID NOs: 2, 4, 6, 8, 10 or 12, or a variant thereof.
  • Recombinant polypeptides can be conveniently prepared using standard protocols as described for example in Sambrook, et ah, (1989, supra), in particular Sections 16 and 17; Ausubel et ah, (1994, supra), in particular Chapters 10 and 16; and Coligan et ah, CURRENT PROTOCOLS IN PROTEIN SCIENCE (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5 and 6.
  • polypeptides agents may be synthesised by chemical synthesis, e.g., using solution synthesis or solid phase synthesis as described, for example, in Chapter 9 of Atherton and Shephard (supra) and in Roberge et al, (Science, 269:202, 1995).
  • the synthesis of conformational ⁇ constrained peptides is described for example in International Publication No. WO 2004106366.
  • the terms "derivative” or the plural “derivatives” and “variant” or “variants” are used interchangeable and, whether in relation to genetic or proteinaceous molecules, include as appropriate parts, mutants, fragments, and analogues as well as hybrid, chimeric or fusion molecules and glycosylation variants.
  • Particularly useful derivatives retain the functional activity of the parent molecule and comprise single or multiple amino acid substitutions, deletions and/or additions to a PRC amino acid sequence.
  • the variants have functional activity or alternatively, modulate a PRC functional activity.
  • portion or fragment of a PRC gene is defined as having a minimal size of at least about 10 nucleotides or preferably about 13 nucleotides or more preferably at least about 20 nucleotides and may have a minimal size of at least about 35 nucleotides.
  • This definition includes all sizes in the range of 10 to 35 as well as greater than 35 nucleotides. Thus, this definition includes nucleic acids of 12,15, 20, 25, 40, 60,
  • nucleic acid molecules having any number of nucleotides between 500 and the number shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14 or 15 or a complementary form thereof.
  • SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 14 or 15 or a complementary form thereof.
  • the same considerations apply mutatis mutandis to any reference herein to a part, portion or fragment of a PRC polypeptide.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein and may be designed to modulate one or more properties of the polypeptide such as stability against proteolytic cleavage without the loss of other functions or properties.
  • Amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. Preferred substitutions are those 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.
  • 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 polypeptide. Since it is the interactive capacity and nature of a protein which defines that protein's biological functional activity, certain amino acid substitutions can be made in a protein sequence and its underlying DNA coding sequence and nevertheless obtain a protein with like properties. In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydrophobic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al, J. MoI. Biol, 757. 105-132, 1982).
  • 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 or structurally related molecules including those from other species.
  • peptide mimetic includes carbohydrate, nucleic acid or polypeptide mimetics and it intended to refer to a substance which has conformational features allowing the substance to perform as a functional analog of at least one biological activity of the reference molecule.
  • a peptide mimetic may be a peptide containing molecule that mimic elements of protein secondary structure (Johnson et al, "Peptide Turn Mimetics” in Biotechnology and Pharmacy, Pezzuto et al, eds Chapman and Hall, New York, 1993).
  • Peptide mimetics may be identified by screening random peptides libraries such as phage display or combinatorial libraries for peptide molecules which mimic a functional activity of a PRC polypeptide.
  • Nucleic acid mimetics include, for example, RNA analogs containing N3' ⁇ P5' phosphoramidate internucleotide linkages which replace the naturally occurring RNA 03'— P5' phosphodiester groups.
  • Enzyme or transcription factor 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 a PRC polypeptide or the expression products of a PRC target gene 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.
  • putative peptide or polypeptide agents may be analyzed by an alanine scan (Wells, Methods Enzymol., 202:2699-2705, 1991). In this technique, an amino acid residue is replaced by Ala and its effect on the peptide's activity is determined. Each of the amino acid residues of the peptide is analyzed in this manner to determine the important regions of the peptide.
  • a target-specific antibody selected by a functional assay and then to solve its crystal structure.
  • this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies (anti-ids) to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of the anti-ids would be expected to be an analog of the original receptor. The anti-id could then be used to identify and isolate peptides from banks of chemically or biologically produced banks of peptides. Selected peptides would then act as the pharmacore. As briefly described, it is possible to design or screen for mimetics which have enhanced activity or stability or are more readily and/or more economically obtained.
  • analogs have enhanced stability and activity or reduced unfavourable pharmacological properties. 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 a subject.
  • analogs of agonist or antagonist agents are contemplated.
  • Analogs of peptide or polypeptide agents 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.
  • Suitable non-proteogenic or non-naturally occurring amino acids may be prepared by side chain modification or by total synthesis.
  • side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 ; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6- trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH 4 .
  • the guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
  • the carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
  • Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 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.
  • Examples of non-proteogenic (non-naturally occurring or unnatural) amino acids contemplated herein are shown in Table 4.
  • Suitable ⁇ -amino acids include, but are not limited to, L- ⁇ -homoalanine, L- ⁇ - homoarginine, L- ⁇ -homoasparagine, L- ⁇ -homoaspartic acid, L- ⁇ -homoglutamic acid, L- ⁇ - homoglutamine, L- ⁇ -homoisoleucine, L- ⁇ -homoleucine, L- ⁇ -homolysine, L- ⁇ - homomethionine, L- ⁇ -homophenylalanine, L- ⁇ -homoproline, L- ⁇ -homoserine, L- ⁇ - homothreonine, L- ⁇ -homotryptophan, L- ⁇ -homotyrosine, L- ⁇ -homovaline, 3-amino- phenylpropionic acid, 3-amino-chlorophenylbutyric acid, 3-amino-fluorophenylbutyric acid, 3-amino-bromopheynyl butyric
  • Sugar amino acids are sugar moieties containing at least one amino group as well as at least one carboxyl group.
  • Sugar amino acids may be based on pyranose sugars or furanose sugars. Suitable sugar amino acids may have the amino and carboxylic acid groups attached to the same carbon atom, ⁇ -sugar amino acids, or attached to adjacent carbon atoms, ⁇ -sugar amino acids. Suitable sugar amino acids include but are not limited to
  • Sugar amino acids may be synthesized starting from commercially available monosaccharides, for example, glucose, glucosamine and galactose.
  • the amino group may be introduced as an azide, cyanide or nitromethane group with subsequent reduction.
  • the carboxylic acid group may be introduced directly as CO 2 , by Wittig reaction with subsequent oxidation or by selective oxidation of a primary alcohol.
  • peptides can be conformationally constrained by, for example, incorporation of C ⁇ and N ⁇ -methylamino acids and the introduction of double bonds between C n and C ⁇ atoms of amino acids.
  • Conformationally constrained peptides are contemplated that modulate the level or activity of a PRC polypeptide or a polypeptide product of a PRC-target gene.
  • the conformation of molecules that bind to a binding site of a PRC protein is stabilized by means of a linker covalently bound between two amino acid residues in the sequence.
  • Agents for use in the present invention such as peptides or small organic or inorganic molecules, carbohydrates, lipids or nucleic acid molecules can readily be conjugated to targeting compounds to allow direct delivery of agents to hematopoietic cells such as in the bone marrow, liver, spleen or lungs.
  • Suitable targeting agents are known to those of skill in the art and include antibodies or antigen-binding fragments thereof.
  • Antibodies and their generation and treatment are well known to those in the art. Exemplary protocols for their production are provided in Coligan et al "Current Protocols in Immunology” (John Wiley & Sons, 1991) and Ausubel et al "Current Protocols in Molecular Biology” (1994-1998).
  • Antibodies may be polyclonal or monoclonal antibodies, fragments include Fv, Fab, Fab 1 and F(ab') 2 portions of immunoglobulin molecules. Synthetic Fv fragments are conveniently employed including synthetic single chain Fv fragments prepared, for example, as described in US Patent No. 5,091,513. Other binding molecules include single variable region domains (referred to as dAbs), or minibodies comprising a single chain comprising the essential elements of a complete antibody as disclosed in US Patent No. 5,837,821.
  • the antigen binding molecule comprises multiple binding sites for one or more antigens (eg multi-scFvs).
  • the antigen binding molecule is a non-immunoglobulin derived protein framework having complementary determining regions selected for a particular antigen such as a platelet surface protein moiety.
  • the small or large chemicals, polypeptides, nucleic acids, antibodies, peptides, modified peptides, chemical analogs, or mimetics of the present invention can be formulated in pharmaceutic compositions which are prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18 th Ed. (1990, Mack Publishing, Company, Easton, PA, U.S.A.).
  • the composition may contain the active agent or pharmaceutically acceptable salts of the active agent.
  • These compositions may comprise, in addition to one of the active substances, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer or other materials well known in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the carrier may take a wide variety of forms depending on the form of preparation desired for administration, e.g. intravenous, oral, intrathecal, epineural or parenteral.
  • the present agents may be used to diagnose, treat, prevent, and/or ameliorate a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia, thrombocytopenia, leukemias, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocytic anemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma, arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis, granulomatous disease, immune deficiency, inflammatory bowel disease, sepsis, neutropenia, neutrophilia, psoriasis, immune reactions to transplanted organs and tissues, systemic lupus erythematosis, hemophilia, hypercoagulation, diabetes mellitus, endocarditis, meningitis,
  • a disease or disorder selected from the group consisting of: anemia, pancytopenia, leukopenia,
  • the agents may promote lymphopoiesis and may be useful in treating or preventing immune disorders such as infection (such as by bacteria, viruses, parasites) inflammation, allergy, autoimmunity, and immunodeficiency including humoral immunodeficiencies.
  • immune disorders such as infection (such as by bacteria, viruses, parasites) inflammation, allergy, autoimmunity, and immunodeficiency including humoral immunodeficiencies.
  • the agents may have commercial utility in the expansion of stem cells and committed progenitors of various blood lineages, and in the differentiation and/or proliferation of various cell types.
  • the subject agents may also be used to determine biological activity, to raise antibodies, as tissue markers, to isolate ligands or receptors, to identify further agents that modulate their interactions, in addition to a use as a nutritional supplement.
  • 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.
  • 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 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 active agent is preferably administered in a therapeutically effective amount.
  • the actual amount administered and the rate and time-course of administration will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc. is within the responsibility of general practitioners or specialists and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences, (supra).
  • targeting therapies may be used to deliver the active agent more specifically to tissues producing or accumulating platelets such as the bone marrow, lung, spleen, vascular system by the use of targeting systems such as antibodies or cell specific ligands or, vectors.
  • Targeting may be desirable for a variety of reasons, e.g. to avoid targeting other areas of the body, 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.
  • these agents could be produced in the target cell, 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 exhibiting a modified PRC genetic sequence may be treated with a genetic composition comprising PRC.
  • the provision of wild type or enhanced PRC function to a cell that carries a mutant or altered form of the gene should in this situation complement the deficiency.
  • the wild type 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 (In: Therapy for Genetic Disease, T. Friedman, Ed., Oxford University Press, pp. 105-121, 1991) or Culver ⁇ Gene Therapy: A Primer for Physicians, 2 nd Ed., Mary Ann Liebert, 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.
  • Non-viral gene transfer methods are known in the art such as chemical techniques including calcium phosphate co-precipitation, mechanical techniques, for example, microinjection, membrane fusion-mediated transfer via liposomes and direct DNA uptake and receptor-mediated DNA transfer.
  • Viral-mediated gene transfer can be combined with direct in vivo gene transfer using liposome delivery.
  • 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 PRC, for example, expression will produce PRC. 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 polylysine.
  • Ligands are chosen on the basis of the presence of the corresponding ligand receptors on the cell surface of the target cell/tissue type. Receptors on the surface of liver cells may be advantageously targeted.
  • 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.
  • the hematopoietic cells is a HSC.
  • the hematopoietic cells is a progenitor cell such as a myeloid or lymphoma committed progenitor cell.
  • the PRC gene is Suzl2 comprises the nucleotide sequence set forth in SEQ ID NO: 3 (human Suzl2).
  • Mutations or other modifications to the gene may cause total or partial gain or loss of PRC function.
  • modification in the gene affects transcription, translation or post-translational processing and so affects the level or activity of a PRC polypeptide.
  • mutation in a PRC gene is in the splice-effector site.
  • 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 thrombocytopenia, leukopenia or HSC defects in a subject 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 a wild-type PRC gene or locus to determine if a modified polypeptide will be produced or if it will be over-produced or under-produced.
  • the term "aberration" in the gene or locus encompasses all forms of mutations including deletions, insertions, point mutations and substitutions in the coding and non-coding regions. It also includes changes in methylation patterns of the gene. Point mutations may result in stop codons, frameshift mutations or amino acid substitutions. Somatic mutations are those which occur only in certain tissues, e.g. in the tumor tissue and are not inherited in the germline. Germline mutations can be found in any of a body's tissues and are inherited.
  • Predisposition to conditions associated with thrombocytopenia or leukopenia or HSC defects can be ascertained by testing any tissue of a human or other mammal for loss of function mutations in a PRC gene.
  • the mutation can be determined by testing DNA from any tissue of a subject's body.
  • pre-natal diagnosis can be accomplished by testing fetal cells, placental cells or amniotic fluid for mutations of a PRC 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.
  • Useful diagnostic techniques to detect aberrations in one or more PRC genes include but are not limited to fluorescent in situ hybridization (FISH), PFGE analysis, Southern blot analysis, dot blot analysis and PCR-SSCP. Also useful is DNA microchip technology. Direct DNA sequencing, either manual sequencing or automated fluorescent sequencing, can detect sequence variation. Another approach is the single-stranded conformation polymorphism assay (SSCP) (Orita et al, Proc. Nat. Acad. Sci. USA, 86:2116-2110, 1989). This method can be optimized to detect most DNA sequence variation. The increased throughput possible with SSCP makes it an attractive, viable alternative to direct sequencing for mutation detection on a research basis.
  • FISH fluorescent in situ hybridization
  • PFGE analysis Southern blot analysis
  • dot blot analysis dot blot analysis
  • PCR-SSCP PCR-SSCP.
  • DNA microchip technology DNA microchip technology.
  • Direct DNA sequencing either manual sequencing or automated fluorescent sequencing, can detect sequence variation.
  • Another approach is the single
  • CDGE clamped denaturing gel electrophoresis
  • HA heteroduplex analysis
  • CMC chemical mismatch cleavage
  • Nucleic acid sequences of a PRC gene which have been amplified by use of PCR or other amplification reactions may also be screened using allele-specific probes. These probes are nucleic acid oligomers, each of which contains a region of the gene sequence harbouring a known mutation. By use of a battery of allele-specific probes, PCR amplification products can be screened to identify the presence of a previously identified mutation in a PRC gene.
  • Hybridization of allele-specific probes with amplified PRC gene sequences can be performed, for example, on a nylon filter. Hybridization to a particular probe under stringent hybridization conditions indicates the presence of the same mutation in the tissue as in the allele-specific probe.
  • 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. It is also possible to study nucleic acid-protein interactions using these nucleic acid microchips.
  • a reporter molecule e.g. fluorescent label
  • nucleic acid-protein interactions using these nucleic acid microchips.
  • the technique is described in a range of publications including Hacia et al. (Nature Genetics, 74:441-447, 1996) and Shoemaker et al. (Nature Genetics, 74:450
  • Alteration of a wild-type PRC gene can also be detected by screening for alteration in the wild-type PRC protein.
  • monoclonal antibodies immunoreactive with a PRC polypeptide can be used to screen sample from a subject. Alteration in the level, size or lack of cognate antigen would indicate a mutation. Antibodies specific for products of mutant alleles could also be used to detect mutant gene product.
  • immunological assays can be done in any convenient format known in the art. These include Western blots, immunohistochemical assays and ELISA and RAPID 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 a PRC polypeptide) or can be done by techniques which are well known to those who are skilled in the art. (See, for example, Douillard et al, Basic Facts about Hybridomas, in Compendium of Immunology Vol. II, ed. by Schwartz, 1981;
  • the present invention provides modified animals or cells for use inter alia in the development or testing of agents as described herein.
  • the genetically modified animals such as Myb, and PRC mutants such as Suzl2 mutants described herein and cells therefrom provide a sensitized system in which to study the effects of a range of agents.
  • the specification provides a genetically modified cell or non-human animal comprising such cells wherein a Suzl2 gene or transcript is modified and the cell or animal produces a substantially reduced level or activity of Suzl2 polypeptide compared to a non-modified cell or animal of the same species.
  • the modification is in one allele of the Suzl2 gene.
  • the cell or organism is, in some embodiments a mammal, a non- human primate, live stock animal, companion animal, laboratory test animal, captive wild animal, reptile, amphibian, fish or bird.
  • the Suzl2 modification may be applied to different genetic backgrounds such as to a genome comprising a modification in the TPO or c-mpl gene.
  • the MpI negative background is particularly useful for sensitising cells to the loss of Suzl2 or other polycomb proteins.
  • Other genetic backgrounds include those of an animal model of a disease or condition.
  • agents that down regulate Suzl2 level or activity, or the activity of complexes comprising Suzl2, such as PRC2 will have efficacy in treating these diseases or conditions in humans.
  • gene or “polynucleotide” is used in its broadest sense and includes cDNA corresponding to the exons of a gene. Reference herein to a “gene” or “polynucleotide” is also taken to include :-
  • a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3'- untranslated sequences of the gene.
  • the genetically modified cell is a prokaryotic, for example a bacterial cell, or a eukaryotic cell such as a human or mammalian, insect or yeast cell.
  • the cell is a stem cell, embryonic cell, hematopoietic cell, bone marrow cell, skin cell, heart cell, bone cell, cartilage cell, liver cell, lung cell, kidney cell, spleen cell, thymus cell or brain cell.
  • the cell is a proliferating cell or a terminally differentiated cell.
  • the cell is an autologous or syngeneic cell suitable for transplantation.
  • the modification is in an exon of a Suzl2 gene, in other embodiemtns, the modification is generated by antisense, co-suppression, gene silencing, induction of RNAi or other such method known to those of skill in the art.
  • modification method is not a limitation of the invention provided that is results in an acceptable level of gene or polypeptide modification.
  • the cell or organism contemplated in accordance with this aspect of the invention is further modified with a modification in the TPO or c-mpl gene.
  • the modification comprises a MpV 1' mutation.
  • Such cells may be used in vitro or in vivo. Cells or constructs may be stored frozen and sold with instructions for use.
  • the modified animals are genetically modified, comprising mutations in one or more PRC genes.
  • RNA DNA RNA
  • Alternations may be single base changes such as a point mutation or may comprise deletion of the entire portions of the gene by techniques such as those using 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 or multiple nucleic acid insertions, deletions, substitutions or combinations thereof resulting in partial loss of function of the gene.
  • Cells and animals which carry one or more modified allele/s can be used as model systems to study the effects of the gene products and/or to test for substances which have potential as therapeutic agents when these function are impaired.
  • Animals for testing therapeutic agents can be selected after mutagenesis of whole animals or after treatment of germline cells or zygotes. After a test substance is applied to the cells, the phenotype of the cell is determined. Any trait of the cells can be assessed. In one embodiment, platlet levels are conveniently moitored.
  • 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 genetic 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.
  • a modified genetic sequence may be introduced using artificial chromosomes.
  • Targeting or other constructs 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, Molecular Cloning: A Laboratory Manual, 3 rd Edition, CSHLP, CSH, NY, 2001; Ausubel (Ed) Current Protocols in Molecular Biology, 5 th Edition, John Wiley & Sons, Inc, NY, 2002.
  • Thrombopoietin is the main cytokine responsible for the stimulation of platelet production in mouse and in humans. Tpo binds to the cell surface receptor MpI and stimulates signalling that enables the expansion of immature hematopoietic progenitors and also cells within the megakaryocyte lineage that are responsible for the generation of platelets.
  • ENU N-Ethyl-N-Nitrosourea
  • mice Male mice were then injected intraperitoneally with one dose of 200 mg/kg, 250 mg/kg, 300 mg/kg, 350 mg/kg or 400 mg/kg, two weekly doses of 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg or 200 mg/kg or three weekly doses of 66 mg/kg, 83 mg/kg, 100 mg/kg, 116 mg/kg or 133 mg/kg.
  • ENU-treated mice were mated with one or two female MpT 1' mice. Following a period of sterility, the length of which increases as the total dose of ENU increases, first-generation (Gl) progeny were produced.
  • mice were mated to untreated female MpT' ' mice to produce first generation (G 1 ) progeny, which are heterozygous for a set of ENU-induced mutations inherited from their father.
  • Gl mice were bled and their peripheral blood platelet count was determined.
  • Gl mice were examined for their platelet levels and those that exhibited platelet counts of more than 3x10 8 /ml, that is they had platelet counts of more than 3 standard deviations from the mean of untreated MpV 1' mice and were hence candidates which might carry an ENU-induced mutation that ameliorated thrombocytopenia (Figure 1). Indeed, the elevation in platelet count was found to be due to a heritable genetic change and the pedigree was designated Platelet 8 (Plt8).
  • the mutant gene responsible for the suppression of thrombocytopenia in PU8/+ mice was identified by a process of genetic mapping and sequencing.
  • Progeny tested mice with inferred genotype C57BL/6 Mpl' ' PU8/+, were crossed with 129/Sv MpT 1' +/+ mice to produce an Fi population. It was assumed that Fj mice with high platelet counts (>1.5 x 10 8 /ml) carried the PU8 mutation, these animals were intercrossed to generate F 2 mice for mapping. Peripheral blood cell analysis was performed at 7 weeks of age, mice were then sacrificed and their livers were removed for isolation of genomic DNA. DNA was isolated from 90 F 2 mice and a genome wide scan was performed with polymorphic microsatellite markers (simply sequence length polymorphisms) spread throughout the genome.
  • EXAMPLE 4 The PU8 mutation is a single base pair deletion ofSuzl2
  • cDNA from bone marrow was used as a template to amplify exon regions.
  • the coding regions of all candidate genes were sequenced from both PU8/+ and wild-type-derived DNA, except for Rhomboid veinlet-like protein 4 for which only part of the coding region was sequenced.
  • the genomic sequence derived from PH8/+ mice was identical with those from wild-type mice with the exception of Suppressor of Zeste 12 protein homolog. In this case, a single base pair deletion was identified in the splice acceptor site of the sixteenth exon of Suzl 2, leading to mis-splicing of the mRNA ( Figures 4 and 5). This mutation was not detected in stock C57BL/6 MpV 1' mice, and was absent in other inbred mouse strains including Balb/c and 129/Sv.
  • mice that carry a genetrap allele ⁇ Suzl2 502gt were analysed in mice that carry a genetrap allele ⁇ Suzl2 502gt ) to verify that the increase in platelet count amongst c-mpV 1' mice was due to impaired Suzl2 function. Similar changes were evident in the peripheral blood of Suzl2 502gl/+ mice (Table 9). Differences in the genetic background of Suzl2 5028t/+ and Suzl2 pltS/+ mice may explain the variation in the magnitude of the increase in platelet and white blood cell counts.
  • Suzl 2 protein expression is reduced in mice that carry the Plt8 mutation (Suzl2 PU8/ *) or a gene trap insertion in the Suzl 2 locus (Suzl2 502gl/+ )
  • An additional mouse model of Suzl2 deficiency was made by generating mice that carry a gene trap insertion in the Suzl2 locus.
  • An embryonic stem cell line was obtained that carries a gene trap vector insertion within the Suzl2 genomic locus.
  • a gene trap vector contains a splice acceptor site which enables the construct to be incorporated into the mature Suzl2 mRNA.
  • the gene trap sequence disrupts the Suzl2 open reading frame, resulting in premature termination of the Suzl2 protein during translation.
  • the loss of function allele is referred to as 502gt which reflects the name given to the original cell line.
  • the embryonic stem cells that carry the Suzl2 gene trap were injected into a developing blastocyst to generate a mouse.
  • mice serve as a control for a mouse that has a 50% reduction in Suzl2 protein.
  • Protein lysates were prepared from sex-matched E 12.5 embyros for analysis by western blotting.
  • Suzl2 and Ezh2 protein levels were reduced in Suzl2 plt8/+ embryos.
  • Suzl2 protein levels were equivalent in Suzl2 plt8/+ embryos and embryos heterozygous for the gene trap allele.
  • mice display enhanced CFU-spleen formation.
  • CFU-S colony- forming units spleen
  • Mature megakaryocyte numbers were determined by microscopic examination of hematoxylin and eosin-stained histological sections of sternal bone marrow and spleen. Megakaryocytes were readily recognisable by their large size and distinctive morphology. Numbers of megakaryocyte progenitor cells are also determined in clonal cultures.
  • 2.5xlO 4 bone marrow or 10 5 spleen cells are plated in 0.3% agar in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% batch-selected fetal or newborn calf serum, and stimulated with a final concentration of 100ng/ml murine SCF, 10ng/ml murine IL-3 (PeproTech, Rocky Hill, NJ) and 4 units/ml human erythropoietin (EPO, Amgen, Thousand Oaks, CA) and incubated for 7 days at 37 0 C in a fully humidified atmosphere of 5% CO2 in air.
  • Cytokines may be obtained from the commercial sources as indicated.
  • Agar cultures are fixed in 2.5% glutaraldehyde, sequentially stained for acetylcholinesterase, Luxol fast Blue and hematoxylin, and the cellular composition of each colony determined by microscopic examination at 100 to 400-fold magnification. These conditions allowed optimal stimulation of neutrophil, neutrophil-macrophage, macrophage, eosinophil, megakaryocyte, erythroid, multilineage and blast cell colony- forming cells (CFC).
  • CFC blast cell colony- forming cells
  • hematopoiesis Analysis of general effects on hematopoiesis were conducted by measurements of hematocrits as well as total peripheral blood white cell counts, the latter by performing manual counts using hemocytometer chambers and/or via automated analysis.
  • the relative numbers of morphologically recognisable precursor cells in hematopoietic organs were assessed by manual 100 to 400 cell leukocyte differential counts of peripheral blood, bone marrow, and spleen following preparation of smears or cytocentrifuge preparations stained with May-Grunwald-Giemsa.
  • the relative numbers of hematopoietic cells expressing lineage-specific cell-surface markers is measured.
  • Single cell suspensions of bone marrow, spleen, peritoneal cells and thymus from adult mice of each genotype are incubated with saturating amounts of 2.4G2 anti-Fc8 receptor antibody to reduce background staining, then with specific monoclonal antibodies to murine cell surface antigens: anti CD4 and CD8, IgM, Ly5-B220, Mac-1, F4/80, Gr-I, Ter-119, and Thyl.2 (Pharmingen, Torrey Pines, CA).
  • Other cell lineage markers are detected, including without limitation, CD34, CD19, CDlO, c-kit, Seal, GrI, Macl, CD19, CD41, CD3, CD4, CD8, CD9, CD42b and CD61.
  • Antibodies may be directly coupled to fluorescein isothiocyanate (FITC) or biotin, the latter being visualised with R-phycoerythrin- streptavidin.
  • FITC fluorescein isothiocyanate
  • biotin biotin
  • Flow cytometric analyses were performed on a FACScan analyser (Becton- Dickinson, Franklin Lakes, NJ) with dead cells and erythrocytes excluded by propidium iodide (lmg/ml) staining and gating of forward angle and side scatter of light.
  • Histological sections of all major organs are prepared by standard techniques, stained with hematoxylin and eosin and examined by light microscopy for evidence of abnormality.
  • CFU-s are enumerated by intravenous injection of bone marrow cells into recipient mice that have been irradiated with 11Gy of gamma-irradiation given in two equal doses given three hours apart from a 137Cs source (Atomic Energy, Ottawa, Canada).
  • Transplanted mice are maintained on oral antibiotic (1.1 g/L neomycin sulfate; Sigma, St. Louis, MO). Spleens are removed after 12 days, fixed in Carnoy's solution (60% ethanol, 30% chloroform, 10% acetic acid), and the numbers of macroscopic colonies were counted.
  • Agar cultures are fixed, sequentially stained for acetylcholinesterase, Luxol Fast Blue and hematoxylin, and the cellular composition of each colony determined at 100 to 400-fold magnification.
  • CFU-E and BFU-E are enumerated using methylcellulose cultures.
  • Spleen (5x10 4 ) or bone marrow (2.5x10 4 ) cells are suspended in 1.5% methylcellulose (Fluka) in IMDM supplemented with 20% fetal calf serum.
  • BFU-E are stimulated with 1 ⁇ g/ml SCF, 2.5 x 103 U/ml IL-3 and 20 U/ml EPO;
  • CFU-E are stimulated with 10 U/ml EPO.
  • Cultures are incubated at 37°C in a fully humidified atmosphere of 5% (v/v) CO 2 in air for 2 days (CFU-E) or 7 days (BFU-E). Colonies are scored as erythroid, myeloid or mixed-erythroid at 35-fold magnification and colonies appearing to contain erythroid cells are verified by staining with diaminofiuorozine.
  • single-cell suspensions of spleen and bone marrow cells are depleted of erythrocytes by lysis with 156mM ammonium chloride (pH 7.3).
  • Cells are stained with a saturating concentration of IgM-FITC and B220-PE, or Terl 19-PE and CD71-FITC (BD Pharmingen, San Diego, CA).
  • Other cell lineage markers are detected, including without limitation, CD34, CD19, CDlO, c-kit, Seal, GrI, Macl, CD19, CD41, CD3, CD4, CD8, CD9, CD42b and CD61.
  • Dead cells are excluded based on propidium iodide (PI) staining.
  • PI propidium iodide
  • the PU4 mutation in c-myb was previously shown to elevate platelet count and megakaryocyte progenitor number in c-mpV' ' mice (Carpinelli et ah, Proc. Natl. Acad. Sci. U.S.A., 707:6553-6558, 2004). A substantial increase in platelet count was evident in c-mpV 1' mice that carry both the Pit 4 and the Plt8 mutations (Table 7). The presence of the PU8 mutation resulted in an approximate two-fold increase in platelet count irrespective of the c-myb genotype (i.e. c-myb +/+ , c-myb p!l4/+ or c-myb PU4/PM ).
  • the cell line became immortalized after extended culture in thrombopoietin, a cytokine known to enhance commitment to the megakaryocyte lineage.
  • the cells have an immature appearance, however, upon re-introduction of Gata-1 GlME cells mature and form megakaryocytes and to a lesser extent mature erythroid cells. This property is pronounced of the situation in vivo, where megakaryocytes and erythroid cells share a common progenitor.
  • monoclonal antibodies directed at Suzl2 and Ezh2 are used to isolate the PRC2 complex, together with binding partners, from nuclear lysates prepared from GlME cells.
  • the bound complex is then dissociated using SDS-PAGE and stained with Coomassie-blue dye to visualize protein bands, individual bands are isolated and proteins are identified.
  • the proteins are conveniently detected by mass spectrometry.
  • target proteins are epitope tagged or expressed with binding moieties that are used to identify the protein using other antibodies determined by the epitope or binding moiety, or other binding partners.
  • Both PRC and c-myb influence lineage specification by regulating the expression of target genes within hematopoietic progenitor cells.
  • PRC2 target genes the expression of specific PRC2 components in GlME cells has been disrupted by RNA-mediated silencing (see for example Dickins et al. 2005) gene expression in these cells subsequently analysed, for example by microarray. Genes that show altered expression in cells that are deficient in Suzl2, Ezh2 or Eed are candidate target genes of PRC.
  • the megakaryocyte cell line GlME was used as a model system to study changes in gene expression that are associated with reduced PRC2 function in hematopoietic cells.
  • shRNAs short hairpin RNAs
  • a target gene Suzl2, Ezh2 or Eed
  • shRNAs short hairpin RNAs
  • Exemplary shRNA for Eed are produced using the following sequence CCGCCCGGACACGCCCACAAAT (SEQ ID NO: 15); for Ezh2 CGCTCTTACTGCTGAGCGTATA (SEQ ID NO: 14); and Suzl2 CCCAAGCACTGTGGTTGAATAA (SEQ ID NO: 13).
  • a scrambled sequence (NONS) and the empty vector (LMP) were used as controls.
  • sequences of the human genes are assessed for suitable target sites for inhibitory RNAs.
  • a wide range of web-based shRNA design programs are available to facilitate the design of suitable agents.
  • GlME cells represent a model system for the study of hematopoietic progenitor cells, it is quite possible that target genes identified in GlME cells are similarly effected in hematopoietic progenitors in Plt8/+ mice. Manipulation of PRC2 function, either through direct inhibition or by modulation of downstream targets, may be an effective way to enhance hematopoietic progenitor activity.
  • Bmil was identified as a potential target of the PRC2 complex (Table 12) and it has been suggested that forced expression of Bmil in hematopoietic progenitors enhances self-renewal activity, as a result these HSC make a greater contribution to mature blood cell lineages upon transplantation (Iwama et ah, Immunity, 27:843-851, 2004). Accordingly, upregulation of Bmil is excluded from the present invention as a means for enhancing self-renewal activity and enhancing transplantation efficiency.
  • Suzl2 deficiency enhances progenitor activity in competitive transplantation assays
  • irradiated C57BL6/Ly5.1 + mice were intravenously injected with approximately 1x10 bone marrow cells from Suzl ⁇ t8/+ mice or compound mutants described herein.
  • 1x10 6 bone marrow cells from C57BL6/Ly5.1 + mice were injected into irradiated Suzl2 Pll8/+ or compound mutant mice. All recipient mice were maintained on oral antibiotics and analysed at least 16 weeks after transplantation by flow cytometry and automated blood cell analysis. Other transplant experiments are carried out to determine the in vivo effects of modified cells.
  • mice (Ly 5.1 + ) are transplanted with test donor cells, either wild type or mutant (both Ly5.2 + ) or with wild type competitor cells (Ly5.1 + ).
  • the competitor marrow shares the same MpI genotypes as the test marrow.
  • competitive transplantation studies were performed to test the ability of Suzl2 p " 8/+ stem cells to repopulate the hematopoietic compartment of lethally irradiated recipients. Irradiated recipient mice (Ly 5.1 + ) were transplanted with an equal number of bone marrow cells from a test marrow (LyS ⁇ + ) and competitor marrow (Ly5.1 + ).
  • shRNA Gene silencing constructs
  • Bone marrow extracted from 5-FU treated mice was infected with either the LMS-Nons or the LMS-Suzl2 virus and transplanted into recipient mice. Thymocytes were isolated 12 weeks after transplantation and fractionated based upon expression of GFP (+ or -); low or intermediate populations were detected in some mice (low). Protein lysates were prepared from sorted cells and Western blotting was performed to detect expression of Suzl2, Ezh2 or histone H3. The results (see Figure 13 and Brief Description of the Figures) show that Suzl2 and Ezh2 protein levels are reduced in cells that are infected with the LMS-Suzl2 virus.
  • Hematopoietic tissue infected with Suzl2-shRNA contributes more to haematopoiesis than cells infected with a control virus
  • Bone marrow extracted from 5-FU treated mice was infected with either the LMS-Nons or the LMS-Suzl2 virus and transplanted into recipient mice. Three independent infections were performed and in each case infected cells were transplanted into five recipient animals. A selection of primary recipients (9-11) were used as donors for secondary transplants, in each case these cells were transplanted into 3-5 recipient mice. The frequency of cells that carried the virus (GFP+) was monitored prior to transplantation (Input) and at 8-12 weeks after transplantation in primary or secondary recipients. To determine the ability of infected cells to contribute to hematopoiesis, the representation of GFP+ cells was compared between donor and recipient populations and a ratio calculated (Recipient GFP%/Donor GFP%).
  • the Suzl2 gene is haploinsufficient, such that two functional copies of the Suzl2 gene are required for production of the appropriate level of Suzl2 protein to maintain PRC2 function.
  • Plt8 mice have a reduced amount of Suzl2 protein which results in functional impairment of the PRC2 complex.
  • Many PRC components are over-expressed in human cancers, particularly in aggressive metastatic disease (Sparmann et al., Nat. Rev. Cancer, 5:846-856, 2006). This finding has lead to the hypothesis that inhibitors of the PRC2 complex may be useful in treating cancer.
  • mice that carry a mutation in Plt8 will allow researchers to determine the effect of reduced PRC2 function in different animal disease models. If disease symptoms are reduced in the presence of the Plt8 mutation then it is likely that inhibition of the PRC2 complex would have efficacy in treating disease in humans.
  • MpI 1' mice provide a method for identifying epigenetic modulators and/or modulators of Suzl2 or a PcG in genetic or proteinaceous form.
  • Non-conventional Code Non-conventional Code amino acid amino acid

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Abstract

L'invention concerne des procédés de modulation du nombre et/ou de l'activité des cellules hématopoïétiques, notamment de l'activité fonctionnelle des cellules-souches hématopoïétiques. L'invention concerne également des procédés de criblage de modulateurs épigénétiques et d'autres agents modulateurs du niveau et/ou de l'activité de protéines Polycomb telles que Suz12. Par ailleurs, des animaux non humains génétiquement modifiés et des cellules génétiquement modifiées déficientes en Suz12 sont également envisagés, ainsi que leur utilisation, et l'utilisation de modèles animaux à déficience hématopoïétique, et notamment plaquettaire, pour développer des agents modificateurs épigénétiques.
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US9920317B2 (en) 2010-11-12 2018-03-20 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10053694B2 (en) 2010-11-12 2018-08-21 The General Hospital Corporation Polycomb-associated non-coding RNAS
US10358644B2 (en) 2010-11-12 2019-07-23 The General Hospital Corporation Polycomb-associated non-coding RNAs
US10174315B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating hemoglobin gene family expression
US10058623B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating UTRN expression
WO2013173637A1 (fr) * 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions et méthodes pour moduler l'expression génique
US10174323B2 (en) 2012-05-16 2019-01-08 The General Hospital Corporation Compositions and methods for modulating ATP2A2 expression
US10059941B2 (en) 2012-05-16 2018-08-28 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
US10655128B2 (en) 2012-05-16 2020-05-19 Translate Bio Ma, Inc. Compositions and methods for modulating MECP2 expression
US10837014B2 (en) 2012-05-16 2020-11-17 Translate Bio Ma, Inc. Compositions and methods for modulating SMN gene family expression
WO2013173635A1 (fr) * 2012-05-16 2013-11-21 Rana Therapeutics, Inc. Compositions et méthodes pour moduler l'expression génique
US11788089B2 (en) 2012-05-16 2023-10-17 The General Hospital Corporation Compositions and methods for modulating MECP2 expression
US10858650B2 (en) 2014-10-30 2020-12-08 The General Hospital Corporation Methods for modulating ATRX-dependent gene repression
US10900036B2 (en) 2015-03-17 2021-01-26 The General Hospital Corporation RNA interactome of polycomb repressive complex 1 (PRC1)
JP2019518801A (ja) * 2016-06-30 2019-07-04 中国科学院上海薬物研究所Ｓｈａｎｇｈａｉ Ｉｎｓｔｉｔｕｔｅ Ｏｆ Ｍａｔｅｒｉａ Ｍｅｄｉｃａ， Ｃｈｉｎｅｓｅ Ａｃａｄｅｍｙ Ｏｆ Ｓｃｉｅｎｃｅｓ 乾癬及び尋常性白斑の治療薬の製造のための、メチル４−［９−（６−アミノプリニル）］−２（ｓ）−ヒドロキシブチラートの使用。
US20230027247A1 (en) * 2019-12-16 2023-01-26 Edigene (Guangzhou) Inc. Small molecule compounds for amplifying hematopoietic stem cells, and combination thereof

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