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  Control of gene expressionUSPTO Application #: 20080026985Title: Control of gene expression Abstract: A method of suppressing the expression of a selected gene in a eukaryotic cell the method comprising introducing into the cell (a) a polypeptide comprising a DNA binding portion which binds to a site at or associated with the selected gene which site is present in a plant or animal genome and a chromatin inactivation portion, or (b) a polynucleotide encoding said polypeptide. (end of abstract) Agent: Nikolai & Mersereau, P.A. - Minneapolis, MN, US Inventors: Lakjaya Buluwela, Simak Ali USPTO Applicaton #: 20080026985 - Class: 514002000 (USPTO) Related Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), Peptide Containing (e.g., Protein, Peptones, Fibrinogen, Etc.) Doai The Patent Description & Claims data below is from USPTO Patent Application 20080026985. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application is a continuation-in-part of application Ser. No. 10/019,520, the entirety of which is deemed incorporated herein by reference, filed Jun. 10, 2002, entitled "CONTROL OF GENE EXPRESSION" and which, in turn, is a continuation of co-pending PCT International Application No. PCT/GB00/02497, having an International filing date of Jun. 26, 2000 and having a priority date of Jun. 30, 1999 and entitled "CONTROL OF GENE EXPRESSION". BACKGROUND OF THE INVENTION [0002] I. Field of the Invention [0003] The present invention relates to the control of gene expression and, in particular, it relates to methods of, and means for, suppressing the expression of a particular, selected gene. [0004] II. Related Art [0005] The ability to selectively suppress the expression of a gene is useful in many areas of biology, for example in methods of treatment where the expression of the gene may be undesirable; in preparing models of disease where lack of expression of a particular gene is associated with the disease; in modifying the phenotype in order to produce desirable properties. Thus, the ability to selectively suppress the expression of a gene may allow the "knockout" of human genes in human cells (whether wild type or mutant) and the knockout of eukaryotic genes in studies of development and differentiation. [0006] Present methods of attempting to suppress the expression of a particular gene fall into three main categories, namely antisense technology, ribozyme technology and targeted gene deletion brought about by homologous recombination. [0007] Antisense techniques rely on the introduction of a nucleic acid molecule into a cell which typically is complementary to a mRNA expressed by the selected gene. The antisense molecule typically suppresses translation of the mRNA molecule and prevents the expression of the polypeptide encoded by the gene. Modifications of the antisense technique may prevent the transcription of the selected gene by the antisense molecule binding to the gene's DNA to form a triple helix. [0008] Ribozyme techniques rely on the introduction of a nucleic acid molecule into a cell which expresses a RNA molecule which binds to, and catalyses the selective cleavage of, a target RNA molecule. The target RNA molecule is typically a mRNA molecule, but it may be, for example, a retroviral RNA molecule. [0009] Antisense- and ribozyme-based techniques have proven difficult to implement and they show varying degrees of success in target gene suppression or inactivation. Furthermore, these two techniques require persistent expression or administration of the gene-inactivating agent. [0010] Targeted gene deletion by homologous recombination requires two gene-inactivating events (one for each allele) and is not easily applicable to primary cells, particularly for example primary human mammary cells which can only be maintained in culture for a few passages. Targeted gene deletion has remained difficult to perform in plants. The cre-lox mediated site-specific integration has been the method of choice although the efficiency of specific integrative events is low (Alberts et al (1995) Plant J. 7, 649-659; Vergunst & Hooykass (1998) Plant Mol. Biol. 38, 393-406; Vergunst et al (1998) Nucl. Acids Res. 26, 2729-2734). [0011] These major shortcomings in existing technology have led us to seek an alternative strategy. [0012] Acute promyelocytic leukaemia (APL) is underlined by the involvement of mutant retinoic acid receptor (RAR) proteins, formed by gene fusions brought about by chromosomal translocations. Molecular analysis of one APL subset has identified a fusion between the RAR.alpha. gene and a Kruppel-like zinc finger gene named promyelocytic leukaemia zinc finger (PLZF). Further investigations have shown that the resulting PLZF-RAR.alpha. fusion protein functions as a gene repressor by targeting histone deacetylation of retinoic acid regulated genes. Several studies have shown that this repression is mediated by the PLZF portion of the fusion protein, which interacts with a complex of proteins which includes the components N-CoR, SMRT, Sin3 and HDAC and which in turn results in the recruitment of the histone deacetylase (HDAC) complex to target genes (see, for example, Grignani et al (1998) Nature 391, 815-818; Chen et al (1993) EMBO J. 12, 1161-1167; Razin (1998) EMBO J. 17, 4905-4908; David et al (1998) Oncogene 16, 2549-2556; and Lin et al (1998) Nature 391, 811-814). HDAC directed gene inactivation, therefore results from the targeted assembly of components, some of which have been identified (eg N-CoR, SMRT, Sin3 etc) making a gene inactivating complex which mediates its effect through histone deacetylation. [0013] Although this work shows that in certain forms of APL fusion proteins are able to recruit histone deacetylase activity which appears to have the effect of inactivating the expression of certain genes, no-one has suggested that a method can be devised based on recruitment of histone deacetylase or other means of inactivating chromatin in order to selectively suppress expression of a chosen target gene or a set of genes. Surprisingly, we have shown that this can be achieved. [0014] RAR.alpha.-PLZF and RAR.alpha.-PML fusion proteins are known from studies of acute promyelocytic leukaemia (APL) and are described in, for example, Grignani et al (1998) Nature 391, 815-818. [0015] Fusions of GAL4 with a portion of PLZF protein, and LexA DNA binding domain fused to various fragment of Sin 3A are described in David et al (1998) Oncogene 16, 2549-2556 which, for the avoidance of doubt, are excluded from the polypeptide of the present invention. [0016] Fusions of the GAL4 DNA binding domain and PLZF-RAR.alpha. are described in Lin et al (1998) Nature 391, 811-814 which, for the avoidance of doubt, are excluded from the polypeptide of the present invention. [0017] Fusions of the GAL4 DNA binding domain with N-CoR or portions thereof, or with the C terminal domain of the T.sub.3R.beta.1 receptor molecule (thyroid hormone receptor molecule), and LexA DNA binding domain fused with the C terminal domain of the T.sub.3R.alpha. or RAR.alpha. (retinoic acid receptor) receptor molecules, which, for the avoidance of doubt, are excluded from the polypeptide of the present invention, are described in Horlein et al (1995) Nature 377, 397-404. Fusions of the GAL4 DNA binding domain with the C terminal domain of vErbA (viral oncogene erbA of the avian erythroblastosis virus (AEV)), T.sub.3R and RAR receptor molecules are also mentioned. These polypeptides are also, for the avoidance of doubt, excluded from the polypeptide of the present invention. [0018] There is no suggestion in David et al (1998) Oncogene 16, 2549-2556, Lin et al (1998) Nature 391, 811-814 or Horlein et al (1995) Nature 377, 397-404 that polypeptides comprising a nucleic acid binding portion and a chromatin inactivation portion can be designed and engineered to bring about the selective suppression of a chosen gene. Rather, David et al (1998) and Lin et al (1998) are both studies of gene repression in acute promyelocytic leukaemia, and Horlein et al (1995) relates to the identification of N-CoR. SUMMARY OF THE INVENTION [0019] A first aspect of the invention provides a polypeptide comprising a nucleic acid binding portion which binds to a site present in a eukaryotic genome and a chromatin inactivation portion provided that when the nucleic acid binding portion is a DNA binding portion of RAR.alpha. the chromatin inactivation portion is not a portion of PLZF protein and is not a portion of PML protein; and provided that when the nucleic acid binding portion is a DNA binding portion of the Saccharomyces cerevisiae GAL4 protein the chromatin inactivation portion is not a portion of PLZF protein, the C-terminal domain of vErbA, T.sub.3R, T.sub.3.beta.1 or RAR, or N-CoR or a portion of N-CoR; and provided that when the nucleic acid binding portion is a DNA binding portion of the Escherichia coli LexA the chromatin inactivation portion is not mSin3, or the C-terminal domain of T.sub.3R.alpha. or RAR.alpha.. [0020] The polypeptides of the invention may be useful in methods and uses provided by further aspects of the invention, discussed in more detail below. In particular, the polypeptides of the invention may be useful in a method of suppressing the expression of a selected gene in a eukaryotic cell the method comprising introducing into the cell (a) a polypeptide comprising a nucleic acid binding portion which binds to a site at or associated with the selected gene which site is present in a eukaryotic genome and a chromatin inactivation portion, or (b) a polynucleotide encoding said polypeptide. [0021] It is preferred if the polypeptides of the invention are hybrid polypeptides which do not occur in nature. For example, it is preferred if the nucleic acid binding portion is derived from one protein and that the chromatin inactivation portion is derived from a different protein and that the molecular configuration does not arise in nature, for example through chromosome translocation events. The proteins from which the nucleic acid binding portion and the chromatin inactivation portion are derived may be from the same species (for example, as is described in more detail below, the nucleic acid binding portion may be a DNA binding portion of a human steroid receptor protein such as oestrogen receptor (ER) and the chromatin inactivation portion may be a portion of human PLZF) or they may be from different species (for example a bacterial DNA binding protein may be fused to a portion of human PLZF). [0022] Thus, in a particular preferred embodiment the polypeptide of the invention is one which is produced by genetic engineering means wherein the nucleic acid binding portion and the chromatin inactivation portion are selected as is described in more detail below. Continue reading... Full patent description for Control of gene expression Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Control of gene expression patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. 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