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Methods and compositions for silencing genes without inducing toxicityRelated Patent Categories: Drug, Bio-affecting And Body Treating Compositions, Designated Organic Active Ingredient Containing (doai), O-glycoside, , Nitrogen Containing Hetero Ring, Polynucleotide (e.g., Rna, Dna, Etc.)Methods and compositions for silencing genes without inducing toxicity description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20070027103, Methods and compositions for silencing genes without inducing toxicity. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a Continuation of, and claims priority from, U.S. patent application Ser. No. 10/425,006, filed Apr. 28, 2003. This application claims the benefit of the filing date of U.S. provisional patent application 60/375,636, filed Apr. 26, 2002. Both applications are incorporated herein by reference. BACKGROUND OF THE INVENTION [0002] In general, the invention relates to novel methods for silencing genes. Desirably, these methods specifically inhibit the expression of one or more target genes in a cell or animal (e.g., a mammal such as a human) without inducing toxicity. [0003] Double stranded RNA (dsRNA) has been shown to induce gene silencing in a number of different organisms. Gene silencing can occur through various mechanisms, one of which is post-transcriptional gene silencing (PTGS). In post-transcriptional gene silencing, transcription of the target locus is not affected, but the RNA half-life is decreased. Transcriptional gene silencing (TGS) is another mechanism by which gene expression can be regulated. In TGS, transcription of a gene is inhibited. Exogenous dsRNA has been shown to act as a potent inducer of PTGS in nematodes, trypanosomes, and insects. Double stranded RNA is also an inducer of TGS. Some current methods for using dsRNA in vertebrate cells to silence genes result in undesirable non-specific cytotoxicity or cell death due to the interferon response that is induced by dsRNA in vertebrate cells. Some methods also result in non-specific or inefficient silencing. [0004] Thus, improved methods are needed for specifically silencing target genes without inducing toxicity or cell death. Desirably, these methods may be used to inhibit gene expression in in vitro samples, cell culture, and intact animals (e.g., vertebrates such as mammals). SUMMARY OF THE INVENTION [0005] In general, the invention features novel methods for silencing genes that produce few, if any, toxic side-effects. In particular, these methods involve administerating to a cell or animal one or more double stranded RNA (dsRNA) molecules that have substantial sequence identity to a region of a target nucleic acid and that specifically inhibit the expression of the target nucleic acid. One or more short dsRNA molecules, which differ from the dsRNA having substantial identity to the target nucleic acid, are also administered to inhibit possible toxic effects or non-specific gene silencing that may otherwise be induced by the former dsRNA. [0006] Accordingly, in a first aspect, the invention features a method for inhibiting the expression of a target nucleic acid in a cell (e.g., an invertebrate cell, a vertebrate cell such as a mammalian or human cell, or a pathogen cell). This method involves introducing into the cell a first agent that provides to the cell a first dsRNA and introducing a second agent that provides to the cell a short, second dsRNA. The first dsRNA has substantial sequence identity to a region of the target nucleic acid and specifically inhibits the expression of the target nucleic acid. The short, second dsRNA differs from the first dsRNA and inhibits the interferon response or dsRNA-mediated toxicity. In some embodiments, the short, second dsRNA binds PKR and inhibits the dimerization and activation of PKR. Exemplary pathogens include bacteria and yeast. In some embodiments, the first dsRNA inhibits the expression of an endogenous nucleic acid in a vertebrate cell or a pathogen cell (e.g., a bacterial or yeast cell) or inhibits the expression of a pathogen nucleic acid in a cell infected with the pathogen. [0007] In another aspect, the invention provides a method for inhibiting the expression of a target nucleic acid in an animal (e.g., an invertebrate or a vertebrate such as a mammal or human). This method involves introducing into the animal a first agent that provides to the animal a first dsRNA and introducing a second agent that provides to the animal a short, second dsRNA. The first dsRNA has substantial sequence identity to a region of the target nucleic acid and specifically inhibits the expression of the target nucleic acid. The short, second dsRNA differs from the first dsRNA and inhibits the interferon response or dsRNA-mediated toxicity. In some embodiments, the short, second dsRNA binds PKR and inhibits the dimerization and activation of PKR. In some embodiments, the first dsRNA inhibits the expression of an endogenous nucleic acid in an animal or inhibits the expression of a pathogen nucleic acid in an animal infected with a pathogen (e.g., a bacterial or yeast cell or a virus). [0008] In yet another aspect, the invention provides a method for treating, stabilizing, or preventing a disease or disorder in an animal (e.g., an invertebrate, a vertebrate such as a mammal or human). This method involves introducing into the animal a first agent that provides to the animal a first dsRNA and a second agent that provides to the animal a short, second dsRNA. The first dsRNA has substantial sequence identity to a region of a target nucleic acid associated with the disease or disorder and specifically inhibits the expression of the target nucleic acid. The short, second dsRNA differs from the first dsRNA and inhibits the interferon response or dsRNA-mediated toxicity. In some embodiments, the short, second dsRNA binds PKR and inhibits the dimerization and activation of PKR. In some embodiments, the target gene is a gene associated with cancer, such as an oncogene, or a gene encoding a protein associated with a disease, such as a mutant protein, a dominant negative protein, or an overexpressed protein. [0009] Exemplary cancers that can be treated, stabilized, or prevented using the above methods include prostate cancers, breast cancers, ovarian cancers, pancreatic cancers, gastric cancers, bladder cancers, salivary gland carcinomas, gastrointestinal cancers, lung cancers, colon cancers, melanomas, brain tumors, leukemias, lymphomas, and carcinomas. Benign tumors may also be treated or prevented using the methods of the present invention. Other cancers and cancer related genes that may be targeted are disclosed in, for example, WO 00/63364, WO 00/44914, and WO 99/32619. [0010] Exemplary endogenous proteins that may be associated with disease include ANA (anti-nuclear antibody) found in SLE (systemic lupus erythematosis), abnormal immunoglobulins including IgG and IgA, Bence Jones protein associated with various multiple myelomas, and abnormal amyloid proteins in various amyloidoses including hereditary amyloidosis and Alzheimer's disease. In Huntington's Disease, a genetic abnormality in the HD (huntingtin) gene results in an expanded tract of repeated glutamine residues. In addition to this mutant gene, HD patients have a copy of chromosome 4 which has a normal sized CAG repeat. Thus, methods of the invention can be used to silence the abnormal gene but not the normal gene. In various embodiments, a nucleic acid encoding a disease-causing protein is silenced using long sRNA, and short dsRNA is used to block the dsRNA stress response that might otherwise be associated with administration of the long dsRNA. [0011] In still another aspect, the invention features a method for treating, stabilizing, or preventing an infection in an animal (e.g., an invertebrate or a vertebrate such as a mammal or human). This method involves introducing into the animal a first agent that provides to the animal a first dsRNA and introducing a second agent that provides to the animal a short, second dsRNA. The first dsRNA has substantial sequence identity to a region of a target nucleic acid in an infectious pathogen (e.g., a virus, bacteria, or yeast) or cell infected with a pathogen and specifically inhibits the expression of the target nucleic acid. The short, second dsRNA differs from the first dsRNA and inhibits the interferon response or dsRNA-mediated toxicity. In some embodiments, the short, second dsRNA binds PKR and inhibits the dimerization and activation of PKR. In various embodiments, the pathogen is an intracellular or extracellular pathogen. In some embodiments, the target nucleic acid is a gene of the pathogen that is necessary for replication and/or pathogenesis. [0012] In a further embodiment of any of the above aspects, the methods of administering a dsRNA or a nucleic acid encoding a dsRNA includes contacting an in-dwelling device with the cell prior to, concurrent with, or following the administration of the in-dwelling device to a patient. In-dwelling devices include, but are not limited to, surgical implants, prosthetic devices, and catheters, i.e., devices that are introduced to the body of an individual and remain in position for an extended time. Such devices include, for example, artificial joints, heart valves, pacemakers, vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinary catheters, and continuous ambulatory peritoneal dialysis (CAPD) catheters. Desirably, the dsRNA prevents the growth of bacteria on the device. In some embodiments, the first dsRNA inhibits the expression of a bacterial nucleic acid in a bacterial cell, a cell infected with a bacteria, or an animal infected with a bacteria. [0013] In other desirable embodiments, the bacterial infection is due to one or more of the following bacteria: Chlamydophila pneumoniae, C. psittaci, C. abortus, Chlamydia trachomatis, Simkania negevensis, Parachlamydia acanthamoebae, Pseudomonas aeruginosa, P. alcaligenes, P. chlororaphis, P. fluorescens, P. luteola, P. mendocina, P. monteilii, P. oryzihabitans, P. pertocinogena, P. pseudalcaligenes, P. putida, P. stutzeri, Burkholderia cepacia, Aeromonas hydrophilia, Escherichia coli, Citrobacter freundii, Salmonella typhimurium, S. typhi, S. paratyphi, S. enteritidis, Shigella dysenteriae, S. flexneri, S. sonnei, Enterobacter cloacae, E. aerogenes, Klebsiella pneumoniae, K. oxytoca, Serratia marcescens, Francisella tularensis, Morganella morganii, Proteus mirabilis, Proteus vulgaris, Providencia alcalifaciens, P. rettgeri, P. stuartii, Acinetobacter calcoaceticus, A. haemolyticus, Yersinia enterocolitica, Y. pestis, Y. pseudotuberculosis, Y. intermedia, Bordetella pertussis, B. parapertussis, B. bronchiseptica, Haemophilus influenzae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, H. ducreyi, Pasteurella multocida, P. haemolytica, Branhamella catarrhalis, Helicobacter pylori, Campylobacter fetus, C. jejuni, C. coli, Borrelia burgdorferi, V. cholerae, V. parahaemolyticus, Legionella pneumophila, Listeria monocytogenes, Neisseria gonorrhea, N. meningitidis, Kingella dentrificans, K. kingae, K. oralis, Moraxella catarrhalis, M. atlantae, M. lacunata, M. nonliquefaciens, M. osloensis, M. phenylpyruvica, Gardnerella vaginalis, Bacteroides fragilis, Bacteroides distasonis, Bacteroides 3452A homology group, Bacteroides vulgatus, B. ovalus, B. thetaiotaomicron, B. uniformis, B. eggerthii, B. splanchnicus, Clostridium difficile, Mycobacterium tuberculosis, M. avium, M. intracellulare, M. leprae, C. diphtheriae, C. ulcerans, C. accolens, C. afermentans, C. amycolatum, C. argentorense, C. auris, C. bovis, C. confusum, C. coyleae, C. durum, C. falsenii, C. glucuronolyticum, C. imitans, C. jeikeium, C. kutscheri, C. kroppenstedtii, C. lipophilum, C. macginleyi, C. matruchoti, C. mucifaciens, C. pilosum, C. propinquum, C. renale, C. riegelii, C. sanguinis, C. singulare, C. striatum, C. sundsvallense, C. thomssenii, C. urealyticum, C. xerosis, Streptococcus pneumoniae, S. agalactiae, S. pyogenes, Enterococcus avium, E. casseliflavus, E. cecorum, E. dispar, E. durans, E. faecalis, E. faecium, E. flavescens, E. gallinarum, E. hirae, E. malodoratus, E. mundtii, E. pseudoavium, E. raffinosus, E. solitarius, Staphylococcus aureus, S. epidermidis, S. saprophyticus, S. intermedius, S. hyicus, S. haemolyticus, S. hominis, and/or S. saccharolyticus. Preferably, a dsRNA is administered in an amount sufficient to prevent, stabilize, or inhibit the growth of a pathogen or to kill the pathogen. In some embodiments, the first dsRNA inhibits the expression of a yeast nucleic acid in a yeast cell, a cell infected with yeast, or an animal infected with yeast. [0014] In desirable embodiments, the viral infection relevant to the methods of the invention is an infection by one or more of the following viruses: Hepatitis B, Hepatitis C, piconarirus, polio, HIV, coxsacchie, herpes simplex virus Type I and 2, St. Louis encephalitis, Epstein-Barr, myxoviruses, JC, coxsakieviruses B, togaviruses, measles, paramyxoviruses, echoviruses, bunyaviruses, cytomegaloviruses, varicella-zoster, mumps, equine encephalitis, lymphocytic choriomeningitis, rhabodoviruses including rabies, simian virus 40, human polyoma virus, parvoviruses, papilloma viruses, primate adenoviruses, coronaviruses, retroviruses, Dengue, yellow fever, Japanese encephalitis virus and/or BK. In some embodiments, the first dsRNA inhibits the expression of a viral nucleic acid in a virus, a cell infected with a virus, or an animal infected with a virus. [0015] In another aspect, the invention features method for reducing or preventing an immune response to a transplant cell, tissue, or organ. The method involves administering to the transplant cell, tissue, or organ a first agent that provides a first dsRNA and a second agent that provides short, second dsRNA. The first dsRNA attenuates the expression of a target nucleic acid in the transplant cell, tissue, or organ that can elicit an immune response in a recipient. The short, second dsRNA differs from the first dsRNA and inhibits the interferon response or dsRNA-mediated toxicity. In some embodiments, the short, second dsRNA binds PKR and inhibits the dimerization and activation of PKR. In some embodiments, an agent that provides a dsRNA molecule is also administered to the recipient to inhibit the expression of an endogenous nucleic acid that would otherwise participate in an adverse immune response to the transplant. [0016] In desirable embodiments of any of the above aspects, the first dsRNA inhibits expression of the target nucleic acid by at least 20, 40, 60, 80, 90, 95, or 100%. In some embodiments, multiple first dsRNA molecules that are substantially identical to different nucleic acids are administered to the cell or animal to inhibit the expression of multiple target nucleic acids. For example, multiple oncogenes or multiple pathogen genes may be simultaneously silenced. [0017] In various embodiments of any of the above aspects, the first agent and/or the second agent is a DNA molecule or DNA vector encoding a dsRNA. In other embodiments, the first agent and/or the second agent is a dsRNA, a single stranded RNA molecule that assumes a double stranded conformation inside the cell or animal (e.g., a hairpin), or a combination of two single stranded RNA molecules that are administered simultaneously or sequentially and that assume a double stranded conformation inside the cell or animal. The first agent may be administered before, during, or after the administration of the second agent. In some embodiments, the first and second agents are the same nucleic acid or the same vector that encodes both dsRNA molecules. In various embodiments, the first agent provides a short dsRNA or a long dsRNA to the cell or animal. [0018] In some embodiments, a cytokine is also administered to the cell or animal. Exemplary cytokines are disclosed in WO 00/63364, filed Apr. 19, 2000. In some embodiments, the expression of the target nucleic acid is increased to promote the amplification of the dsRNA, resulting in more dsRNA to silence the target gene. For example, a vector containing the target nucleic acid can be administered to the cell or animal before, during, or after the administration of the first and/or second agent. [0019] The invention also features high throughput methods of using dsRNA-mediated gene silencing to identify a nucleic acid that confers or modulates a detectable phenotype. A detectable phenotype may include, for example, any outward physical manifestation, such as molecules, macromolecules, structures, metabolism, energy utilization, tissues, organs, reflexes, and behaviors, as well as anything that is part of the detectable structure, function, or behavior of a cell, tissue, or living organism. Particularly useful in the methods of the invention are dsRNA mediated changes, wherein the detectable phenotype derives from modulation of the function of a cell, modulation of expression of a target nucleic acid, or modulation of the biological activity of a target polypeptide through dsRNA effects on a target nucleic acid. For example, see the dsRNA mediated methods of determining gene function in EP 1229134 A2 and WO 00/01846, the teachings of which are hereby incorporated by reference. The method involves the use of specially constructed cDNA libraries derived from a cell, for example, a primary cell or a cell line that has an observable phenotype or biological activity, (e.g., an activity mediated by a target polypeptide or altered gene expression), that are transfected into cells to inhibit gene expression. In addition, a short dsRNA or a nucleic acid (e.g., a vector) encoding a short dsRNA is administered to the cell to inhibit potential dsRNA mediated toxicity, including adverse effects due to the possible induction of the interferon response by the dsRNA expression library. The inhibition of gene expression by the present methods alters a detectable phenotype, e.g., the function of a cell, gene expression of a target nucleic acid, or the biological activity of a target polypeptide and allows the nucleic acid responsible for the modulation of the detectable phenotype to be readily identified. While less desirable, the method may also utilize randomized nucleic acid sequences or a given sequence for which the function is not known, as described, e.g., in U.S. Pat. No. 5,639,595, the teaching of which is hereby incorporated by reference. [0020] Accordingly, in one aspect, the invention features a method for identifying a nucleic acid sequence that modulates the function of a cell. The method involves (a) transforming a population of cells with a dsRNA expression library, where at least two cells of the population of cells are each transformed with a different nucleic acid from the dsRNA expression library, and where at least one encoded dsRNA specifically inhibits the expression of a target nucleic acid in at least one cell (b) transforming the cells with a short dsRNA or a nucleic acid encoding a short dsRNA; (c) optionally selecting for a cell in which the nucleic acid is expressed in the cell; and (d) assaying for a modulation in the function of the cell, wherein a modulation identifies a nucleic acid sequence that modulates the function of a cell. The short dsRNA differs from at least one or all of the dsRNA molecules produced by the expression library that specifically inhibit the expression of a target nucleic acid in a cell or differs from all of the dsRNA molecules produced by the expression library. The short dsRNA inhibits the interferon response or dsRNA-mediated toxicity. In some embodiments, the short, dsRNA binds PKR and inhibits the dimerization and activation of PKR. [0021] In a desirable embodiment of the above aspect of the invention, assaying for a modulation in the function of a cell comprises measuring cell motility, apoptosis, cell growth, cell invasion, vascularization, cell cycle events, cell differentiation, cell dedifferentiation, neuronal cell regeneration, or the ability of a cell to support viral replication. Continue reading about Methods and compositions for silencing genes without inducing toxicity... 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