Methods and compositions for silencing genes without inducing toxicity

Abstract

The present invention provides methods of post-transcriptional gene silencing which involve the use of a first dsRNA having substantial sequence identity to a target nucleic acid and a short, second dsRNA which inhibits dsRNA-mediated toxicity. These methods can be used to prevent or treat a disease or infection by silencing a gene associated with the disease or infection. The invention also provides methods for identifying nucleic acid sequences that modulate a detectable phenotype, including the function of a cell, the expression of a gene, or the biological activity of a target polypeptide.

1 . A method for inhibiting the expression of a target nucleic acid in a cell, said method comprising introducing into said cell a first agent that provides to said cell a first double stranded RNA (dsRNA) and a second agent that provides to said cell a short, second dsRNA, wherein said first dsRNA has substantial sequence identity to a region of said target nucleic acid and specifically inhibits said expression of said target nucleic acid, and wherein said short, second dsRNA inhibits dsRNA-mediated toxicity.  
     
     
         2 . The method of  claim 1 , wherein the double stranded region in said second dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         3 . The method of  claim 1 , wherein the double stranded region in said first dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         4 . The method of  claim 1 , wherein the double stranded region in said first dsRNA contains over 30 nucleotides.  
     
     
         5 . The method of  claim 4 , wherein the double stranded region in said first dsRNA contains over 200 nucleotides.  
     
     
         6 . The method of  claim 1 , wherein said first and/or second agent is a nucleic that encodes a dsRNA.  
     
     
         7 . The method of  claim 1 , wherein said cell is a vertebrate cell.  
     
     
         8 . The method of  claim 1 , wherein said cell is a mammalian cell.  
     
     
         9 . The method of  claim 8 , wherein said cell is a human cell.  
     
     
         10 . A method for inhibiting the expression of a target nucleic acid in an animal, said method comprising introducing into said animal a first agent that provides to said animal a first dsRNA and a second agent that provides to said animal a short, second dsRNA, wherein said first dsRNA has substantial sequence identity to region of said target nucleic acid and specifically inhibits said expression of said target nucleic acid, and wherein said short, second dsRNA inhibits dsRNA-mediated toxicity.  
     
     
         11 . The method of  claim 10 , wherein the double stranded region in said second dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         12 . The method of  claim 10 , wherein the double stranded region in said first dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         13 . The method of  claim 10 , wherein the double stranded region in said first dsRNA contains over 30 nucleotides.  
     
     
         14 . The method of  claim 13 , wherein the double stranded region in said first dsRNA contains over 200 nucleotides.  
     
     
         15 . The method of  claim 10 , wherein said first and/or second agent is a nucleic acid that encodes a dsRNA.  
     
     
         16 . The method of  claim 10 , wherein said animal is a vertebrate.  
     
     
         17 . The method of  claim 10 , wherein said animal is a mammal.  
     
     
         18 . The method of  claim 17 , wherein said animal is a human.  
     
     
         19 . A method for treating, stabilizing, or preventing a disease, disorder, or infection in an animal, said method comprising introducing into said animal a first agent that provides to said animal a first dsRNA and a second agent that provides to said animal a short, second dsRNA, wherein said first dsRNA has substantial sequence identity to a region of a target nucleic acid associated with said disease, disorder, or infection and specifically inhibits said expression of said target nucleic acid, and wherein said short, second dsRNA inhibits dsRNA-mediated toxicity.  
     
     
         20 . The method of  claim 19 , wherein the double stranded region in said second dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         21 . The method of  claim 19 , wherein the double stranded region in said first dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         22 . The method of  claim 19 , wherein the double stranded region in said first dsRNA contains over 30 nucleotides.  
     
     
         23 . The method of  claim 22 , wherein the double stranded region in said first dsRNA contains over 200 nucleotides.  
     
     
         24 . The method of  claim 19 , wherein said first and/or second agent is a nucleic acid that encodes a dsRNA.  
     
     
         25 . The method of  claim 19 , wherein said animal is a vertebrate.  
     
     
         26 . The method of  claim 19 , wherein said animal is a mammal.  
     
     
         27 . The method of  claim 26 , wherein said animal is a human.  
     
     
         28 . The method of  claim 19 , wherein said target nucleic acid is associated with a pathogen.  
     
     
         29 . The method of  claim 28 , wherein said pathogen is a virus, bacterium, yeast, or infectious agent.  
     
     
         30 . A method for identifying a nucleic acid that modulates a detectable phenotype in a cell, said method comprising said steps of: 
 (a) transforming a population of cells with a dsRNA expression library, wherein at least two cells of said population of cells are each transformed with a different nucleic acid from said dsRNA expression library;    (b) transforming said cells with a short dsRNA or a nucleic acid encoding a short dsRNA; and    (c) assaying for a modulation in said detectable phenotype, wherein said modulation identifies a nucleic acid that is associated with said phenotype.    
     
     
         31 . The method of  claim 30 , wherein said modulation in a detectable phenotype is a modulation in the function of a cell, a modulation in the biological activity of a polypeptide, or a modulation in the expression of a target nucleic acid.  
     
     
         32 . The method of  claim 30 , further comprising: 
 (d) identifying said nucleic acid by amplifying said nucleic acid and sequencing said amplified nucleic acid.    
     
     
         33 . The method of  claim 30 , wherein said dsRNA expression library comprises cDNAs derived from said cells.  
     
     
         34 . The method of  claim 30 , wherein the double stranded region in said second dsRNA contains between 11 and 30 nucleotides, inclusive.  
     
     
         35 . The method of  claim 30 , wherein the double stranded region in said first dsRNA or said dsRNA encoded by said library contains between 11 and 30 nucleotides, inclusive.  
     
     
         36 . The method of  claim 30 , wherein the double stranded region in said first dsRNA or said dsRNA encoded by said library contains over 30 nucleotides.  
     
     
         37 . The method of  claim 36 , wherein the double stranded region in said first dsRNA or said dsRNA encoded by said library contains over 200 nucleotides.  
     
     
         38 . The method of  claim 30 , wherein said first and/or second agent is a nucleic acid which encodes a dsRNA.  
     
     
         39 . The method of  claim 30 , wherein said cell is a vertebrate cell.  
     
     
         40 . The method of  claim 30 , wherein said cell is a mammalian cell.  
     
     
         41 . The method of  claim 40 , wherein said cell is a human cell.