Methods and compositions for RNA interference

Abstract

The present invention provides methods for attenuating gene expression in a cell using gene-targeted double stranded RNA (dsRNA). The dsRNA contains a nucleotide sequence that hybridizes under physiologic conditions of the cell to the nucleotide sequence of at least a portion of the gene to be inhibited (the “target” gene).

We claim:  
     
         1 . A method for attenuating expression of a target gene in cultured cells, comprising introducing double stranded RNA (dsRNA) into the cells in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.  
     
     
         2 . A method for attenuating expression of a target gene in a mammalian cell, comprising 
 (i) activating one or both of a Dicer activity or an Argonaut activity in the cell, and    (ii) introducing into the cell a double stranded RNA (dsRNA) in an amount sufficient to attenuate expression of the target gene, wherein the dsRNA comprises a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.    
     
     
         3 . The method of  claim 2 , wherein the cell is suspended in culture.  
     
     
         4 . The method of  claim 2 , wherein the cell is in a whole animal, such as a non-human mammal.  
     
     
         5 . The method of  claim 1  or  2 , wherein is engineered with (i) a recombinant gene encoding a Dicer activity, (ii) a recombinant gene encoding an Argonaut activity, or (iii) both.  
     
     
         6 . The method of  claim 5 , wherein the recombinant gene encodes a protein which includes an amino acid sequence at least 50 percent identical to SEQ ID No. 2 or 4 or the Argonaut sequence shown in FIG. 24.  
     
     
         7 . The method of  claim 5 , wherein the recombinant gene includes a coding sequence hybridizes under wash conditions of 2×SSC at 22° C. to SEQ ID No. 1 or 3.  
     
     
         8 . The method of  claim 1  or  2 , wherein an endogenous Dicer gene or Argonaut gene is activated.  
     
     
         9 . The method of  claim 1  or  2 , wherein the target gene is an endogenous gene of the cell.  
     
     
         10 . The method of  claim 1  or  2 , wherein the target gene is an heterologous gene relative to the genome of the cell, such as a pathogen gene.  
     
     
         11 . The method of  claim 1  or  2 , wherein the cell is treated with an agent that inhibits protein kinase RNA-activated (PKR) apoptosis, such as by treatment with agents which inhibit expression of PKR, cause its destruction, and/or inhibit the kinase activity of PKF.  
     
     
         12 . The method of  claim 1  or  2 , wherein the cell is a primate cell, such as a human cell.  
     
     
         13 . The method of  claim 1  or  2 , wherein the dsRNA is at least 20 nucleotides in length.  
     
     
         14 . The method of  claim 13 , wherein the dsRNA is at least 100 nucleotides in length.  
     
     
         15 . The method of  claim 1  or  2 , wherein expression of the target gene is attenuated by at least 10 fold.  
     
     
         16 . An assay for identifying nucleic acid sequences responsible for conferring a particular phenotype in a cell, comprising 
 (i) constructing a variegated library of nucleic acid sequences from a cell in an orientation relative to a promoter to produce double stranded DNA;    (ii) introducing the variegated dsRNA library into a culture of target cells, which cells have an activated Dicer activity or Argonaut activity;    (iii) identifying members of the library which confer a particular phenotype on the cell, and identifying the sequence from a cell which correspond, such as being identical or homologous, to the library member.    
     
     
         17 . A method of conducting a drug discovery business comprising: 
 (i) identifying, by the assay of  claim 16 , a target gene which provides a phenotypically desirable response when inhibited by RNAi;    (ii) identifying agents by their ability to inhibit expression of the target gene or the activity of an expression product of the target gene;    (iii) conducting therapeutic profiling of agents identified in step (b), or further analogs thereof, for efficacy and toxicity in animals; and    (iv) formulating a pharmaceutical preparation including one or more agents identified in step (iii) as having an acceptable therapeutic profile.    
     
     
         18 . The method of  claim 17 , including an additional step of establishing a distribution system for distributing the pharmaceutical preparation for sale, and may optionally include establishing a sales group for marketing the pharmaceutical preparation.  
     
     
         19 . A method of conducting a target discovery business comprising: 
 (i) identifying, by the assay of  claim 16 , a target gene which provides a phenotypically desirable response when inhibited by RNAi;    (ii) (optionally) conducting therapeutic profiling of the target gene for efficacy and toxicity in animals; and    (iii). licensing, to a third party, the rights for further drug development of inhibitors of the target gene.    
     
     
         20 . A method for attenuating expression of a target gene in a cell, comprising introducing into the cell a hairpin nucleic acid in an amount sufficient to attenuate expression of the target gene, wherein the hairpin nucleic acid comprises an inverted repeat of a nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene.  
     
     
         21 . A hairpin nucleic acid for inhibiting expression of a target gene, comprising a first nucleotide sequence that hybridizes under stringent conditions to a nucleotide sequence of the target gene, and a second nucleotide sequence which is an complementary inverted repeat of said first nucleotide sequence and hybridizes to said first nucleotide sequence to form a hairpin structure.  
     
     
         22 . The method of  claim 20  or the hairpin nucleic acid of  claim 21 , wherein the hairpin nucleic is RNA.  
     
     
         23 . A non-human transgenic mammal having germline and/or somatic cells comprising a transgene encoding a dsRNA construct.  
     
     
         24 . The transgenic animal of  claim 23 , which is chimeric for said transgene.  
     
     
         25 . The transgenic animal of  claim 23 , wherein said transgene is chromosomally incorporated.