Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna

Abstract

Introduction of double stranded RNA into cells, cell culture, organs and tissues, and whole organisms, particularly vertebrates, specifically attenuates gene expression.

1 - 61 . (canceled) 
     
     
         62 . A method for attenuating the expression of a target gene in a vertebrate cell ex vivo comprising:
 explanting a vertebrate cell from a vertebrate organism;   supplying the cell with at least one double stranded RNA in an amount sufficient to specifically attenuate expression of the target gene and not induce interferon-α/β mediated toxicity, wherein one of the strands of the double stranded RNA is capable of hybridizing to the target gene in vitro in 400 mM NaCl, 40 mM PIPES ph 6.4, and 1 mM EDTA, at 50° C.; and   implanting the cell into a vertebrate organism, wherein expression of the target gene is attenuated in said vertebrate cell.   
     
     
         63 . The method of  claim 62 , wherein the cell is implanted back into the vertebrate organism from which it was explanted. 
     
     
         64 . The method of  claim 62 , wherein the double stranded RNA has a length of less than about 200 bases. 
     
     
         65 . The method of  claim 62 , wherein the double stranded RNA comprises a nucleotide sequence that is complementary to a region of at least about 25 bases of the target gene. 
     
     
         66 . The method of  claim 62 , wherein the double stranded RNA is supplied to the cell by delivery to the cell of the double stranded RNA. 
     
     
         67 . The method of  claim 66 , wherein the double stranded RNA is purified in the absence of phenol or chloroform. 
     
     
         68 . The method of  claim 62 , wherein the target gene is an endogenous gene. 
     
     
         69 . The method of  claim 62 , wherein the target gene is a foreign gene. 
     
     
         70 . The method of  claim 62 , wherein the target gene is a chromosomal gene. 
     
     
         71 . The method of  claim 62 , wherein the target gene is an extrachromosomal gene. 
     
     
         72 . The method of  claim 62 , wherein the double stranded RNA is supplied in an amount to completely inhibit expression of the target gene. 
     
     
         73 . The method of  claim 62 , wherein the double stranded RNA comprises a single strand comprising self-complementary portions that are essentially identical to the target gene. 
     
     
         74 . The method of  claim 62 , wherein the double stranded RNA comprises two separate complementary strands. 
     
     
         75 . The method of  claim 66 , wherein the double stranded RNA is treated with RNase prior to delivery to the cell. 
     
     
         76 . The method of  claim 74 , wherein the two strands of the double stranded RNA are annealed in the presence of potassium chloride prior to delivery. 
     
     
         77 . The method of  claim 62 , wherein the function of the target gene is unknown. 
     
     
         78 . The method of  claim 62 , further comprising identifying a phenotypic change in the vertebrate cell associated with attenuated expression of the target gene. 
     
     
         79 . The method of  claim 62 , wherein the target gene is associated with a disease. 
     
     
         80 . The method of  claim 62 , wherein the target gene is associated with a pathogen. 
     
     
         81 . The method of  claim 80 , wherein the pathogen is selected from the group consisting of a virus, bacterium, fungus or protozoan. 
     
     
         82 . A method for attenuating the expression of a target gene in a vertebrate cell in vitro comprising:
 supplying the cell with at least one double stranded RNA in an amount sufficient to specifically attenuate expression of the target gene and not induce interferon-α/β mediated toxicity, wherein one of the strands of the double stranded RNA is capable of hybridizing to the target gene in vitro in 400 mM NaCl, 40 mM PIPES pH 6.4, and 1 mM EDTA, at 50° C.   
     
     
         83 . The method of  claim 82 , wherein the double stranded RNA has a length of less than about 200 bases. 
     
     
         84 . The method of  claim 82 , wherein the double stranded RNA comprises a nucleotide sequence that is complementary to a region of at least about 25 bases of the target gene. 
     
     
         85 . The method of  claim 82 , wherein the double stranded RNA is supplied to the cell by delivery to the cell of the double stranded RNA. 
     
     
         86 . The method of  claim 85 , wherein the double stranded RNA is purified in the absence of phenol or chloroform. 
     
     
         87 . The method of  claim 82 , wherein the target gene is an endogenous gene. 
     
     
         88 . The method of  claim 82 , wherein the target gene is a foreign gene. 
     
     
         89 . The method of  claim 82 , wherein the target gene is a chromosomal gene. 
     
     
         90 . The method of  claim 82 , wherein the target gene is an extrachromosomal gene. 
     
     
         91 . The method of  claim 82 , wherein the double stranded RNA is supplied in an amount to completely inhibit expression of the target gene. 
     
     
         92 . The method of  claim 82 , wherein the double stranded RNA comprises a single strand comprising self-complementary portions that are essentially identical to the target gene. 
     
     
         93 . The method of  claim 82 , wherein the double stranded RNA comprises two separate complementary strands. 
     
     
         94 . The method of  claim 85 , wherein the double stranded RNA is treated with RNase prior to delivery to the cell. 
     
     
         95 . The method of  claim 93 , wherein the two strands of the double stranded RNA are annealed in the presence of potassium chloride prior to delivery. 
     
     
         96 . The method of  claim 82 , wherein the function of the target gene is unknown. 
     
     
         97 . The method of  claim 82 , further comprising identifying a phenotypic change in the vertebrate cell associated with attenuated expression of the target gene. 
     
     
         98 . The method of  claim 82 , wherein the target gene is associated with a disease. 
     
     
         99 . The method of  claim 82 , wherein the target gene is associated with a pathogen. 
     
     
         100 . The method of  claim 99 , wherein the pathogen is selected from the group consisting of a virus, bacterium, fungus or protozoan. 
     
     
         101 . The method of  claim 82 , wherein the cell is a zebrafish cell, a murine cell, or a chick cell. 
     
     
         102 . The method of  claim 101 , wherein the murine cell is a rat cell. 
     
     
         103 . The method of  claim 82 , wherein the cell is an embryonic cell. 
     
     
         104 . The method of  claim 62 , wherein the vertebrate cell is a murine cell.