Methods and compositions for the specific inhibition of gene expression by double-stranded rna

Abstract

The invention provides compositions and methods for selectively reducing the expression of a gene product from a desired target gene, as well as treating diseases caused by expression of the gene. The method involves introducing into the environment of a cell an amount of a double-stranded RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the cytoplasm of the cell to cause a reduction in the expression of the target gene. The dsRNA has a first oligonucleotide sequence that is between 26 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of from about 19 to about 23 nucleotides is complementary to a nucleotide sequence of the RNA produced from the target gene.

1 . A method for reducing expression of a target gene in a mammalian cell comprising contacting a mammalian cell in vitro with an isolated double stranded ribonucleic acid (dsRNA) at a concentration of less than 200 picomolar in the environment of said cell, wherein said dsRNA comprises first and second oligonucleotide strands, each strand having a 5′ terminus and a 3′ terminus, wherein said dsRNA comprises blunt ends, wherein each of said first and said second oligonucleotide strands consists of the same number of nucleotide residues and is 25-30 nucleotides in length and wherein said second oligonucleotide strand of said isolated dsRNA comprises a sequence complementary to a target RNA of a target gene and said isolated double stranded nucleic acid reduces target gene expression when introduced into said mammalian cell. 
     
     
         2 . The method of  claim 1 , wherein said second oligonucleotide strand of said isolated dsRNA is complementary to said target RNA along at least 19 nucleotides of said second oligonucleotide strand length. 
     
     
         3 . The method of  claim 1 , wherein said isolated dsRNA reduces target gene expression at a concentration of less than 50 picomolar in the environment of said cell. 
     
     
         4 . The method of  claim 1 , wherein said isolated dsRNA is cleaved endogenously in said cell to produce a double-stranded nucleic acid of a length in the range of 19-23 nucleotides that reduces target gene expression. 
     
     
         5 . The method of  claim 1 , wherein said isolated dsRNA is a substrate of Dicer in said cell. 
     
     
         6 . The method of  claim 1 , wherein target gene expression is reduced by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95% and at least 99%. 
     
     
         7 . The method of  claim 1 , wherein target gene expression is reduced by at least 90% at least 8 days after said cell is contacted with said dsRNA. 
     
     
         8 . The method of  claim 1 , wherein target gene expression is reduced by at least 70% at least 10 days after said cell is contacted with said dsRNA. 
     
     
         9 . The method of  claim 1 , wherein said dsRNA reduces target gene expression in said cell for at least twice as long as a 21mer siRNA directed to said target RNA sequence when assayed at an effective concentration in the environment of said cell. 
     
     
         10 . The method of  claim 1 , wherein the isolated dsRNA comprises a modified nucleotide selected from the group consisting of a deoxyribonucleotide, a dideoxyribonucleotide, an acyclonucleotide, a 3′-deoxyadenosine (cordycepin), a 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxyinosine (ddI), a 2′,3′-dideoxy-3′-thiacytidine (3TC), a 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a monophosphate nucleotide of 3′-azido-3′-deoxythymidine (AZT), a 2′,3′-dideoxy-3′-thiacytidine (3TC) and a monophosphate nucleotide of 2′,3′-didehydro-2′,3′-dideoxythymidine (d4T), a 4-thiouracil, a 5-bromouracil, a 5-iodouracil, a 5-(3-aminoallyl)-uracil, a 2′-O-alkyl ribonucleotide, a 2′-O-methyl ribonucleotide, a 2′-amino ribonucleotide, a 2′-fluoro ribonucleotide, and a locked nucleic acid. 
     
     
         11 . The method of  claim 1 , wherein the isolated dsRNA comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate, and a phosphotriester. 
     
     
         12 . The method of  claim 1 , wherein position 1 of said 3′ terminus of said first oligonucleotide strand is a deoxyribonucleotide. 
     
     
         13 . The method of  claim 1 , wherein said 5′ terminus of each of said first and said second oligonucleotide strands comprises a 5′ phosphate. 
     
     
         14 . The method of  claim 1 , wherein each of said first and said second oligonucleotide strands has a length which is at least 26 and at most 30 nucleotides. 
     
     
         15 . The method of  claim 1 , wherein said first and second oligonucleotide strands are, independently, 27 nucleotide residues in length. 
     
     
         16 . The method of  claim 1 , wherein said first and second oligonucleotide strands are joined by a chemical linker. 
     
     
         17 . The method of  claim 1 , wherein said 3′ terminus of said first oligonucleotidide strand and said 5′ terminus of said second oligonucleotidide strand are joined by a chemical linker. 
     
     
         18 . The method of  claim 1 , wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs. 
     
     
         19 . The method of  claim 1 , wherein the ultimate and penultimate residues of said 5′ terminus of said first strand and the ultimate and penultimate residues of said 3′ terminus of said second strand form one or two mismatched base pairs. 
     
     
         20 . The mammalian cell of  claim 1 , wherein said second strand of said double stranded nucleic acid is fully complementary to said target RNA. 
     
     
         21 . A method for reducing expression of a target gene in a mammalian cell comprising contacting a mammalian cell with an isolated double stranded ribonucleic acid (dsRNA), wherein said dsRNA comprises first and second oligonucleotide strands, each strand having a 5′ terminus and a 3′ terminus and wherein said dsRNA comprises blunt ends, wherein each of said first and said second oligonucleotide strands consists of the same number of nucleotide residues and is 25-30 nucleotides in length, wherein said second oligonucleotide strand of said isolated dsRNA comprises a sequence complementary to a target RNA sequence of a target gene and said isolated double stranded nucleic acid reduces target gene expression when introduced into said mammalian cell, and wherein said dsRNA reduces target gene expression in said cell for at least twice as long as a 21mer siRNA directed to said target RNA sequence when said dsRNA and said 21mer siRNA are assayed at an effective concentration in the environment of said cell, thereby reducing expression of the target gene in said mammalian cell. 
     
     
         22 . The method of  claim 21 , wherein target gene expression is reduced by at least 90% at least 8 days after said cell is contacted with said dsRNA. 
     
     
         23 . The method of  claim 21 , wherein target gene expression is reduced by at least 70% at least 10 days after said cell is contacted with said dsRNA. 
     
     
         24 . The method of  claim 21 , wherein said dsRNA is cleaved endogenously in said cell to produce a double-stranded nucleic acid of a length in the range of 19-23 nucleotides that reduces target gene expression. 
     
     
         25 . The method of  claim 21 , wherein said isolated dsRNA is a substrate of Dicer in said cell. 
     
     
         26 . The method of  claim 21 , wherein expression of said target gene is reduced by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95% and at least 99%. 
     
     
         27 . The method of  claim 21 , wherein said cell is contacted in vitro. 
     
     
         28 . The method of  claim 21 , wherein said effective concentration is selected from the group consisting of less than 1 nanomolar, less than 200 picomolar and less than 50 picomolar in the environment of said cell. 
     
     
         29 . The method of  claim 21 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide. 
     
     
         30 . The method of  claim 21 , wherein said 5′ terminus of each of said first and said second oligonucleotide strands comprises a 5′ phosphate. 
     
     
         31 . The method of  claim 21 , wherein each of said first and said second oligonucleotide strands has a length which is at least 26 and at most 30 nucleotides. 
     
     
         32 . The method of  claim 21 , wherein said first and second oligonucleotide strands are, independently, 27 nucleotide residues in length. 
     
     
         33 . The method of  claim 21 , wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs. 
     
     
         34 . The method of  claim 21 , wherein the ultimate and penultimate residues of said 5′ terminus of said first strand and the ultimate and penultimate residues of said 3′ terminus of said second strand form one or two mismatched base pairs.