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 formulation comprising an isolated double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus, wherein said double stranded nucleic acid comprises blunt ends and each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, 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, and wherein said second oligonucleotide strand 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 a mammalian cell,
 wherein said double stranded nucleic acid is present in said formulation in an amount effective to reduce target gene expression when introduced into a mammalian cell by at least 10% relative to a control mammalian cell.   
     
     
         2 . The formulation of  claim 1 , wherein said second oligonucleotide strand of said isolated double stranded nucleic acid is complementary to said target RNA along at least 19 nucleotides of said second oligonucleotide strand length. 
     
     
         3 . The formulation of  claim 1 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         4 . The formulation of  claim 1 , wherein each of said first and said second strands of said isolated double stranded nucleic acid has a length which is at least 26 and at most 30 nucleotides. 
     
     
         5 . The formulation of  claim 1 , wherein said first and second strands of said isolated double stranded nucleic acid are, independently, 27 nucleotide residues in length. 
     
     
         6 . The formulation 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 of said isolated double stranded nucleic acid form two mismatched base pairs. 
     
     
         7 . The formulation of  claim 1 , wherein said 5′ terminus of each of said first and said second strands of said isolated double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         8 . The formulation of  claim 1 , wherein said second strand of said isolated double stranded nucleic acid is fully complementary to said target RNA. 
     
     
         9 . The formulation of  claim 1 , wherein the isolated double stranded nucleic acid 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. 
     
     
         10 . The formulation of  claim 1 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         11 . The formulation of  claim 1 , wherein said double stranded nucleic acid reduces target gene expression in a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 50% and at least 80%. 
     
     
         12 . The formulation of  claim 1 , wherein said double stranded nucleic acid is cleaved endogenously in a mammalian cell to produce a double stranded nucleic acid of a length in the range of 19-23 nucleotides in length that reduces target gene expression. 
     
     
         13 . The formulation of  claim 1 , wherein the first and second strands of said isolated double stranded nucleic acid are joined by a chemical linker. 
     
     
         14 . The formulation of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand of said isolated double stranded nucleic acid are joined by a chemical linker. 
     
     
         15 . A formulation comprising an isolated double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus, wherein said double stranded nucleic acid comprises blunt ends and each of said first and said second strands is 27 nucleotides in length, 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 two mismatched base pairs, and wherein said second oligonucleotide strand 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 a mammalian cell,
 wherein said double stranded nucleic acid is present in said formulation in an amount effective to reduce target gene expression when introduced into a mammalian cell by at least 10% relative to a control mammalian cell.   
     
     
         16 . The formulation of  claim 15 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         17 . The formulation of  claim 15 , wherein the isolated double stranded nucleic acid 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. 
     
     
         18 . The formulation of  claim 15 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         19 . The formulation of  claim 15 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         20 . A formulation comprising an isolated double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus, wherein said double stranded nucleic acid comprises blunt ends and each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, 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, and wherein said second oligonucleotide strand 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 a mammalian cell,
 wherein said double stranded nucleic acid is present in said formulation in an amount effective to reduce target gene expression when introduced into a mammalian cell by at least 10% relative to a control mammalian cell.   
     
     
         21 . The formulation of  claim 20 , 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 of said isolated double stranded nucleic acid form two mismatched base pairs. 
     
     
         22 . The formulation of  claim 20 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         23 . The formulation of  claim 20 , wherein the isolated double stranded nucleic acid 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. 
     
     
         24 . The formulation of  claim 20 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         25 . The formulation of  claim 20 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         26 . A formulation comprising an isolated double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus, wherein said double stranded nucleic acid comprises blunt ends and each of said first and said second strands is 27 nucleotides in length, 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 two mismatched base pairs, and wherein said second oligonucleotide strand 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 a mammalian cell,
 wherein said double stranded nucleic acid is present in said formulation in an amount effective to reduce target gene expression when introduced into a mammalian cell by at least 10% relative to a control mammalian cell.   
     
     
         27 . The formulation of  claim 26 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         28 . The formulation of  claim 26 , wherein the isolated double stranded nucleic acid 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. 
     
     
         29 . The formulation of  claim 26 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         30 . The formulation of  claim 26 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.