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

Abstract

The invention is directed to compositions and methods for selectively reducing the expression of a gene product from a desired target gene in a cell, as well as for treating diseases caused by the expression of the gene. More particularly, the invention is directed to compositions that contain double stranded RNA (“dsRNA”), and methods for preparing them, that are capable of reducing the expression of target genes in eukaryotic cells. The dsRNA has a first oligonucleotide sequence that is between 25 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 at least 19 nucleotides is sufficiently complementary to a nucleotide sequence of the RNA produced from the target gene to trigger the destruction of the target RNA by the RNAi machinery.

1 . A mammalian cell containing 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 starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide, wherein said first strand has a length which is at least 25 and at most 30 nucleotides and wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand and forms a blunt end at its 5′ terminus with said 3′ terminus of said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 25 nucleotides in length, 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 in said mammalian cell. 
     
     
         2 . The mammalian cell 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 mammalian cell of  claim 1 , wherein said first strand of said double stranded nucleic acid has a length which is at least 26 nucleotides. 
     
     
         4 . The mammalian cell of  claim 1 , wherein said double stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length. 
     
     
         5 . The mammalian cell of  claim 1 , wherein said second strand of said double stranded nucleic acid is 2 nucleotides longer at its 3′ terminus than said first strand. 
     
     
         6 . The mammalian cell of  claim 1 , wherein said first strand of said double stranded nucleic acid is 25 nucleotides in length and said second strand is 27 nucleotides in length. 
     
     
         7 . The mammalian cell of  claim 1 , wherein said blunt end formed by said 3′ terminus of said first strand and said 5′ terminus of said second strand of said double stranded nucleic acid is a base-paired blunt end. 
     
     
         8 . The mammalian cell of  claim 1 , wherein said modified nucleotide residue of said 3′ terminus of said first strand of said double stranded nucleic acid is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         9 . The mammalian cell of  claim 1 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand of said double stranded nucleic acid is a deoxyribonucleotide. 
     
     
         10 . The mammalian cell of  claim 1 , wherein positions 1 and 2 of said 3′ terminus of the first oligonucleotide strand of said double stranded nucleic acid are deoxyribonucleotides. 
     
     
         11 . The mammalian cell 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 that reduces target gene expression. 
     
     
         12 . The mammalian cell of  claim 1 , wherein said second strand of said double stranded nucleic acid is fully complementary to the target RNA. 
     
     
         13 . The mammalian cell of  claim 1 , wherein the relative length in nucleotide residues of said second and first strands of said double stranded nucleic acid is selected from the group consisting of: second strand 26-29 nucleotide residues in length and said first strand 25 nucleotide residues in length, second strand 27-30 nucleotide residues in length and said first strand 26 nucleotide residues in length, second strand 28-30 nucleotide residues in length and first strand 27 nucleotide residues in length, second strand 29-30 nucleotide residues in length and first strand 28 nucleotide residues in length, and second strand 30 nucleotide residues in length and first strand 29 nucleotide residues in length. 
     
     
         14 . The mammalian cell of  claim 1 , wherein said 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. 
     
     
         15 . The mammalian cell 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. 
     
     
         16 . The mammalian cell of  claim 1 , wherein the concentration of said double stranded nucleic acid sufficient to reduce expression of the target gene is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less in the environment of said cell. 
     
     
         17 . The mammalian cell 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 10%, at least 50% and at least 80%. 
     
     
         18 . A mammalian cell containing 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 first strand has a length which is at least 25 and at most 29 nucleotides and said second strand has a length which is at most 30 nucleotides, wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a blunt end and said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, 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 in said mammalian cell. 
     
     
         19 . The mammalian cell of  claim 18 , 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 that reduces target gene expression. 
     
     
         20 . The mammalian cell of  claim 18 , wherein starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand of said double stranded nucleic acid, position 1, 2 and/or 3 is substituted with a modified nucleotide. 
     
     
         21 . The mammalian cell of  claim 20 , wherein said modified nucleotide residue of said 3′ terminus of said first strand of said double stranded nucleic acid is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         22 . The mammalian cell of  claim 18 , wherein said double stranded nucleic acid comprises a duplex region of at least 25 nucleotides in length. 
     
     
         23 . A mammalian cell containing 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 starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide and wherein said first strand has a length which is at least 25 and at most 29 nucleotides and said second strand has a length which is at most 30 nucleotides, is at least one and at most four nucleotides longer at its 3′ terminus than said first strand and forms a blunt end at its 5′ terminus with said 3′ terminus of said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 25 nucleotides in length, 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 in said mammalian cell. 
     
     
         24 . The mammalian cell of  claim 23 , 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 that reduces target gene expression. 
     
     
         25 . The mammalian cell of  claim 23 , wherein said modified nucleotide residue of said 3′ terminus of said first strand of said double stranded nucleic acid is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         26 . A mammalian cell containing 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 first strand is 25 nucleotide residues in length, and wherein starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide, and said second strand is 27 nucleotide residues in length and is 2 nucleotides longer at its 3′ terminus than said first strand, 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 in said mammalian cell. 
     
     
         27 . The mammalian cell of  claim 26 , 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 that reduces target gene expression. 
     
     
         28 . The mammalian cell of  claim 26 , wherein said modified nucleotide residue of said 3′ terminus of said first strand of said double stranded nucleic acid is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         29 . The mammalian cell of  claim 26 , wherein said second strand of said double stranded nucleic acid forms a base-paired blunt end at its 5′ terminus with said 3′ terminus of said first strand.