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 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 first oligonucleotide strand has a length which is at least 25 and at most 30 nucleotides and starting from the first nucleotide (position 1) at the 3′ terminus of said first oligonucleotide strand, position 1, 2 and/or 3 is substituted with a modified nucleotide, 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 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 said first strand of said double stranded nucleic acid has a length which is at least 26 nucleotides. 
     
     
         5 . The formulation 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 formulation of  claim 1 , wherein said double-stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length. 
     
     
         7 . The formulation 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. 
     
     
         8 . The formulation of  claim 1 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand of said double stranded nucleic acid is a deoxyribonucleotide. 
     
     
         9 . The formulation 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. 
     
     
         10 . The formulation of  claim 1 , wherein said second oligonucleotide strand has a length which is at least 26 and at most 30 nucleotides. 
     
     
         11 . The formulation of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand of said double stranded nucleic acid form a blunt end. 
     
     
         12 . The formulation of  claim 11 , 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. 
     
     
         13 . The formulation 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. 
     
     
         14 . 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 that reduces target gene expression. 
     
     
         15 . The formulation of  claim 1 , wherein said second strand of said double stranded nucleic acid is fully complementary to said target RNA. 
     
     
         16 . The formulation of  claim 1 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         17 . The formulation 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. 
     
     
         18 . The formulation of  claim 1 , wherein the first and second strands of said double stranded nucleic acid are joined by a chemical linker. 
     
     
         19 . The formulation of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand of said double stranded nucleic acid are joined by a chemical linker. 
     
     
         20 . The formulation of  claim 1 , wherein a nucleotide of said second or first strand of said double stranded nucleic acid is substituted with a modified nucleotide that directs the orientation of Dicer cleavage. 
     
     
         21 . The formulation 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. 
     
     
         22 . 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. 
     
     
         23 . 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%. 
     
     
         24 . 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 first oligonucleotide strand has a length which is at least 25 and at most 29 nucleotides, and wherein said second oligonucleotide strand has a length which is at most 30 nucleotides and 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, 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.   
     
     
         25 . The formulation of  claim 24 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         26 . The formulation of  claim 24 , wherein starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand of said isolated double stranded nucleic acid, position 1, 2 and/or 3 is substituted with a modified nucleotide. 
     
     
         27 . The formulation 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. 
     
     
         28 . The formulation of  claim 24 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         29 . The formulation of  claim 24 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         30 . 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 first oligonucleotide strand has a length which is at least 25 and at most 29 nucleotides and 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 second oligonucleotide strand has a length which is at most 30 nucleotides and 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 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.   
     
     
         31 . The formulation of  claim 30 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         32 . The formulation of  claim 30 , 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. 
     
     
         33 . The formulation of  claim 30 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         34 . The formulation of  claim 30 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         35 . 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 first oligonucleotide strand is 25 nucleotides in length and 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 second oligonucleotide strand is 27 nucleotides in length and 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, 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.   
     
     
         36 . The formulation of  claim 35 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         37 . The formulation of  claim 35 , 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. 
     
     
         38 . The formulation of  claim 35 , wherein said 5′ terminus of each of said first and said second strands of said double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         39 . The formulation of  claim 35 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.