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 method of selecting a double stranded nucleic acid possessing increased potency in reducing expression of a target gene in a mammalian expression system, said method comprising:
 (a) determining the amount of reduction in expression of a target gene in a mammalian cell at a transfecting concentration of a first double stranded nucleic acid of 50 nanomolar or less, relative to a control transfection, wherein said first double stranded nucleic acid comprises 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 30 nucleotides, and wherein said second oligonucleotide strand of said first double stranded nucleic acid has a length which is at least 25 and at most 30 nucleotides and comprises a sequence complementary to a target RNA of a target gene and said first double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell;   (b) comparing said reduction in target gene expression of said step (a) to the reduction in said target gene expression in a mammalian cell of a second double stranded nucleic acid at said transfecting concentration, said second double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides, having a 5′ terminus and a 3′ terminus, and consisting of 21-23 nucleotides, wherein said second oligonucleotide strand of said second double stranded nucleic acid comprises said sequence complementary to said target RNA, and   (c) whereby said comparing of said step (b) identifies at least a 10% greater reduction in said target gene expression by said first double stranded nucleic acid, as determined in step (a), relative to said second double stranded nucleic acid, thereby permitting selection of a double stranded nucleic acid possessing increased potency.   
     
     
         2 . The method of  claim 1 , wherein said second oligonucleotide strand of said first double stranded nucleic acid 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 transfecting concentration is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         4 . The method of  claim 1 , wherein said comparing of said step (b) identifies a level of greater reduction in said target gene expression by said first double stranded nucleic acid selected from at least 50%, at least 80% and at least 90% greater reduction. 
     
     
         5 . The method of  claim 1 , wherein said first strand of said first double stranded nucleic acid has a length which is at least 26 nucleotides. 
     
     
         6 . The method of  claim 1 , wherein each of said first and second strands of said first double stranded nucleic acid has a length which is at least 26 nucleotides. 
     
     
         7 . The method of  claim 1 , wherein each of said first and said second strands of said first double stranded nucleic acid consists of 26-30 nucleotides. 
     
     
         8 . The method of  claim 1 , wherein said second strand of said first double stranded nucleic acid is 1-4 nucleotides longer at its 3′ terminus than said first strand of said first double stranded nucleic acid. 
     
     
         9 . The method of  claim 1 , wherein said second strand of said first double stranded nucleic acid is 2 nucleotides longer at its 3′ terminus than said first strand of said first double stranded nucleic acid. 
     
     
         10 . The method of  claim 1 , wherein said first double stranded nucleic acid comprises a duplex region of at least 25 nucleotides in length. 
     
     
         11 . The method of  claim 1 , wherein said first double stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length. 
     
     
         12 . The method of  claim 1 , wherein said first and second strands of said first double stranded nucleic acid comprise the same number of nucleotide residues. 
     
     
         13 . The method of  claim 1 , wherein starting from the first nucleotide (position 1) at the 3′ terminus of the first oligonucleotide strand of said first double stranded nucleic acid, position 1, 2 and/or 3 is substituted with a modified nucleotide. 
     
     
         14 . The method of  claim 13 , wherein said modified nucleotide residue of said 3′ terminus of said first strand of said first double stranded nucleic acid is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         15 . The method of  claim 13 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand of said first double stranded nucleic acid is a deoxyribonucleotide. 
     
     
         16 . The method of  claim 13 , wherein positions 1 and 2 of said 3′ terminus of the first oligonucleotide strand of said first double stranded nucleic acid are deoxyribonucleotides. 
     
     
         17 . The method of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand of said first double stranded nucleic acid form a blunt end. 
     
     
         18 . The method of  claim 17 , wherein said blunt end formed by said 3′ terminus of said first strand and said 5′ terminus of said second strand of said first double stranded nucleic acid is a base-paired blunt end. 
     
     
         19 . The method of  claim 1 , wherein said first strand of said first double stranded nucleic acid is 25 nucleotides in length and said second strand of said first double stranded nucleic acid is 27 nucleotides in length. 
     
     
         20 . The method of  claim 1 , wherein said first double stranded nucleic acid is cleaved endogenously in a mammalian cell by Dicer. 
     
     
         21 . The method of  claim 1 , wherein said first 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. 
     
     
         22 . The method of  claim 1 , wherein said second strand of said first double stranded nucleic acid is fully complementary to the target RNA. 
     
     
         23 . The method of  claim 1 , wherein said 5′ terminus of each of said first and said second strands of said first double stranded nucleic acid comprises a 5′ phosphate. 
     
     
         24 . The method of  claim 1 , wherein the relative length in nucleotide residues of said second and first strands of said first 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. 
     
     
         25 . The method of  claim 1 , wherein the first and second strands of said first double stranded nucleic acid are joined by a chemical linker. 
     
     
         26 . The method of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand of said first double stranded nucleic acid are joined by a chemical linker. 
     
     
         27 . The method of  claim 1 , wherein a nucleotide of said second or first strand of said first double stranded nucleic acid is substituted with a modified nucleotide that directs the orientation of Dicer cleavage. 
     
     
         28 . The method of  claim 1 , wherein said first 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 method of  claim 1 , wherein said first double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         30 . A method of selecting a double stranded nucleic acid possessing increased potency in reducing expression of a target gene in a mammalian expression system, said method comprising:
 (a) determining the amount of reduction in expression of a target gene in a mammalian cell at a transfecting concentration of a first double stranded nucleic acid of 50 nanomolar or less, relative to a control transfection, wherein said first double stranded nucleic acid comprises 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 30 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, 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 first 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 first double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell;   (b) comparing said reduction in target gene expression of said step (a) to the reduction in said target gene expression in a mammalian cell of a second double stranded nucleic acid at said transfecting concentration, said second double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides, having a 5′ terminus and a 3′ terminus, and consisting of 21-23 nucleotides, wherein said second oligonucleotide strand of said second double stranded nucleic acid comprises said sequence complementary to said target RNA, and   (c) whereby said comparing of said step (b) identifies at least a 10% greater reduction in said target gene expression by said first double stranded nucleic acid, as determined in step (a), relative to said second double stranded nucleic acid, thereby permitting selection of a double stranded nucleic acid possessing increased potency.   
     
     
         31 . The method of  claim 30 , wherein said transfecting concentration is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         32 . The method of  claim 30 , wherein said first 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. 
     
     
         33 . The method of  claim 30 , wherein said first double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         34 . A method of selecting a double stranded nucleic acid possessing increased potency in reducing expression of a target gene in a mammalian expression system, said method comprising:
 (a) determining the amount of reduction in expression of a target gene in a mammalian cell at a transfecting concentration of a first double stranded nucleic acid of 50 nanomolar or less, relative to a control transfection, wherein said first double stranded nucleic acid comprises 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 first double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell;   (b) comparing said reduction in target gene expression of said step (a) to the reduction in said target gene expression in a mammalian cell of a second double stranded nucleic acid at said transfecting concentration, said second double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides, having a 5′ terminus and a 3′ terminus, and consisting of 21-23 nucleotides, wherein said second oligonucleotide strand of said second double stranded nucleic acid comprises said sequence complementary to said target RNA, and   (c) whereby said comparing of said step (b) identifies at least a 10% greater reduction in said target gene expression by said first double stranded nucleic acid, as determined in step (a), relative to said second double stranded nucleic acid, thereby permitting selection of a double stranded nucleic acid possessing increased potency.   
     
     
         35 . The method of  claim 34 , wherein said transfecting concentration is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         36 . The method of  claim 34 , wherein said first 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. 
     
     
         37 . The method of  claim 34 , wherein said first double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         38 . A method of selecting a double stranded nucleic acid possessing increased potency in reducing expression of a target gene in a mammalian expression system, said method comprising:
 (a) determining the amount of reduction in expression of a target gene in a mammalian cell at a transfecting concentration of a first double stranded nucleic acid of 50 nanomolar or less, relative to a control transfection, wherein said first double stranded nucleic acid comprises 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, 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, wherein said first 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 first double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell;   (b) comparing said reduction in target gene expression of said step (a) to the reduction in said target gene expression in a mammalian cell of a second double stranded nucleic acid at said transfecting concentration, said second double stranded nucleic acid comprising first and second oligonucleotide strands, each strand comprising ribonucleotides, having a 5′ terminus and a 3′ terminus, and consisting of 21-23 nucleotides, wherein said second oligonucleotide strand of said second double stranded nucleic acid comprises said sequence complementary to said target RNA, and   (c) whereby said comparing of said step (b) identifies at least a 10% greater reduction in said target gene expression by said first double stranded nucleic acid, as determined in step (a), relative to said second double stranded nucleic acid, thereby permitting selection of a double stranded nucleic acid possessing increased potency.   
     
     
         39 . The method of  claim 38 , wherein said transfecting concentration is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less and 50 picomolar or less. 
     
     
         40 . The method of  claim 38 , wherein said first 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. 
     
     
         41 . The method of  claim 38 , wherein said first double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester.