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 pharmaceutical composition comprising an isolated double stranded nucleic acid and a pharmaceutically acceptable carrier, wherein the 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 double stranded nucleic acid comprises blunt ends, 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, wherein said second strand comprises a sequence complementary to a target RNA of a target gene, and wherein said double stranded nucleic acid is present in said pharmaceutical composition in an amount effective to reduce target gene expression when said pharmaceutical composition is administered to a mammal. 
     
     
         2 . The pharmaceutical composition of  claim 1 , wherein said second oligonucleotide strand of said isolated double stranded nucleic acid is complementary to the target RNA along at least 19 nucleotides of said second oligonucleotide strand length. 
     
     
         3 . The pharmaceutical composition 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. 
     
     
         4 . The pharmaceutical composition of  claim 1 , wherein said first and second strands of said isolated double stranded nucleic acid are, independently, 27 nucleotide residues in length. 
     
     
         5 . The pharmaceutical composition 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 two mismatched base pairs. 
     
     
         6 . The pharmaceutical composition of  claim 1 , wherein said effective amount of said double stranded nucleic acid in said pharmaceutical composition is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.10 to 2.5 micrograms per kilogram. 
     
     
         7 . The pharmaceutical composition 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. 
     
     
         8 . The pharmaceutical composition of  claim 1 , wherein said second strand of said double stranded nucleic acid is fully complementary to the target RNA. 
     
     
         9 . The pharmaceutical composition 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. 
     
     
         10 . The pharmaceutical composition of  claim 1 , wherein the first and second strands of said double stranded nucleic acid are joined by a chemical linker. 
     
     
         11 . The pharmaceutical composition 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. 
     
     
         12 . The pharmaceutical composition 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. 
     
     
         13 . The pharmaceutical composition 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. 
     
     
         14 . The pharmaceutical composition 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. 
     
     
         15 . A pharmaceutical composition comprising an isolated double stranded nucleic acid and a pharmaceutically acceptable carrier, wherein the double stranded nucleic acid comprises first and second oligonucleotide strands, each strand comprising ribonucleotides, having a 5′ terminus and a 3′ terminus and consisting of 27 nucleotides, wherein said double stranded nucleic acid comprises blunt ends and 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 strand comprises a sequence complementary to a target RNA of a target gene, and wherein said double stranded nucleic acid is present in said pharmaceutical composition in an amount effective to reduce target gene expression when said pharmaceutical composition is administered to a mammal. 
     
     
         16 . The pharmaceutical composition of  claim 15 , wherein said effective amount of said double stranded nucleic acid in said pharmaceutical composition is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.10 to 2.5 micrograms per kilogram. 
     
     
         17 . The pharmaceutical composition of  claim 15 , wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate. 
     
     
         18 . The pharmaceutical composition of  claim 15 , wherein the 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. 
     
     
         19 . The pharmaceutical composition of  claim 15  comprising a modification of the phosphate backbone of said double stranded nucleic acid selected from the group consisting of a phosphonate, a phosphorothioate, and a phosphotriester. 
     
     
         20 . A pharmaceutical composition comprising an isolated double stranded nucleic acid and a pharmaceutically acceptable carrier, wherein the 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 double stranded nucleic acid comprises blunt ends, 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, wherein said second strand comprises a sequence complementary to a target RNA of a target gene, and wherein said double stranded nucleic acid is present in said pharmaceutical composition in an amount effective to reduce target gene expression when said pharmaceutical composition is administered to a mammal. 
     
     
         21 . The pharmaceutical composition 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 pharmaceutical composition of  claim 20 , wherein said effective amount of said double stranded nucleic acid in said pharmaceutical composition is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.10 to 2.5 micrograms per kilogram. 
     
     
         23 . The pharmaceutical composition of  claim 20 , wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate. 
     
     
         24 . The pharmaceutical composition of  claim 20 , wherein the 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. 
     
     
         25 . The pharmaceutical composition of  claim 20  comprising a modification of the phosphate backbone of said double stranded nucleic acid selected from the group consisting of a phosphonate, a phosphorothioate, and a phosphotriester. 
     
     
         26 . A pharmaceutical composition comprising an isolated double stranded nucleic acid and a pharmaceutically acceptable carrier, wherein the double stranded nucleic acid comprises first and second oligonucleotide strands, each strand comprising ribonucleotides, having a 5′ terminus and a 3′ terminus and consisting of 27 nucleotides, wherein said double stranded nucleic acid comprises blunt ends and 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 strand comprises a sequence complementary to a target RNA of a target gene, and wherein said double stranded nucleic acid is present in said pharmaceutical composition in an amount effective to reduce target gene expression when said pharmaceutical composition is administered to a mammal. 
     
     
         27 . The pharmaceutical composition of  claim 26 , wherein said effective amount of said double stranded nucleic acid in said pharmaceutical composition is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram, 0.001 to 0.25 milligrams per kilogram, 0.01 to 20 micrograms per kilogram, 0.01 to 10 micrograms per kilogram, 0.10 to 5 micrograms per kilogram, and 0.10 to 2.5 micrograms per kilogram. 
     
     
         28 . The pharmaceutical composition of  claim 26 , wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate. 
     
     
         29 . The pharmaceutical composition of  claim 26 , wherein the 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. 
     
     
         30 . The pharmaceutical composition of  claim 26  comprising a modification of the phosphate backbone of said double stranded nucleic acid selected from the group consisting of a phosphonate, a phosphorothioate, and a phosphotriester.