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 method for preparing an isolated double stranded nucleic acid that corresponds to a selected target sequence of a target gene, comprising:
 synthesizing first and second oligonucleotide strands, wherein each of said first and said second strands has a 5′ terminus and a 3′ terminus, consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein said first and second strands are complementary to each other such that they form a duplex of at least 25 nucleotides, 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 one or two mismatched base pairs when said duplex is formed, and wherein said second strand comprises a nucleotide sequence complementary to the selected target sequence such that it anneals to said target sequence under biological conditions, and   annealing said first and said second oligonucleotide strands to form a double stranded nucleic acid, wherein said double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell,   thereby preparing said isolated double stranded nucleic acid.   
     
     
         2 . The method of  claim 1 , wherein said biological conditions are conditions as found in the cytoplasm of a mammalian cell. 
     
     
         3 . The method of  claim 1 , wherein said double stranded nucleic acid is then formulated into a pharmaceutically acceptable excipient. 
     
     
         4 . The method of  claim 1 , wherein the selected target sequence of said target gene comprises at least 19 nucleotides. 
     
     
         5 . The method of  claim 1 , wherein each of said first and second strands of said isolated double stranded nucleic acid has a length which is at least 26 and at most 30 nucleotides. 
     
     
         6 . The method of  claim 1 , wherein said first and second strands of said isolated double stranded nucleic acid are, independently, 27 nucleotide residues in length. 
     
     
         7 . The method 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. 
     
     
         8 . The method 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. 
     
     
         9 . The method of  claim 1 , wherein said second strand is fully complementary to the selected target sequence. 
     
     
         10 . The method of  claim 1 , wherein said 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. 
     
     
         11 . The method 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. 
     
     
         12 . The method 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. 
     
     
         13 . The method of  claim 1 , wherein said double stranded nucleic acid reduces target gene expression when introduced into 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%. 
     
     
         14 . The method of  claim 1 , wherein the first and second strands of said isolated double stranded nucleic acid are joined by a chemical linker. 
     
     
         15 . The method 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. 
     
     
         16 . A method for preparing an isolated double stranded nucleic acid that corresponds to a selected target sequence of a target gene, comprising:
 synthesizing first and second oligonucleotide strands, wherein each of said first and said second strands has a 5′ terminus and a 3′ terminus and is 27 nucleotides in length, wherein said first and second strands are complementary to each other such that they form a duplex, 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 when said duplex is formed, and wherein said second strand comprises a nucleotide sequence complementary to the selected target sequence such that it anneals to said target sequence under biological conditions, and   annealing said first and said second oligonucleotide strands to form a double stranded nucleic acid, wherein said double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell,   thereby preparing said isolated double stranded nucleic acid.   
     
     
         17 . The method of  claim 16 , wherein said 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 method of  claim 16 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         19 . The method of  claim 16 , wherein said double stranded nucleic acid reduces target gene expression when introduced into 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%. 
     
     
         20 . A method for preparing an isolated double stranded nucleic acid that corresponds to a selected target sequence of a target gene, comprising:
 synthesizing first and second oligonucleotide strands, wherein each of said first and said second strands has a 5′ terminus and a 3′ terminus, consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein said first and second strands are complementary to each other such that they form a duplex of at least 25 nucleotides, 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 one or two mismatched base pairs when said duplex is formed, and wherein said second strand comprises a nucleotide sequence complementary to the selected target sequence such that it anneals to said target sequence under biological conditions, and   annealing said first and said second oligonucleotide strands to form a double stranded nucleic acid, wherein said double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell,   thereby preparing said isolated double stranded nucleic acid.   
     
     
         21 . The method 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 form two mismatched base pairs. 
     
     
         22 . The method of  claim 20 , wherein said 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. 
     
     
         23 . The method 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. 
     
     
         24 . The method of  claim 20 , wherein said double stranded nucleic acid reduces target gene expression when introduced into 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%. 
     
     
         25 . A method for preparing an isolated double stranded nucleic acid that corresponds to a selected target sequence of a target gene, comprising:
 synthesizing first and second oligonucleotide strands, wherein each of said first and said second strands has a 5′ terminus and a 3′ terminus and is 27 nucleotides in length, wherein said first and second strands are complementary to each other such that they form a duplex, 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 when said duplex is formed, and wherein said second strand comprises a nucleotide sequence complementary to the selected target sequence such that it anneals to said target sequence under biological conditions, and   annealing said first and said second oligonucleotide strands to form a double stranded nucleic acid, wherein said double stranded nucleic acid reduces target gene expression when introduced into a mammalian cell,   thereby preparing said isolated double stranded nucleic acid.   
     
     
         26 . The method of  claim 25 , wherein said 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. 
     
     
         27 . The method of  claim 25 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         28 . The method of  claim 25 , wherein said double stranded nucleic acid reduces target gene expression when introduced into 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%.