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 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   said first oligonucleotide strand has a 5′ terminus and a 3′ terminus and comprises 25-30 nucleotides, wherein 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   said second oligonucleotide strand comprises 26 or more nucleotides, wherein said first and second strands are complementary to each other such that they form a duplex of at least 25 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said 5′ terminus of said first strand and said 5′ terminus of said second strand and said 3′ terminus of said first strand form a blunt end 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 said first strand of said double stranded nucleic acid has a length which is at least 26 nucleotides. 
     
     
         6 . The method of  claim 1 , wherein said double stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length. 
     
     
         7 . The method of  claim 1 , wherein said second strand is 2 nucleotides longer at its 3′ terminus than said first strand. 
     
     
         8 . The method 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. 
     
     
         9 . The method of  claim 1 , wherein the blunt end formed by said 3′ terminus of said first strand and said 5′ terminus of said second strand is a base-paired blunt end. 
     
     
         10 . The method of  claim 1 , wherein said modified nucleotide residue of said 3′ terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         11 . The method of  claim 1 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide. 
     
     
         12 . The method of  claim 1 , wherein positions 1 and 2 of said 3′ terminus of the first oligonucleotide strand are deoxyribonucleotides. 
     
     
         13 . 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. 
     
     
         14 . The method of  claim 1 , wherein said second strand is fully complementary to the selected target sequence. 
     
     
         15 . The method of  claim 1 , wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate. 
     
     
         16 . The method of  claim 1 , wherein the relative length in nucleotide residues of said second and first strands 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 ucleotide residues in length. 
     
     
         17 . The method of  claim 1 , further comprising joining the first and second strands by a chemical linker. 
     
     
         18 . The method of  claim 1 , further comprising joining the 3′ terminus of said first strand and said 5′ terminus of said second strand by a chemical linker. 
     
     
         19 . The method of  claim 1 , wherein a nucleotide of said second or first strand is substituted with a modified nucleotide that directs the orientation of Dicer cleavage. 
     
     
         20 . The method 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. 
     
     
         21 . 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. 
     
     
         22 . 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%. 
     
     
         23 . 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   said first oligonucleotide strand has a 5′ terminus and a 3′ terminus and comprises 25-29 nucleotides, and   said second oligonucleotide strand comprises 26-30 nucleotides, wherein said first and second strands are complementary to each other such that they form a duplex, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said 5′ terminus of said first strand and said 5′ terminus of said second strand and said 3′ terminus of said first strand form a blunt end 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.   
     
     
         24 . The method of  claim 23 , 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. 
     
     
         25 . The method of  claim 23 , wherein said double-stranded nucleic acid comprises a duplex region of at least 25 nucleotides in length. 
     
     
         26 . The method of  claim 23 , wherein said modified nucleotide residue of said 3′ terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         27 . The method of  claim 23 , 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 23 , 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%. 
     
     
         29 . 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   said first oligonucleotide strand has a 5′ terminus and a 3′ terminus and comprises 25-29 nucleotides, wherein 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 said second oligonucleotide strand comprises 26-30 nucleotides, wherein said first and second strands are complementary to each other such that they form a duplex of at least 25 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said 5′ terminus of said first strand and said 5′ terminus of said second strand and said 3′ terminus of said first strand form a blunt end 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.   
     
     
         30 . The method of  claim 29 , wherein said modified nucleotide residue of said 3′ terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         31 . The method of  claim 29 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         32 . The method of  claim 29 , 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%. 
     
     
         33 . 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   said first oligonucleotide strand has a 5′ terminus and a 3′ terminus and consists of 25 nucleotides, wherein 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   said second oligonucleotide strand consists of 27 nucleotides, wherein said first and second strands are complementary to each other such that they form a duplex, wherein said second strand is two nucleotides longer at its 3′ terminus than said 5′ terminus of said first strand and said 5′ terminus of said second strand and said 3′ terminus of said first strand form a blunt end 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.   
     
     
         34 . The method of  claim 33 , 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. 
     
     
         35 . The method of  claim 33 , wherein said modified nucleotide residue of said 3′ terminus of said first strand is selected from the group consisting of a deoxyribonucleotide, an acyclonucleotide and a fluorescent molecule. 
     
     
         36 . The method of  claim 33 , wherein said double stranded nucleic acid comprises a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         37 . The method of  claim 33 , 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%.