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 . 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 strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 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. 
     
     
         2 . The isolated double stranded nucleic acid of  claim 1 , wherein said second oligonucleotide strand is complementary to said target RNA along at least 19 nucleotides of said second oligonucleotide strand length. 
     
     
         3 . The isolated double stranded nucleic acid of  claim 1 , wherein said second strand is 2 nucleotides longer at its 3′ terminus than said first strand. 
     
     
         4 . The isolated double stranded nucleic acid of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a blunt end. 
     
     
         5 . The isolated double stranded nucleic acid of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a base-paired blunt end. 
     
     
         6 . The isolated double stranded nucleic acid of  claim 1 , 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. 
     
     
         7 . The isolated double stranded nucleic acid of  claim 6 , 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. 
     
     
         8 . The isolated double stranded nucleic acid of  claim 6 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide. 
     
     
         9 . The isolated double stranded nucleic acid of  claim 6 , wherein positions 1 and 2 of said 3′ terminus of the first oligonucleotide strand are deoxyribonucleotides. 
     
     
         10 . The isolated double stranded nucleic acid of  claim 1 , wherein said double stranded nucleic acid is cleaved endogenously in a mammalian cell by Dicer. 
     
     
         11 . The isolated double stranded nucleic acid 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. 
     
     
         12 . The isolated double stranded nucleic acid of  claim 1 , wherein said second strand is fully complementary to the target RNA. 
     
     
         13 . The isolated double stranded nucleic acid of  claim 1 , wherein said 5′ terminus of each of said first and said second strands comprises a 5′ phosphate. 
     
     
         14 . The isolated double stranded nucleic acid of  claim 1 , wherein the first and second strands are joined by a chemical linker. 
     
     
         15 . The isolated double stranded nucleic acid of  claim 1 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand are joined by a chemical linker. 
     
     
         16 . The isolated double stranded nucleic acid 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. 
     
     
         17 . The isolated double stranded nucleic acid of  claim 1  comprising 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 isolated double stranded nucleic acid of  claim 1  comprising a phosphate backbone modification selected from the group consisting of a phosphonate, a phosphorothioate and a phosphotriester. 
     
     
         19 . The isolated double stranded nucleic acid 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%. 
     
     
         20 . A method for reducing expression of a target gene in a mammalian cell comprising: contacting a mammalian cell in vitro with an isolated double stranded nucleic acid in an amount sufficient to reduce expression of a target gene in said cell, wherein said double stranded nucleic acid comprises a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein said first strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is 1-4 nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 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, thereby reducing expression of the target gene in said mammalian cell. 
     
     
         21 . The method of  claim 20 , wherein said second oligonucleotide strand is complementary to a target RNA along at least 19 nucleotides of said second oligonucleotide strand length. 
     
     
         22 . The method of  claim 20 , 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 
     
     
         23 . The method of  claim 20 , 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. 
     
     
         24 . The method of  claim 20 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide. 
     
     
         25 . The method of  claim 20 , wherein positions 1 and 2 of said 3′ terminus of the first oligonucleotide strand are deoxyribonucleotides. 
     
     
         26 . The method of  claim 20 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a blunt end. 
     
     
         27 . The method of  claim 20 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a base-paired blunt end. 
     
     
         28 . The method of  claim 20 , wherein said isolated double stranded nucleic acid is cleaved endogenously in said cell by Dicer. 
     
     
         29 . The method of  claim 20 , wherein target gene expression is reduced in the mammalian cell by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80%. 
     
     
         30 . A method for reducing expression of a target gene in a mammal comprising: administering an isolated double stranded nucleic acid to a mammal in an amount sufficient to reduce expression of a target gene in the mammal, wherein said double stranded nucleic acid comprises a first oligonucleotide strand having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand having a 5′ terminus and a 3′ terminus, wherein said first strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is 1-4 nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length, and wherein said second oligonucleotide strand of said isolated double stranded nucleic acid 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, thereby reducing expression of the target gene in the mammal. 
     
     
         31 . The method of  claim 30 , 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. 
     
     
         32 . The method of  claim 30 , 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. 
     
     
         33 . The method of  claim 30 , wherein position 1 of said 3′ terminus of the first oligonucleotide strand is a deoxyribonucleotide. 
     
     
         34 . The method of  claim 30 , wherein positions 1 and 2 of said 3′ terminus of the first oligonucleotide strand are deoxyribonucleotides. 
     
     
         35 . The method of  claim 30 , wherein said 3′ terminus of said first strand and said 5′ terminus of said second strand form a blunt end. 
     
     
         36 . The method of  claim 30 , wherein said 3′ terminus of said first strand and said 5′terminus of said second strand form a base-paired blunt end. 
     
     
         37 . The method of  claim 30 , wherein said isolated double stranded nucleic acid is administered at a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of body weight 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 0.1 to 2.5 micrograms per kilogram. 
     
     
         38 . The method of  claim 30 , wherein said administering step comprises a mode selected from the group consisting of intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral and inhaled delivery. 
     
     
         39 . The method of  claim 30 , wherein said isolated double stranded nucleic acid is cleaved endogenously in said cell to a length in the range of 19-23 nucleotides. 
     
     
         40 . The method of  claim 30 , wherein target gene expression is reduced in the mammal by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80%. 
     
     
         41 . 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, said first strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length, 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. 
     
     
         42 . 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, said first strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 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.   
     
     
         43 . 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 has a length which is at least 26 and at most 30 nucleotides and   said second oligonucleotide strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 nucleotides in length 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.   
     
     
         44 . A mammalian cell containing 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, said first strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 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 in said mammalian cell. 
     
     
         45 . 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, said first strand has a length which is at least 26 and at most 30 nucleotides and said second strand has a length which is at least 27 and at most 30 nucleotides, wherein said second strand is at least one and at most four nucleotides longer at its 3′ terminus than said first strand, wherein said double-stranded nucleic acid comprises a duplex region of at least 26 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.