RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)

Abstract

The present invention concerns methods and reagents useful in modulating gene expression in a variety of applications, including use in therapeutic, diagnostic, target validation, and genomic discovery applications. Specifically, the invention relates to synthetic chemically modified small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules capable of mediating RNA interference (RNAi) against target nucleic acid sequences. The small nucleic acid molecules are useful in the treatment of any disease or condition that responds to modulation of gene expression or activity in a cell, tissue, or organism.

1 . A method of synthesizing a double stranded nucleic acid molecule consisting of a sense strand and an antisense strand, wherein: 
 (a) each strand of said double stranded nucleic acid molecule is 19 to 29 nucleotides in length;    (b) at least 19 nucleotides of the sense strand are complementary to the antisense strand    (c) the antisense strand of said double stranded nucleic acid molecule has complementarity to a target RNA encoded by a human gene;    (d) at least 90% of the nucleotides of each strand of said double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and    (e) at least two of said sugar modifications are different from each other;    the method comprising (i) synthesizing two strands of nucleic acid molecules that have the structural features described in parts (a)-(e), and (ii) annealing the two complementary strands under conditions suitable to obtain a double-stranded nucleic acid molecule.    
     
     
         2 . The method of  claim 1 , wherein said double stranded nucleic acid molecule comprises no ribonucleotides.  
     
     
         3 . The method of  claim 1 , wherein said double stranded nucleic acid molecule comprises ribonucleotides.  
     
     
         4 . The method of  claim 1 , wherein pyrimidine nucleotides in the sense strand are 2′-O-methyl pyrimidine nucleotides.  
     
     
         5 . The method of  claim 1 , wherein purine nucleotides in the sense strand are 2′-deoxy purine nucleotides.  
     
     
         6 . The method of  claim 1 , wherein the pyrimidine nucleotides present in the sense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.  
     
     
         7 . The method of  claim 1 , wherein the sense strand has a terminal cap moiety at the 5′-end, the 3′-end, or both of the 5′ and 3′ ends.  
     
     
         8 . The method of  claim 7 , wherein said terminal cap moiety is an inverted deoxy abasic moiety.  
     
     
         9 . The method of  claim 1 , wherein the pyrimidine nucleotides of said antisense strand are 2′-deoxy-2′-fluoro pyrimidine nucleotides.  
     
     
         10 . The method of  claim 1 , wherein the purine nucleotides of said antisense strand are 2′-O-methyl purine nucleotides.  
     
     
         11 . The method of  claim 1 , wherein the purine nucleotides present in said antisense strand comprise 2′-deoxy-purine nucleotides.  
     
     
         12 . The method of  claim 10 , wherein said antisense strand comprises a phosphorothioate internucleotide linkage at the 3′ end.  
     
     
         13 . The method of  claim 1 , wherein each of the two strands of said double stranded nucleic acid molecule is 21 nucleotides in length.  
     
     
         14 . The method of  claim 13 , wherein at least two 3′ terminal nucleotides of each strand of the double stranded nucleic acid molecule are not base-paired to the nucleotides of the other strand of the double stranded nucleic acid molecule.  
     
     
         15 . The method of  claim 14 , wherein each of the two 3′ terminal nucleotides of each strand of the double stranded nucleic acid molecule are 2′-deoxy-pyrimidines.  
     
     
         16 . The method of  claim 15 , wherein said 2′-deoxy-pyrimidine is 2′-deoxythymidine.  
     
     
         17 . The method of  claim 13 , wherein all 21 nucleotides of each strand of the double stranded nucleic acid molecule are base-paired to the complementary nucleotides of the other strand of the double stranded nucleic acid molecule.  
     
     
         18 . The method of  claim 13 , wherein 19 nucleotides of the antisense strand are base-paired to the target RNA.  
     
     
         19 . The method of  claim 13 , wherein 21 nucleotides of the antisense strand are base-paired to the target RNA.  
     
     
         20 . The method of  claim 1 , wherein the 5′-end of the antisense strand includes a phosphate group.  
     
     
         21 . The method of  claim 1 , wherein said sugar modifications are selected from the group consisting of 2′-H, 2′-O-alkyl, 2′-O-CF 3  and 2′-deoxy-2′-fluoro.  
     
     
         22 . The method of  claim 1 , wherein each strand of said double stranded nucleic acid molecule is about 19 to 23 nucleotides in length and at least about 19 nucleotides of the sense strand are complementary to the antisense strand 23. A method of synthesizing a double stranded nucleic acid molecule consisting of a sense strand and an antisense strand, wherein: 
 (a) each strand of said double stranded nucleic acid molecule is 18 to 27 nucleotides in length;    (b) 18-23 nucleotides of the sense strand are complementary to the antisense strand    (c) the antisense strand of said double stranded nucleic acid molecule has complementarity to a target RNA encoded by a human gene;    (d) at least 90% of the nucleotides of each strand of said double stranded nucleic acid molecule are modified nucleosides having a sugar modification; and    (e) at least two of said sugar modifications are different from each other; and    the method comprising (i) synthesizing two strands of nucleic acid molecules that have the structural features described in parts (a)-(e), and (ii) annealing the two complementary strands under conditions suitable to obtain a double-stranded nucleic acid molecule.