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 - 10 . (canceled) 
     
     
         11 . A double-stranded short interfering nucleic acid (siNA) molecule comprising a sense strand and an antisense strand, wherein:
 a) the antisense strand is complementary to the nucleotide sequence of SEQ ID NO:13;   b) each strand of said double-stranded siNA molecule is 19 to 23 nucleotides in length;   c) each strand of said double-stranded siNA molecule comprises at least 19 nucleotides that are complementary to the nucleotides of the other strand;   d) said siNA molecule comprises no ribonucleotides; and   e) the sense strand comprises a terminal cap moiety at the 3′ end of the sense strand,   wherein the siNA molecule downregulates expression of a Hepatitis B virus target gene.   
     
     
         12 . The siNA molecule of  claim 11 , wherein said terminal cap moiety is an inverted deoxy abasic moiety. 
     
     
         13 . The siNA molecule of  claim 11 , wherein said antisense region comprises a phosphorothioate internucleotide linkage at the 3′ end of said antisense region. 
     
     
         14 . The siNA molecule of  claim 11 , wherein each of the two strands of said siNA molecule are 21 nucleotides in length. 
     
     
         15 . The siNA molecule of  claim 14 , wherein all 21 nucleotides of the sense strand of the siNA molecule are base-paired to the complementary nucleotides of the antisense strand of the siNA molecule. 
     
     
         16 . The siNA molecule of  claim 14 , wherein at least 19 nucleotides of the antisense strand are base-paired to the nucleotide sequence or a portion thereof of the RNA encoded by the target gene. 
     
     
         17 . The siNA molecule of  claim 11 , wherein:
 (a) the sense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides;   (b) the antisense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides; and   (c) 10 or more pyrimidine nucleotides of the sense and/or antisense strand are 2′-deoxy, 2′-O-methyl or 2′-deoxy-2′-fluoro nucleotides.   
     
     
         18 . A pharmaceutical composition comprising the siNA molecule of claim  1  in an acceptable carrier or diluent. 
     
     
         19 . A double-stranded short interfering nucleic acid (siNA) molecule comprising a sense strand and an antisense strand, wherein:
 a) the antisense strand is complementary to the nucleotide sequence of SEQ ID NO: 13;   b) each strand of said double-stranded siNA molecule is 21 nucleotides in length;   c) all 21 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule;   (d) the sense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides;   (e) the antisense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides; and   (f) 10 or more pyrimidine nucleotides of the sense and/or antisense strand are 2′-deoxy, 2′-O-methyl or 2′-deoxy-2′-fluoro nucleotides; and   wherein the siNA molecule downregulates expression of a Hepatitis B virus target gene.   
     
     
         20 . A pharmaceutical composition comprising the siNA molecule of  claim 19  in an acceptable carrier or diluent. 
     
     
         21 . A double-stranded short interfering nucleic acid (siNA) molecule comprising a sense strand and an antisense strand, wherein:
 a) the antisense strand is complementary to the nucleotide sequence of SEQ ID NO: 13;   b) each strand of said double-stranded siNA molecule is 21 nucleotides in length;   c) all 21 nucleotides of each strand of the siNA molecule are base-paired to the complementary nucleotides of the other strand of the siNA molecule;   d) said siNA molecule comprises no ribonucleotides;   e) said siNA molecule comprises an inverted deoxy abasic cap moiety; and   wherein the siNA molecule inhibits replication of a Hepatitis B virus.   
     
     
         22 . The siNA molecule of  claim 21 , wherein:
 (a) the sense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides;   (b) the antisense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides; and   (c) 10 or more pyrimidine nucleotides of the sense and/or antisense strand are 2′-deoxy, 2′-O-methyl or 2′-deoxy-2′-fluoro nucleotides.   
     
     
         23 . A pharmaceutical composition comprising the siNA molecule of  claim 22  in an acceptable carrier or diluent.