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

A chemically modified double-stranded short interfering nucleic acid molecule comprising a sense strand and an antisense strand that mediates RNA interference (RNAi) against a target RNA sequence, wherein:

• a) the antisense strand of the nucleic acid molecule is complementary to the target RNA sequence;
• b) each strand of the nucleic acid molecule is independently 18 to 24 nucleotides in length;
• c) the sense strand comprises 10 or more modified nucleotides chosen from 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides, or a combination thereof;
• d) the antisense strand comprises 10 or more modified nucleotides chosen from 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides, or a combination thereof;
• and
• e) 10 or more pyrimidine nucleotides of either the sense strand or the antisense strand, or both the sense strand and the antisense strand, are chemically modified with 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotides, or a combination thereof,
• wherein at least one strand of the nucleic acid molecule comprises a ligand.

A conjugate molecule comprising a chemically modified double-stranded short interfering nucleic acid molecule, said nucleic acid molecule comprising a sense strand and an antisense strand that mediates RNA interference (RNAi) against a target RNA sequence, wherein:

• a) the antisense strand of the nucleic acid molecule is complementary to the target RNA sequence;
• b) each strand of the nucleic acid molecule is independently 18 to 24 nucleotides in length;
• c) the sense strand comprises 10 or more modified nucleotides chosen from 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides, or a combination thereof;
• d) the antisense strand comprises 10 or more modified nucleotides chosen from 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, or universal base modified nucleotides, or a combination thereof;
• and
• e) 10 or more pyrimidine nucleotides of either the sense strand or the antisense strand, or both the sense strand and the antisense strand, are chemically modified with 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotides, or a combination thereof.

A conjugate molecule comprising:

• a double-stranded short interfering nucleic acid molecule comprising a sense strand and an antisense strand that mediates RNA interference (RNAi) against a target RNA sequence, wherein:
• a) the antisense strand of the nucleic acid molecule is complementary to the target RNA sequence;
• b) each strand of the nucleic acid molecule is independently 18 to 24 nucleotides in length;
• c) the sense strand comprises 10 or more modified nucleotides chosen from 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof;
• d) the antisense strand comprises 10 or more modified nucleotides chosen from 2′-O-methyl or 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof;
• and
• e) 10 or more pyrimidine nucleotides of either the sense strand or the antisense strand, or both the sense strand and the antisense strand, are 2′-O-methyl or 2′-deoxy-2′-fluoro nucleotides, or a combination thereof,
• f) the 3′ end of the antisense strand comprises a nucleotide overhang of 2 nucleotides in length;
• g) the antisense strand comprises one or more phosphorothioate internucleotide linkages at the 3′-end;
• and
• f) the 3′ end of the sense strand is blunt-ended and comprises a tri-antennary N-acetylgalactosamine.

A short interfering RNA (siRNA) molecule comprising a sense strand and an antisense strand that mediates RNA interference, wherein:

• (a) each strand is independently between 18 and 24 nucleotides in length;
• (b) the sense strand comprises 10 or more modified nucleotides chosen from 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof;
• (c) the antisense strand comprises 10 or more modified nucleotides chosen from 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof;
• (d) 10 or more pyrimidine nucleotides of the sense strand, the antisense strand, or both the sense and antisense strand, are 2′-deoxy, 2′-O-methyl, or 2′-deoxy-2′-fluoro nucleotides, or a combination thereof;
• and
• (e) at least one strand comprises a galactosamine moiety.