RNA INTERFERENCE MEDIATED INHIBITION OF GENE EXPRESSION USING CHEMICALLY MODIFIED SHORT INTERFERING NUCLEIC ACID (siNA)
The present invention concerns methods and reagents useful 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, issue, or organism.
1 - 10 . (canceled)
11 . A method for synthesis of a chemically modified siNA molecule for inhibiting expression of a target gene, the method comprising:
(a) providing a sense strand and antisense strand of the siNA molecule;
(b) annealing the sense strand and antisense strand together under conditions suitable to form a double-stranded siNA molecule,
wherein the antisense strand comprises a 19 nucleotide sequence complementary to RNA of the target gene and the sense strand;
wherein the sense strand and the antisense strand of the siNA molecule are each no more than 100 nucleotides in length;
wherein the sense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof; and the antisense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof; and
wherein the sense strand or the antisense strand comprises a ligand.
12 . The method of claim 11 , wherein the ligand comprises a carbohydrate and the sense strand is conjugated to the carbohydrate.
13 . The method of claim 12 , wherein the carbohydrate comprises a galactosamine.
14 . The method of claim 13 , wherein the galactosamine comprises N-acetylgalactosamine.
15 . The method of claim 11 , wherein the ligand comprises a mono-antennary galactosamine, bi-antennary galactosamine, or a tri-antennary galactosamine.
16 . The method of claim 11 , wherein the siNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages in the sense strand.
17 . The method of claim 11 , wherein the siNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages in the antisense strand.
18 . The method of claim 11 , wherein the sense strand comprises 10 or more 2′-deoxy modified pyrimidines, 2′-O-methyl modified pyrimidines, 2′-deoxy-2′-fluoro modified pyrimidines; or a combination thereof.
19 . The method of claim 11 , wherein the antisense strand comprises 10 or more 2′-deoxy modified pyrimidines, 2′-O-methyl modified pyrimidines, 2′-deoxy-2′-fluoro modified pyrimidines; or a combination thereof.
20 . The method of claim 11 , wherein the sense strand is no more than 80 nucleotides in length.
21 . The method of claim 11 , wherein the sense strand is no more than 50 nucleotides in length.
22 . The method of claim 11 , wherein the antisense strand is no more than 80 nucleotides in length.
23 . The method of claim 11 , wherein the antisense strand is no more than 50 nucleotides in length.
24 . The method of claim 11 , wherein the sense strand and the antisense strand each comprise at least 21 nucleotides.
25 . A method of inhibiting expression of a gene within a cell comprising introducing a siNA molecule into a cell under conditions suitable to inhibit expression of the gene in the cell, wherein the siNA molecule comprises a sense strand and an antisense strand,
wherein the antisense strand comprises a 19 nucleotide sequence complementary to RNA of the gene and the sense strand;
wherein the sense strand and the antisense strand of the siNA molecule are each no more than 100 nucleotides in length;
wherein the sense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof; and the antisense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof; and
wherein the sense strand or the antisense strand comprises a ligand.
26 . The method of claim 25 , wherein the cell in an organism.
27 . An siNA molecule made by the method of claim 11 .
28 . A method for synthesis of a chemically modified siNA molecule for inhibiting expression of a target gene, the method comprising:
(a) synthesizing a sense strand and antisense strand of the siNA molecule;
(b) conjugating a ligand to the sense strand or the antisense stand;
(c) annealing the sense strand and antisense strand together under conditions suitable to form a double-stranded siNA molecule,
wherein the antisense strand comprises a 19 nucleotide sequence complementary to RNA of the target gene and the sense strand;
wherein the sense strand and the antisense strand of the siNA molecule are each no more than 100 nucleotides in length; and
wherein the sense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof; and the antisense strand comprises 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro modified nucleotides, or a combination thereof.