RNA interference mediated inhibition of gene expression using chemically modified short interfering nucleic acid (siNA)
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 compound comprising:
(I) a short interfering nucleic acid (siNA) molecule capable of inhibiting expression of a human transthyretin (TTR) gene, said siNA molecule consisting of a single-stranded polynucleotide comprising a nucleotide sequence complementary to a TTR RNA, wherein said single-stranded polynucleotide:
i) is about 18 to 20 nucleotides in length;
ii) comprises 10 or more phosphorothioate internucleotide linkages;
iii) comprises 10 or more modified pyrimidines chosen from 2′ deoxy pyrimidine, 2′-O-methyl pyrimidine, 2′-deoxy-2′-fluoro pyrimidine, locked nucleic acid (LNA) pyrimidine, 2′-methoxyethoxy (MOE) pyrimidine, or a combination thereof; and
(II) a galactosamine.
2. The compound of claim 1 , wherein the single-stranded polynucleotide comprises 10 or more 2′-deoxy pyrimidines.
3. The compound of claim 1 , wherein the single-stranded polynucleotide comprises one or more 2′-methoxyethoxy (MOE) pyrimidines.
4. The compound of claim 1 , wherein the galactosamine comprises N-acetylgalactosamine which is a mono-antennary galactosamine, bi-antennary galactosamine, or a tri-antennary galactosamine.
5. The compound of claim 1 , wherein the galactosamine is attached at the 5′-end or the 3′-end of the single-stranded polynucleotide.
6. The compound of claim 1 , wherein the single stranded polynucleotide is 20 nucleotides long.
7. The compound of claim 1 , wherein the 10 or more modified pyrimidines comprise 4 or more 2′ deoxy pyrimidines.
8. A short interfering nucleic acid (siNA) molecule capable of inhibiting expression of a human transthyretin (TTR) gene, said siNA molecule consisting of a single-stranded polynucleotide comprising a nucleotide sequence complementary to a human TTR RNA, wherein said single-stranded polynucleotide:
i) is about 18 to 20 nucleotides in length;
ii) comprises 10 or more phosphorothioate internucleotide linkages;
iii) comprises 10 or more modified pyrimidines chosen from 2′ deoxy pyrimidine, 2′-O-methyl pyrimidine, 2′-deoxy-2′-fluoro pyrimidine, 2′-methoxyethoxy (MOE) pyrimidine, or a combination thereof.
9. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide further comprises a ligand chosen from a galactosamine or a cholesterol.
10. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide further comprises a ligand chosen from a mono-antennary galactosamine, bi-antennary galactosamine, or a tri-antennary galactosamine.
11. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide comprises one or more 2′-methoxyethoxy (MOE) pyrimidines.
12. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide comprises 1, 2, 3, 4, 5 or more locked nucleic acid (LNA) pyrimidines, wherein said LNA pyrimidines is/are located at the 5′-end, the 3′-end, both of the 5′ and 3′-ends, of the single-stranded polynucleotide.
13. The siNA molecule of claim 8 , wherein the 10 or more modified pyrimidines comprise 4 or more 2′ deoxy pyrimidines.
14. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide comprises 10 or more 2′-deoxy nucleotides.
15. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide comprises one or more 5-methylcytosines.
16. The siNA molecule of claim 8 , wherein the single-stranded polynucleotide comprises 5 or more 5-methylcytosines.
17. A short interfering nucleic acid (siNA) molecule capable of inhibiting expression of a human transthyretin (TTR) gene, said siNA molecule consisting of a single-stranded polynucleotide comprising a nucleotide sequence complementary to a human TTR RNA, wherein said single-stranded polynucleotide:
i) is 20 nucleotides in length;
ii) comprises 10 or more phosphorothioate internucleotide linkages;
iii) comprises 10 or more modified pyrimidines chosen from 2′ deoxy pyrimidine, 2′-O-methyl pyrimidine, 2′-deoxy-2′-fluoro pyrimidine, 2′-methoxyethoxy (MOE) pyrimidine, or a combination thereof; and
iv) comprises one or more 5-methylcytosines.
18. The siNA molecule of claim 17 , which comprises 5 or more 5-methylcytosine nucleotides.
19. The siNA molecule of claim 17 , wherein the single-stranded polynucleotide comprises one or more 2′-methoxyethoxy (MOE) pyrimidines.
20. The siNA molecule of claim 17 , wherein the 10 or more modified pyrimidines comprise 4 or more 2′ deoxy pyrimidines.
21. The siNA molecule of claim 17 , wherein the single-stranded polynucleotide comprises 10 or more modified pyrimidines chosen from 2′ deoxy pyrimidine or 2′-methoxyethoxy (MOE) pyrimidine, or a combination thereof.