Extended dicer substrate agents and methods for the specific inhibition of gene expression
The invention provides compositions and methods for reducing expression of a target gene in a cell, involving contacting a cell with an isolated double stranded nucleic acid (dsNA) in an amount effective to reduce expression of a target gene in a cell. The dsNAs of the invention possess a single stranded extension (in most embodiments, the single stranded extension comprises at least one modified nucleotide and/or phosphate back bone modification). Such single stranded extended Dicer-substrate siRNAs (DsiRNAs) were demonstrated to be effective RNA inhibitory agents compared to corresponding double stranded DsiRNAs.
1. A method for reducing expression of a target RNA in a mammalian cell, the method comprising:
contacting said mammalian cell with a double stranded nucleic acid (dsNA) in an amount effective to reduce expression of said target RNA in said mammalian cell, wherein said dsNA comprises:
an antisense strand that comprises a 3′ terminus and a 5′ terminus, wherein said antisense strand is 21 nucleotides in length, wherein at least 13 of the nucleotides comprise a 2′-O-methyl modified sugar moiety, wherein at least one of the nucleotides comprises a 2′-fluoro modified sugar moiety, and wherein a phosphate backbone of said antisense strand comprises at least one phosphorothioate modification,
a sense strand that comprises a 3′ terminus and a 5′ terminus, wherein said sense strand is 21 nucleotides in length, wherein at least 18 of the nucleotides comprise a 2′-O-methyl modified sugar moiety, wherein at least one of the nucleotides comprises a 2′-fluoro modified sugar moiety, wherein a phosphate backbone of said sense strand comprises at least one phosphorothioate modification, wherein said sense strand comprises one or more inverted abasic residues, and wherein said sense strand is conjugated to a non-nucleic acid moiety or an organic compound,
wherein said antisense strand is annealed to said sense strand to form a duplex region of 21 nucleotides in length, and
wherein said antisense strand is sufficiently complementary to said target RNA to reduce said target RNA expression in said mammalian cell.
2. The method of claim 1 , wherein an organism comprises said mammalian cell.
3. The method of claim 2 , wherein said organism is human.
4. The method of claim 2 , wherein said contacting step comprises administering said dsNA to said organism via a route of administration selected from the group consisting of: parenteral, intravenous, intradermal, subcutaneous, oral, transdermal, transmucosal, and rectal administration.
5. The method of claim 2 , wherein said contacting step comprises administering said dsNA to said organism in a pharmaceutical composition that comprises a pharmaceutically acceptable carrier.
6. The method of claim 1 , wherein at least a fragment of said antisense strand is associated with an RNA-induced silencing complex (RISC), which fragment is complementary to and binds said target RNA, and promotes cleavage of said target RNA by said RISC.
7. The method of claim 1 , wherein one or both of said antisense and sense strands comprise a 5′ phosphate.
8. The method of claim 1 , wherein said antisense strand comprises one or more patterns of alternating nucleotides that comprise said 2′-O-methyl modified sugar moiety.
9. The method of claim 1 , wherein said antisense strand comprises 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides that comprise said 2′-O-methyl modified sugar moiety.
10. The method of claim 1 , wherein said antisense strand comprises one or more patterns of alternating nucleotides that comprise said 2′-O-methyl modified sugar moiety and said 2′-fluoro modified sugar moiety.
11. The method of claim 1 , wherein said sense strand comprises 19, 20, or 21 nucleotides that comprise said 2′-O-methyl modified sugar moiety.
12. The method of claim 1 , wherein said antisense strand is 100% complementary to said target RNA.
13. The method of claim 1 , wherein said dsNA forms one or more blunt ends.
14. The method of claim 1 , wherein said antisense strand comprises one or more patterns of alternating nucleotides that comprise said 2′-O-methyl modified sugar moiety and said 2′-fluoro modified sugar moiety, and wherein said sense strand comprises a 5′ phosphate.
15. The method of claim 1 , wherein said sense strand comprises one or more patterns of alternating nucleotides that comprise said 2′-O-methyl modified sugar moiety.
16. The method of claim 1 , wherein pyrimidines of said antisense strand are 2′-fluoro modified.
17. The method of claim 1 , wherein purines of said antisense strand are 2′-fluoro modified.
18. The method of claim 1 , wherein pyrimidines of said sense strand are 2′-fluoro modified.
19. The method of claim 1 , wherein purines of said sense strand are 2′-fluoro modified.
20. The method of claim 1 , wherein purines of said antisense strand are 2′-O-methyl modified.
21. The method of claim 1 , wherein purines of said sense strand are 2′-O-methyl modified.
22. The method of claim 1 , wherein said phosphorothioate modification of said antisense strand is a 3′-terminal phosphorothioate linkage.
23. The method of claim 1 , wherein said phosphorothioate modification of said sense strand is a 3′-terminal phosphorothioate linkage.