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 double stranded nucleic acid capable of reducing expression of a target gene comprising:
a first oligonucleotide strand of 25 to 30 nucleotides in length comprising a 3′ terminus and a 5′ terminus, and
a second oligonucleotide strand comprising 19-21 nucleotides and a 3′ terminus and a 5′ terminus,
wherein said second strand is annealed to said first strand to form a duplex region of 19 to 21 nucleotides in length,
wherein the 5′ terminus of said first strand consists of 4-30 unpaired nucleotides that overhang the 3′ terminus of the second strand, wherein each of the 4-30 unpaired nucleotides is a modified nucleotide, wherein the modified nucleotide comprises a modified sugar moiety, and wherein the modified sugar moiety comprises a 2′-O-methyl and/or 2′-fluoro; and
wherein said second strand is sufficiently complementary to a target RNA along at least 19 nucleotides of said second strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.
2. The double stranded nucleic acid of claim 1 , wherein the 5′ terminus of said first strand consists of 4 to 15 unpaired nucleotides that overhang the 3′ terminus of the second strand.
3. The double stranded nucleic acid of claim 1 , wherein the 5′ or 3′ terminus of said first strand is conjugated to a non-nucleic acid moiety.
4. The double stranded nucleic acid of claim 3 , wherein the non-nucleic acid moiety is a peptide or an organic compound.
5. The double stranded nucleic acid of claim 3 , wherein the non-nucleic acid moiety increases cellular uptake.
6. The double stranded nucleic acid of claim 3 , wherein the non-nucleic acid moiety possesses enhanced cellular targeting of the double stranded nucleic acid, increases stability of the double stranded nucleic acid, facilitates tracking of the double stranded nucleic acid, increases the binding affinity of the double stranded nucleic acid, or decreases the immunogenicity of the double stranded nucleic acid.
7. The double stranded nucleic acid of claim 1 , wherein said double stranded nucleic acid is cleaved endogenously to produce a double stranded nucleic acid that reduces target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.
8. The double stranded nucleic acid of claim 7 , wherein said double stranded nucleic acid is cleaved by Dicer.
9. The double stranded nucleic acid of claim 1 , wherein each of the first and second strands comprise at least one modified nucleotide.
10. The double stranded nucleic acid of claim 1 , wherein all of the nucleotides of the double stranded nucleic acid are modified.
11. The double stranded nucleic acid of claim 9 , wherein the at least one modified nucleotide comprises a modified base, a modified phosphate backbone, and/or a modified sugar moiety.
12. The double stranded nucleic acid of claim 11 , wherein the modified sugar moiety is selected from the group consisting of 2′-O-methyl, 2′-methoxyethoxy, 2′-fluoro, 2′-allyl, 2′-O[2-(methylamino)-2-oxoethyl], 4′-thio, 4′-CH2-O-2′-bridge, 4′-(CH2)2-O-2′-bridge, 2′-LNA, 2′-amino and 2′-O—(N-methylcarbamate).
13. The double stranded nucleic acid molecule of claim 10 , wherein all of the nucleotides of the double stranded nucleic acid comprise a modified sugar moiety and the modified sugar moiety comprises a 2′-O-methyl and/or 2′-fluoro.
14. The double stranded nucleic acid of claim 11 , wherein the modified phosphate backbone is selected from the group consisting of: a phosphonate, a phosphorothioate and a phosphotriester.
15. The double stranded nucleic acid molecule of claim 13 , further comprising at least one phosphorothioate in each of the first and second strands.
16. The double stranded nucleic acid of claim 1 , wherein the first strand has a nucleotide sequence that is at least 80%, 90%, 95% or 100% complementary to the second strand nucleotide sequence.
17. The double stranded nucleic acid of claim 1 , wherein the nucleic acid reduces target gene expression in a mammalian cell in vitro by at least 50%.
18. The double stranded nucleic acid of claim 1 , wherein the duplex region comprises a mismatch.
19. The double stranded nucleic acid of claim 18 , wherein the duplex region comprises a base analog selected from the group consisting of: hypoxanthine (I), xanthine (X), 3β-D-ribofuranosyl-(2,6-diaminopyrimidine) (K), 3β-D-ribofuranosyl-(1-methyl-pyrazolo [4,3-d]pyrimidine-5,7(4H, 6H)-dione) (P), iso-cytosine (iso-C), iso-guanine (iso-G), 1-β-D-ribofuranosyl-(5-nitroindole), 1-β-D-ribofuranosyl-(3-nitropyrrole), 5-bromouracil, 2-aminopurine, 4-thio-dT, 7-(2-thienyl)-imidazo[4,5-b]pyridine (Ds), pyrrole-2-carbaldehyde (Pa), 2-amino-6-(2-thienyl)purine (S), 2-oxopyridine (Y), difluorotolyl, 4-fluoro-6-methylbenzimidazole, 4-methylbenzimidazole, 3-methyl isocarbostyrilyl, 5-methyl isocarbostyrilyl, 3-methyl-7-propynyl isocarbostyrilyl, 7-azaindolyl, 6-methyl-7-azaindolyl, imidizopyridinyl, 9-methyl-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl, 7-propynyl isocarbostyrilyl, propynyl-7-azaindolyl, 2,4,5-trimethylphenyl, 4-methylindolyl, 4,6-dimethylindolyl, phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl, stilbenzyl, tetracenyl and pentacenyl.
20. The double stranded nucleic acid of claim 19 , wherein the base analog comprises a universal base.
21. A method for reducing expression of a target gene in a cell, comprising: contacting a cell with the double stranded nucleic acid of claim 1 in an amount effective to reduce expression of a target gene in a cell in comparison to a reference nucleic acid.
22. A method for reducing expression of a target gene in an animal, comprising: administering the double stranded nucleic acid of claim 1 to an animal with in an amount effective to reduce expression of a target gene in a cell.
23. A pharmaceutical composition for reducing expression of a target gene in a cell of a subject comprising the double stranded nucleic acid of claim 1 and a pharmaceutically acceptable carrier.