Reduction of off-target RNA interference toxicity
The present invention is directed to RNA interference (RNAi) molecules targeted against a nucleic acid sequence, and methods of using these RNAi molecules to reduce off-target toxicity.
1. An isolated nucleic acid comprising miSCA1-1399 (SEQ ID NO: 209).
2. An isolated nucleic acid consisting of miSCA1-1399 (SEQ ID NO: 209).
3. An expression cassette comprising a promoter linked to a sequence encoding the nucleic acid of claim 1 .
4. The expression cassette of claim 3 , wherein the promoter is a polII or polIII promoter.
5. The expression cassette of claim 4 , wherein the polIII promoter is a U6 promoter.
6. The expression cassette of claim 4 , wherein the polIII promoter is a mouse U6 promoter.
7. The expression cassette of claim 3 , wherein the promoter is a polII promoter.
8. The expression cassette of claim 3 , wherein the promoter is a tissue-specific promoter.
9. The expression cassette of claim 3 , wherein the promoter is an inducible promoter.
10. The expression cassette of claim 3 , further comprising a marker gene.
11. A vector comprising the expression cassette of claim 3 .
12. The vector of claim 11 , wherein the vector is an adeno-associated virus (AAV) vector.
13. A non-human animal comprising the nucleic acid of claim 1 .
14. A method of delivering the nucleic acid of claim 1 to a cell of a subject, comprising administering the nucleic acid to the subject.
15. A nucleic acid encoding an artificial primary miRNA transcript (pri-miRNA) consisting of, in order of position, a 5′-flanking region, a first siRNA region, a loop region, a second siRNA region, and a 3′-flanking region, wherein the 5′-flanking region comprises:
(a) a 5′-joining sequence contiguously linked to the first siRNA region, wherein the 5′-joining sequence consists of 5-7 nucleotides;
(b) a 5′-bulge sequence positioned upstream from the 5′-joining sequence, wherein the 5′-bulge sequence consists of 1-10 nucleotides; and
(c) a 5′-spacer sequence positioned upstream from the 5′-bulge sequence, wherein the 5′-spacer sequence consists of 10-12 nucleotides; and
(d) a 5′-upstream sequence positioned upstream from the 5′-spacer sequence, wherein the 5′-upstream sequence is about 30-2000 nucleotides in length;
and wherein the 3′-flanking region comprises:
(e) a 3′-joining sequence contiguously linked to the second siRNA region, wherein the 3′-joining sequence consists of 5-7 nucleotides;
(f) a 3′-bulge sequence positioned downstream from the 3′-joining sequence, wherein the 3′-bulge sequence consists of 1-10 nucleotides;
(g) a 3′-spacer sequence positioned downstream from the 3′-bulge sequence, wherein the 3′-spacer sequence consists of 10-12 nucleotides; and
(h) a 3′-downstream sequence positioned downstream from the 3′-spacer sequence, wherein the 3′-downstream sequence is about 30-2000 nucleotides in length.
16. A vector comprising the nucleic acid of claim 15 .
17. The vector of claim 16 , wherein the vector is an adeno-associated virus (AAV) vector.
18. A method of delivering the vector of claim 17 to a cell of a subject, comprising administering the vector to the subject.
19. The method of claim 18 , wherein the vector is an adeno-associated virus (AAV) vector.
20. A vector comprising the nucleic acid of claim 1 .
21. The vector of claim 20 , wherein the vector is an adeno-associated virus (AAV) vector.
22. A method of delivering the vector of claim 20 to a cell of a subject, comprising administering the vector to the subject.
23. The method of claim 22 , wherein the vector is an adeno-associated virus (AAV) vector.
24. A method of delivering the vector of claim 11 to a cell of a subject, comprising administering the nucleic acid to the subject.
25. The method of claim 24 , wherein the vector is an adeno-associated virus (AAV) vector.