Reduction of off-target RNA interference toxicity

Patent metadata

Field	Value
Publication number	US-9650631-B2
Application number	US-201314011539-A
Country	US
Kind code	B2
Filing date	Aug 27, 2013
Priority date	Apr 26, 2007
Publication date	May 16, 2017
Grant date	May 16, 2017

Abstract

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.

First claim

What is claimed is:

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.

Assignees

• Univ Iowa Res Found

Inventors

• Davidson Beverly L
• Mas Monteys Alejandro
• Mcbride Jodi L
• Boudreau Ryan

Classifications

• C12N2310/141

  MicroRNAs, miRNAs · CPC title

• C12N15/113 Primary

  Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)} · CPC title

• C12N2310/14

  interfering nucleic acids [NA] · CPC title

• C12N2320/30

  Special therapeutic applications · CPC title

• C12N15/111 Primary

  General methods applicable to biologically active non-coding nucleic acids · CPC title