Therapeutic compounds
The present invention is directed to RNA interference (RNAi) molecules targeted against a Huntington's disease nucleic acid sequence, and methods of using these RNAi molecules to treat Huntington's disease.
1. A nucleic acid encoding an artificial primary miRNA transcript (pri-miRNA) consisting of, in order of position, a 5′-flanking region, a non-guide region, a loop region, a guide region, and a 3′-flanking region, wherein the guide region comprises a sequence 100% identical to cgaccaugcgagccagca (miHDS.1 guide. SEQ ID NO:7) and the non-guide region is at least 80% complementary to the guide region.
2. The nucleic acid of claim 1 , wherein the guide region consists of 18-30 nucleotides.
3. The nucleic acid of claim 1 , wherein the 5′-flanking region comprises a 5′-joining sequence contiguously linked to the non-guide region.
4. The nucleic acid of claim 3 , wherein the 5′ joining sequence consists of 5-8 nucleotides.
5. The nucleic acid of claim 2 , wherein the 5′-flanking region further comprises
(i) a 5′-bulge sequence positioned upstream from the 5′ joining sequence;
(ii) a 5′-spacer sequence positioned upstream from the 5′-bulge sequence; and/or
(iii) a 5′-upstream sequence positioned upstream from the 5′-spacer sequence.
6. The nucleic acid of claim 5 , wherein the 5′-bulge sequence consists of 1-10 nucleotides, the 5′-spacer sequence consists of 10-12 nucleotides, and/or the 5′-upstream sequence consists of 30-2000 nucleotides.
7. The nucleic acid of claim 1 , wherein the 3′-flanking region comprises a 3′-joining sequence contiguously linked to the guide region.
8. The nucleic acid of claim 7 , wherein the 3′ joining sequence consists of 5-8 nucleotides.
9. The nucleic acid of claim 8 , wherein the 3′-joining sequence is at least about 85% complementary to the 5′-joining sequence.
10. The nucleic acid of claim 7 , wherein the 3′-flanking region further comprises
(i) a 3′-bulge sequence positioned downstream from the 3′ joining sequence;
(ii) a 3′-spacer sequence positioned downstream from the 3′-bulge sequence; and/or
(iii) a 3′-downstream sequence positioned downstream from the 3′-spacer sequence.
11. The nucleic acid of claim 10 , wherein the 3′-bulge sequence consists of 1-10 nucleotides, the 3′-spacer sequence consists of 10-12 nucleotides, and/or the 3′-downstream sequence is about 30-2000 nucleotides in length.
12. The nucleic acid of claim 1 , wherein the loop region is from 15-25 nucleotides in length.
13. An expression cassette comprising a promoter contiguously linked to the nucleic acid of claim 1 .
14. A vector comprising the expression cassette of claim 13 .
15. The vector of claim 14 , wherein the vector is an adeno-associated virus (AAV) vector.
16. The vector of claim 15 , wherein the AAV is AAV1, AAV2, AAV4, AAV5, or AAV2/1.
17. An isolated microRNA molecule comprising the nucleic acid of claim 1 .
18. A method of inducing RNA interference comprising administering to a subject an effective amount of the nucleic acid of claim 1 .
19. A method of treating a subject with Huntington's Disease, comprising administering to the subject the nucleic acid of claim 1 so as to treat the Huntington's Disease.
20. A nucleic acid encoding an artificial primary miRNA transcript (pri-miRNA) consisting of, in order of position, a 5′-flanking region, a non-guide region, a loop region, a guide region, and a 3′-flanking region, wherein the guide region comprises a sequence at least 90% identical to agucgcugaugaccggga (miHDS.2 guide, SEQ ID NO:8) or acgucguaaacaagagga (miHDS.5 guide, SEQ ID NO:9) and the non-guide region is at least 80% complementary to the guide region.