Huntington's disease therapeutic compounds

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 consists of SEQ ID NO: 37 (miHDss3), SEQ ID NO:6 (miHDS1v5U) or SEQ ID NO:7 (miHDS1v6A), and the non-guide region is at least 80% complementary to the guide region.

2. The nucleic acid of claim 1 , wherein the 5′-flanking region comprises a 5′-joining sequence contiguously linked to the non-guide region.

3. The nucleic acid of claim 2 , wherein the 5′-joining sequence consists of 5-8 nucleotides.

4. The nucleic acid of claim 2 , wherein the 5′-joining sequence encodes GUGAGCGA (SEQ ID NO:13) or GUGAGCGC (SEQ ID NO:14).

5. The nucleic acid of claim 2 , wherein the 5′-flanking region further comprises a 5′-bulge sequence positioned upstream from the 5′ joining sequence.

6. The nucleic acid of claim 2 , wherein the 3′-flanking region comprises a 3′ joining sequence contiguously linked to the guide region, and wherein the 3′-joining sequence is at least 85% complementary to the 5′-joining sequence.

7. The nucleic acid of claim 5 , wherein the 5′-bulge sequence comprises a cloning site.

8. The nucleic acid of claim 5 , wherein the 5′-bulge sequence consists of about 1-10 nucleotides.

9. The nucleic acid of claim 5 , wherein the 5′-bulge sequence encodes UAAACUCGA (SEQ ID NO:15).

10. The nucleic acid of claim 5 , wherein the 5′-flanking region further comprises a 5′-spacer sequence positioned upstream from the 5′-bulge sequence.

11. The nucleic acid of claim 5 , wherein the 3′-flanking region comprises a 3′ joining sequence contiguously linked to the guide region, wherein the 3′-flanking region further comprises a 3′-bulge sequence positioned downstream from the 3′-joining sequence, and wherein the 5′-bulge sequence is complementary to the 3′-bulge sequence at only one nucleotide at each end of the 5′-bulge sequence.

12. The nucleic acid of claim 10 , wherein the 5′-spacer sequence consists of 10-12 nucleotides.

13. The nucleic acid of claim 10 , wherein the 5′-spacer sequence encodes UGGUACCGUU (SEQ ID NO:16).

14. The nucleic acid of claim 10 , wherein the 5′-flanking region further comprises a 5′-upstream sequence positioned upstream from the 5′-spacer sequence.

15. The nucleic acid of claim 10 , wherein the 3′-flanking region comprises a 3′-joining sequence contiguously linked to the guide region, wherein the 3′-flanking region further comprises a 3′-bulge sequence positioned downstream from the 3′-joining sequence, wherein the 3′-flanking region further comprises a 3′-spacer sequence positioned downstream from the 3′-bulge sequence, and wherein the 3′-spacer sequence is at least 70% complementary to the 5′-spacer sequence.

16. The nucleic acid of claim 14 , wherein the 5′-upstream sequence is about 30-2000 nucleotides in length.

17. The nucleic acid of claim 14 , wherein the 3′-flanking region comprises a 3′-joining sequence contiguously linked to the guide region, wherein the 3′-flanking region further comprises a 3′-bulge sequence positioned downstream from the 3′-joining sequence, wherein the 3′-flanking region further comprises a 3′-spacer sequence positioned downstream from the 3′-bulge sequence, wherein the 3′-flanking region further comprises a 3′-downstream sequence positioned downstream from the 3′-spacer sequence, and wherein the 5′-upstream sequence is less than 20% homologous to the 3′-downstream sequence.

18. The nucleic acid of claim 1 , wherein the 3′-flanking region comprises a 3′-joining sequence contiguously linked to the guide region.

19. The nucleic acid of claim 18 , wherein the 3′-joining sequence consists of 5-8 nucleotides.

20. The nucleic acid of claim 18 , wherein the 3′-joining sequence encodes CGCCUAC (SEQ ID NO:18).

21. The nucleic acid of claim 18 , wherein the 3′-flanking region further comprises a 3′-bulge sequence positioned downstream from the 3′-joining sequence.

22. The nucleic acid of claim 21 , wherein the 3′-bulge sequence comprises a cloning site.

23. The nucleic acid of claim 21 , wherein the 3′-bulge sequence consists of about 1-10 nucleotides.

24. The nucleic acid of claim 21 , wherein 3′-bulge sequence encodes UAG (SEQ ID NO:30).

25. The nucleic acid of claim 21 , wherein the 3′-flanking region further comprises a 3′-spacer sequence positioned downstream from the 3′-bulge sequence.

26. The nucleic acid of claim 25 , wherein the 3′-spacer sequence consists of 10-12 nucleotides.

27. The nucleic acid of claim 25 , wherein the 3′-spacer sequence encodes AGCGGCCGCCA (SEQ ID NO:19).

28. The nucleic acid of claim 25 , wherein the 3′-flanking region further comprises a 3′-downstream sequence positioned downstream from the 3′-spacer sequence.

29. The nucleic acid of claim 28 , wherein the 3′-downstream sequence is about 30-2000 nucleotides in length.

30. The nucleic acid of claim 1 , wherein the loop region is from 15-25 nucleotides in length.

31. An expression cassette encoding the isolated nucleic acid described in claim 1 operably linked to a promoter.

32. The expression cassette of claim 31 , wherein the promoter is a tissue-specific promoter.

33. The expression cassette of claim 31 , wherein the promoter is an inducible promoter.

34. A vector comprising the expression cassette of claim 31 .

35. The vector of claim 34 , wherein the vector is an adeno-associated virus (AAV) vector.

36. The vector of claim 35 , wherein the AAV is AAV1, AAV2, AAV5, AAV6 and/or AAV9.

37. The vector of claim 36 , wherein the AAV is AAV2.

38. An isolated microRNA molecule comprising the nucleic acid of claim 1 having an overhang at the 3′ end.

39. The isolated microRNA molecule of claim 38 , wherein the overhang is a 2 to 5-nucleotide repeat.

40. The isolated microRNA of claim 38 , wherein the overhang is a UU (SEQ ID NO:24), UUU (SEQ ID NO:25) or UUUU (SEQ ID NO:26) sequence.

41. The isolated microRNA of claim 38 , wherein the overhang is a CUU (SEQ ID NO:27), CUUU (SEQ ID NO:28) or CUUUU (SEQ ID NO:29) sequence.

42. An isolated nucleic acid between 80-4000 nucleotides in length, comprising 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 consists of SEQ ID NO: 37 (miHDss3), SEQ ID NO:6 (miHDS1v5U) or SEQ ID NO:7 (miHDS1v6A), and the non-guide region is at least 80% complementary to the guide region.

43. An isolated nucleic acid consisting of Pri-miHDS1v5U (SEQ ID NO:8), Pri-miHDS1v6A (SEQ ID NO:9), Pre-miHDS1v5U (SEQ ID NO:10), or Pre-miHDS1v6A (SEQ ID NO:11).

44. An isolated RNA duplex comprising a guide region of nucleic acid and a non-guide region of nucleic acid, wherein the guide region consists of SEQ ID NO: 37 (miHDss3), SEQ ID NO:6 (miHDS1v5U) or SEQ ID NO:7 (miHDS1v6A) and the non-guide region is at least 80% complementary to the guide region.

45. The isolated RNA duplex of claim 44 , wherein the duplex is between 19-30 base pairs in length.

46. A method of inducing RNA interference comprising administering to a subject an effective amount of the nucleic acid of claim 1 .

47. A method of inducing low-toxicity RNA interference comprising administering to a subject the nucleic acid of claim 1 .

48. 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.

49. The method of claim 46 , wherein the nucleic acid is administered to the brain of the subject either directly or via the bloodstream.

50. The method of claim 49 , wherein the nucleic acid is administered intra-cranially.

51. The method of claim 50 , wherein the nucleic acid is administered to the subject's cisterna magna, striatum, cortex or ventricle, subarachnoid space and/or intrathecal space.

52. The method of claim 49 , wherein the subject is human.

53. The method of claim 49 , wherein the nucleic acid is injected at 1-5 locations in the CNS.