Methods and sequences to suppress primate huntington gene expression in vivo

• US 20100325746A9
• Filed: 08/09/2006
• Published: 12/23/2010
• Est. Priority Date: 05/06/2005
• Status: Active Grant

• Current Assignee: Medtronic Incorporated (Medtronic Plc)
• Original Assignee: Medtronic Incorporated (Medtronic Plc)
• Inventors: Kaemmerer, William F., Kaytor, Michael D.
• Granted Patent: US 7,902,352 B2
• Time in Patent Office: Days
• US Class Current: 800/14
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: C12N 15/113 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 Class Codes: C12N 2310/111 spanning the whole gene, or a large part of it
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14171 Demonstrated in vivo effect

Abstract

Disclosed herein are sequences, molecules and methods used to suppress the expression of HD genes encoding for huntingtin protein in primates including Macaca mulatta and Homo sapiens. These sequences, molecules and methods aid in the study of the pathogenesis of HD and can also provide a treatment for this disease by reducing HD mRNA without causing death, locomotor impairment or cellular alterations of the Macaca mulatta and Homo sapiens.

Claims

1. An isolated nucleic acid duplex comprising a first strand of nucleic acid and a second strand of nucleic acid, wherein the first strand comprises at least 19 contiguous nucleotides encoded by the group consisting of SEQ ID NO:

1, and SEQ ID NO;

4 and wherein the second strand is complementary to at least 15 contiguous nucleotides of the first strand.

2. The nucleic acid duplex according to claim 1, wherein said duplex is between 19 and 30 base pairs in length.

3. The nucleic acid duplex according to claim 1, wherein said first and/or said second strand further comprises an overhang region.

4. The nucleic acid duplex according to claim 3, wherein said overhang region comprises a 3′

overhang region, a 5′

overhang region, or both 3′ and

5′

overhang regions.

5. The nucleic acid duplex according to claim 3, wherein said overhang region is from 1 to 10 nucleotides in length.

6. The nucleic acid duplex according to claim 1, wherein said first strand and said second strand are operably linked by means of a nucleic acid loop strand to form a hairpin structure comprising a duplex structure and a loop structure.

7. The nucleic acid duplex according to claim 6, wherein said loop structure contains from 4 to 10 nucleotides.

8. An expression cassette comprising a nucleic acid encoding at least one strand of the nucleic acid duplex of claim 1.

9. The expression cassette according to claim 8, further comprising a promoter.

10. The expression cassette according to claim 9, wherein said promoter is a regulatable promoter.

11. The expression cassette according to claim 9, wherein said promoter is a constitutive promoter.

12. The expression cassette according to claim 11, wherein said promoter is a CMV, RSV, pol II or pol III promoter.

13. The expression cassette according to claim 8, wherein said expression cassette further comprises a polyadenylation signal.

14. The expression cassette according to claim 13, wherein said polyadenylation signal is a synthetic minimal polyadenylation signal.

15. The expression cassette according to claim 8, further comprising a marker gene.

16. A vector comprising the expression cassette of claim 8.

17. A vector comprising two expression cassettes, a first expression cassette comprising a nucleotide sequence encoding said first strand of said nucleic acid duplex of claim 1 and a second expression cassette comprising a nucleotide sequence encoding the second strand of the nucleic acid duplex of claim 1.

18. The vector of claim 17, wherein the first strand comprises SEQ ID. NO:

4.

19. The vector of claim 17, wherein said vector is a viral vector.

20. The vector according to claim 19, wherein said vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpes simplex virus, feline immunodeficiency virus or murine Maloney-based viral vector.

21. The vector of claim 20, wherein the viral vector is the adeno-associated viral (AAV) vector.

22. The vector of claim 17, further comprising a promoter.

23. The vector of claim 22, wherein said promoter is an inducible promoter.

24. A cell comprising the expression cassette of claim 8.

25. A cell of claim 24, wherein said cell is a mammalian cell.

26. A non-human mammal comprising the expression cassette of claim 8.

27. The non-human mammal of claim 26, which is a primate.

28. A method of preventing cytotoxic effects of Huntington'"'"'s disease (“

HD”

) in a Macaca mulatta or Homo sapiens comprising introducing the isolated nucleic acid duplex of claim 1 into a cell in an amount sufficient to suppress accumulation of a protein associated with HD, and wherein the nucleic acid duplex prevents cytotoxic effects of HD.

29. A method of inhibiting expression of huntingtin in a Macaca mulatta or Homo sapiens comprising:

introducing the isolated nucleic acid duplex of claim 1 into a cell in an amount sufficient to inhibit expression of huntingtin wherein said nucleic acid duplex inhibits expression of the protein associated with HD.

30. The method of claim 28 or 29, wherein the isolated nucleic acid duplex comprises SEQ. ID. NO:

4.

31. The method of claim 28 or 29, wherein said isolated nucleic acid duplex is included within a vector.

32. The method of claim 31, wherein the vector is a viral vector.

33. The method of claim 32, wherein the viral vector is the adeno-associated viral (AAV) vector.

34. The method of claim 28 or 29, wherein said Macaca mulatta or Homo sapiens survive for at least four weeks after the step of introducing the nucleic acid complex.

35. The method of claim 28 or 29, wherein said Macaca mulatta or Homo sapiens do not exhibit an impairment in fine locomotor activity.

36. The method of claim 28 or 29, wherein said nucleic acid duplex is introduced into at least one cell located in a brain structure selected from the group consisting of putamen, caudate nucleus, corona radiata, and striatum.

37. The method of claim 36, wherein expression of said nucleic acid duplex does not impair an endoplasmic reticulum of the at least one cell.

38. The method of claim 36, wherein expression of said nucleic acid duplex does not impair expression or distribution of calnexin.

39. The method of claim 36, wherein expression of said nucleic acid duplex does not impair expression or distribution of protein disulfide isomerase (PDI).