RNA interference suppresion of neurodegenerative diseases and methods of use thereof

• US 20050042646A1
• Filed: 06/02/2004
• Published: 02/24/2005
• Est. Priority Date: 08/05/2002
• Status: Abandoned Application

• Current Assignee: University of Iowa Research Foundation
• Original Assignee: University of Iowa Research Foundation
• Inventors: Mao, Qinwen, Xia, Haibin, Davidson, Beverly L., Paulson, Henry, Boudreau, Ryan L.
• Application Number:
  US10/859,751
• Publication Number:
  US 20050042646A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• 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 2310/53 partially self-complementary or closed
• CPC Class Codes: C12N 2330/30 chemically synthesised
• CPC Class Codes: C12N 2799/021 for the expression of a heterologous nucleic acid
• CPC Class Codes: C12N 2799/022 where the vector is derived from an adenovirus
• CPC Class Codes: C12Y 302/01031 Beta-glucuronidase (3.2.1.31)
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention is directed to small interfering RNA molecules (siRNA) targeted against nucleic acid sequence that encodes huntingtin or ataxin-1, and methods of using these siRNA molecules.

Claims

1. An isolated RNA duplex comprising a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by shSCA1.F10 (SEQ ID NO:

13) or shSCA1.F11 (SEQ ID NO;

14), and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand.

2. The RNA duplex of claim 1, wherein the first strand of RNA is encoded by shSCA1.F10.

3. The RNA duplex of claim 1, wherein the second strand of RNA is encoded by shSCA1.F11.

12. The RNA duplex of claim 1, wherein the duplex is between 15 and 30 base pairs in length.

13. The RNA duplex of claim 1, wherein the duplex is between 19 and 25 base pairs in length.

14. The RNA duplex of claim 1, wherein the first and/or second strand further comprises an overhang region.

15. The RNA duplex of claim 1, wherein the first and/or second strand further comprises a 3′

overhang region, a 5′

overhang region, or both 3′ and

5′

overhang regions.

16. The RNA duplex of claim 14, wherein the overhang region is from 1 to 10 nucleotides in length.

17. The RNA duplex of claim 1, wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure.

18. The RNA duplex of claim 17, wherein the loop structure contains from 4 to 10 nucleotides.

19. The RNA duplex of claim 17, wherein the loop structure contains 4, 5 or 6 nucleotides.

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

21. The expression cassette of claim 20, further comprising a promoter.

22. The expression cassette of claim 21, wherein the promoter is a regulatable promoter.

23. The expression cassette of claim 21, wherein the promoter is a constitutive promoter.

24. The expression cassette of claim 21, wherein the promoter is a CMV, RSV, pol II or pol III promoter.

25. The expression cassette of claim 20, wherein the expression cassette further comprises a polyadenylation signal.

26. The expression cassette of claim 25, wherein the polyadenylation signal is a synthetic minimal polyadenylation signal.

27. The expression cassette of claim 20, further comprising a marker gene.

28. A vector comprising the expression cassette of claim 20.

29. A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding the first strand of the RNA duplex of claim 1 and a second expression cassette comprising a nucleic acid encoding the second strand of the RNA duplex of claim 1.

30. A cell comprising the expression cassette of claim 20.

31. The cell of claim 30, wherein the cell is a mammalian cell.

32. A non-human mammal comprising the expression cassette of claim 20.

33. A method of suppressing the accumulation of huntingtin or ataxin-1 in a cell comprising introducing a ribonucleic acid (RNA) of claim 1 into the cell in an amount sufficient to suppress accumulation of huntingtin or ataxin-1 in the cell.

34. The method of claim 1 in which accumulation of huntingtin or ataxin-1 is suppressed by at least 10%.

35. A method of preventing cytotoxic effects of mutant huntingtin or ataxin-1 in a cell comprising introducing a ribonucleic acid (RNA) of claim 1 into the cell in an amount sufficient to suppress accumulation of huntingtin or ataxin-1, and wherein the RNA prevents cytotoxic effects of huntingtin or ataxin-1 in the cell.

36. A method to inhibit expression of a huntingtin or ataxin-1 gene in a cell comprising introducing a ribonucleic acid (RNA) of claim 1 into the cell in an amount sufficient to inhibit expression of the huntingtin or ataxin-1, and wherein the RNA inhibits expression of the huntingtin or ataxin-1 gene.

37. A method to inhibit expression of a huntingtin or ataxin-1 gene in a mammal comprising:

(a) providing a mammal containing a neuronal cell, wherein the neuronal cell contains the huntingtin or ataxin-1 gene and the neuronal cell is susceptible to RNA interference, and the huntingtin or ataxin-1 gene is expressed in the neuronal cell; and

(b) contacting the mammal with a ribonucleic acid (RNA) of claim 1 or a vector of claim 27, thereby inhibiting expression of the huntingtin or ataxin-1 gene.

38. The method of claim 36 or 37, in which expression of huntingtin or ataxin-1 is inhibited by at least 10%.

39. The method of claim 37, wherein the mammal is a human.

40. The method of any of claims 35 or 36, wherein the cell is located in vivo in a mammal.

50. The vector of claim 40, wherein the target sequence is a sequence encoding ataxin-1 or huntingtin.

51. A method of preventing cytotoxic effects of neurodegenerative disease in a mammal in need thereof, comprising introducing the vector of claim 40 into a cell in an amount sufficient to suppress accumulation of a protein associated with the neurodegenerative disease, and wherein the RNA prevents cytotoxic effects of neurodegenerative disease.

52. A method to inhibit expression of a protein associated with the neurodegenerative disease in a mammal in need thereof, comprising introducing the vector of claim 40 into a cell in an amount sufficient to inhibit expression of the protein associated with the neurodegenerative disease, wherein the RNA inhibits expression of the protein associated with the neurodegenerative disease.

4. An RNA duplex comprising a first strand of RNA and a second strand of RNA, wherein the first strand comprises at least 15 contiguous nucleotides encoded by

(a) shHDEx2.1 (SEQ ID NO:

7),

(b) shHDEx2.2 19 nt (SEQ ID NO;

8),

(c) shHDEx2.2 21 nt (SEQ ID NO;

9),

(d) shHDEx3.1 19 nt (SEQ ID NO;

10),

(e) shHDEx3.1 21 nt (SEQ ID NO;

11),

(f) siEX58#1 (SEQ ID NO;

12), or

(g) siEX58#2 (SEQ ID NO;

17),

and wherein the second strand is complementary to at least 12 contiguous nucleotides of the first strand.

5. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by shHDEx2.1.

6. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by shHDEx2.2 19 nt.

7. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by shHDEx2.2 21 nt.

8. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by shHDEx3.1 19 nt.

9. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by shHDEx3.1 21 nt.

10. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by siEX58#2.

11. The RNA duplex of claim 4, wherein the first strand of RNA is encoded by siEX58#1.

41. A viral vector comprising a promoter and an miRNA shuttle containing an embedded siRNA that specificly targets a sequence associated with a condition amenable to siRNA therapy.

42. The vector of claim 41, wherein the promoter is an inducible promoter.

43. The vector of claim 41, wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector.

44. The vector of claim 41, wherein the vector is an adenoviral viral vector.

45. The vector of claim 44, wherein the condition amenable to siRNA therapy is a neurodegenerative disease.

46. The vector of claim 45, wherein the neurodegenerative disease is a trinucleotide-repeat disease.

47. The vector of claim 46, wherein the trinucleotide-repeat disease is a disease associated with polyglutamine repeats.

48. The vector of claim 47, wherein the trinucleotide-repeat disease is Huntington'"'"'s disease or a spinocerebellar ataxia (SCA).

49. The vector of claim 48, wherein the SCA is SCA1, SCA2, SCA3, SCA6, SCA7,or SCA17.

53. A method to inhibit expression of huntingtin or ataxin-1 in a mammal in need thereof comprising:

(a) providing a mammal containing a neuronal cell, wherein the neuronal cell contains the huntingtin or ataxin-1 gene and the neuronal cell is susceptible to RNA interference, and the huntingtin or ataxin-1 gene is expressed in the neuronal cell; and

(b) contacting the mammal the vector of claim 41, thereby inhibiting expression of the huntingtin or ataxin-1 gene.