RNA INTERFERENCE SUPPRESSION OF NEURODEGENERATIVE DISEASES AND METHODS OF USE THEREOF

• US 20110212520A1
• Filed: 12/09/2010
• Published: 09/01/2011
• Est. Priority Date: 08/05/2002
• Status: Active Grant

• Current Assignee: University of Iowa Research Foundation
• Original Assignee: University of Iowa Research Foundation
• Inventors: Mao, Qinwen, Harper, Scott, Xia, Haibin, Davidson, Beverly L., Paulson, Henry, Boudreau, Ryan
• Granted Patent: US 8,481,710 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• 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: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• 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 15/86 Viral vectors
• 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 2310/531 Stem-loop; Hairpin
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• 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: C12N 2799/025 where the vector is derived from a parvovirus
• 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:

102), shSCA1.F11 (SEQ ID NO;

103), SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

63, SEQ ID NO;

66, SEQ ID NO;

68, SEQ ID NO;

70, SEQ ID NO;

72, SEQ ID NO;

74, SEQ ID NO;

76, SEQ ID NO;

78, SEQ ID NO;

80, SEQ ID NO;

82, SEQ ID NO;

84, SEQ ID NO;

86, or SEQ ID NO;

88, SEQ ID NO;

90, SEQ ID NO;

96 through SEQ ID NO;

101 or SEQ ID NO;

106 through SEQ ID NO;

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

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

5. 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.

6. The RNA duplex of claim 5, wherein the overhang region is from 1 to 10 nucleotides in length.

7. 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.

8. The RNA duplex of claim 7, wherein the loop structure contains from 4 to 10 nucleotides.

10. The RNA duplex of claim 7, wherein the loop structure corresponds to SEQ ID NO:

61 or SEQ ID NO;

64.

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

12. The expression cassette of claim 11, further comprising a promoter.

16. The expression cassette of claim 11, wherein the expression cassette further comprises a polyadenylation signal.

19. A vector comprising the expression cassette of claim 11.

20. 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.

21. A cell comprising the vector of claim 19.

24. The cell of claim 21, wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector.

2. (canceled)

4. (canceled)

9. (canceled)

13-15. -15. (canceled)

17-18. -18. (canceled)

22-23. -23. (canceled)

25-33. -33. (canceled)

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

36. The vector of claim 34, wherein the vector is an adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murine Maloney-based viral vector.

38. The vector of claim 34, wherein the condition amenable to siRNA therapy is a neurodegenerative disease.

41. The vector of claim 38, wherein the condition amenable to siRNA therapy is Huntington'"'"'s disease or a spinocerebellar ataxia (SCA).

43. The vector of claim 34, wherein the target sequence is a sequence encoding ataxin-1 or huntingtin.

35. (canceled)

37. (canceled)

39-40. -40. (canceled)

42. (canceled)

44-46. -46. (canceled)

47. An AAV-1 expressed siRNA comprising an isolated first strand of RNA of 15 to 30 nucleotides in length and an isolated second strand of RNA of 15 to 30 nucleotides in length, wherein the first or second strand comprises a sequence that is complementary to a nucleotide sequence encoding a mutant Huntington'"'"'s Disease protein, wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences the expression of the nucleotide sequence encoding the mutant Huntington'"'"'s Disease protein in the cell.

48. The siRNA of claim 47, wherein the first and/or second strand further comprises a 3′

overhang region, a 5′

overhang region, or both 3′ and

5′

overhang regions.

50. The siRNA of claim 47, 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.

51. The siRNA of claim 50, wherein the loop structure contains from 4 to 10 nucleotides.

52. The siRNA of claim 50, wherein the loop structure corresponds to SEQ ID NO:

61 or SEQ ID NO;

64.

53. The siRNA of claim 47, wherein the first strand corresponds to SEQ ID NO:

60 and the second strand corresponds to SEQ ID NO;

62.

49. (canceled)

54. A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding a first strand of an RNA duplex corresponding to SEQ ID NO:

60 and a second expression cassette comprising a nucleic acid encoding a second strand of the RNA duplex corresponding to SEQ ID NO;

62.

55-61. -61. (canceled)