RAAV-BASED COMPOSITIONS AND METHODS FOR TREATING AMYOTROPHIC LATERAL SCLEROSIS

• US 20170114340A1
• Filed: 03/18/2015
• Published: 04/27/2017
• Est. Priority Date: 03/18/2014
• Status: Active Grant

• Current Assignee: University Of Massachusetts
• Original Assignee: University Of Massachusetts
• Inventors: MUELLER, Christian, BROWN, JR., Robert H.
• Granted Patent: US 10,280,418 B2
• Time in Patent Office: Days
• CPC Class Codes: A61P 21/02 Muscle relaxants, e.g. for tetanus or cramps
• 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 15/86 Viral vectors
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/141 MicroRNAs, miRNAs
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: C12N 2330/51 Specially adapted vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12Y 115/01001 Superoxide dismutase (1.15.1.1)

Abstract

The invention relates to inhibitory nucleic acids and rAAV-based compositions, methods and kits useful for treating Amyotrophic Lateral Sclerosis.

Claims

1. A method of inhibiting C9orf72 expression in a cell, the method comprising:

delivering to the cell an inhibitory nucleic acid that targets both pre-mRNA and mRNA encoded by a C9orf72 gene.

2. The method of claim 1, wherein the cell expresses C9orf72 having G₄C₂ expansions of up to 50, up to 90, up to 160, or up to 200 repeats.

3. The method of claim 1, wherein the level of a mRNA encoding isoform B of C9orf72 in the cell is greater than the level of a mRNA encoding isoform A of C9orf72 protein in the cell.

4. The method of any one of claims 1 to 3, wherein the cell is a cell of the central nervous system.

5. The method of claim 4, wherein the cell is a neuron.

6. The method of any one of claims 1 to 5, wherein, prior to being exposed to the inhibitory nucleic acid, the cell contains intranuclear G₄C₂ foci.

7. The method of claim 6, wherein delivery of the inhibitory nucleic acid to the cell results in a reduction in intranuclear G₄C₂ foci.

8. The method of any one of claims 1 to 7, wherein, prior to being exposed to the inhibitory nucleic acid, the cell contains C9 RAN proteins.

9. The method of claim 8, wherein delivery of the inhibitory nucleic acid to the cell results in a reduction in C9 RAN protein levels.

10. The method of any one of claims 1 to 8, wherein the cell is in vivo.

11. The method of any one of claims 1 to 8, wherein the cell is in vitro.

12. The method of any one of claim 1 to 11, wherein the cell is of a subject having one or more symptoms of FTD or ALS.

13. The method of any one of claim 1 to 12, wherein the cell is of a subject suspected of having FTD or ALS.

21. The method of any one of claims 1 to 20, wherein the inhibitory nucleic acid comprises a region of complementarity that is complementary with at least 5 consecutive nucleotides within exon 3 of C9orf72.

22. The method of claim 21, wherein the at least 5 consecutive nucleotides are within nucleotides 220 to 241 of C9orf72.

23. The method of any one of claims 1 to 22, wherein the inhibitory nucleic acid targets mRNA encoding isoform A and mRNA encoding isoform B of C9orf72 protein.

24. The method of any one of claims 1 to 22, wherein the inhibitory nucleic acid targets C9orf72 variants V1 (SEQ ID NO:

18), V2 (SEQ ID NO;

     19), and V3 (SEQ ID NO;

     20).

25. The method of any one of claims 1 to 22, wherein the inhibitory nucleic acid targets C9orf72 variants V1 (SEQ ID NO:

18) and V3 (SEQ ID NO;

     20), but not V2 (SEQ ID NO;

     19).

26. The method of any one of claims 1 to 22, wherein the inhibitory nucleic acid targets C9orf72 variant V1 (SEQ ID NO:

18), but not V2 (SEQ ID NO;

     19) and V3 (SEQ ID NO;

     20).

27. The method of any one of claims 1 to 23, wherein the inhibitory nucleic acid reduces levels of C9orf72 mRNA in a cell by at least 50%.

28. The method of any one of claims 1 to 24, wherein the inhibitory nucleic acid reduces levels of C9orf72 pre-mRNA in a cell by at least 50%.

29. The method of any one of claims 1 to 25, wherein the inhibitory nucleic acid comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence as set forth in any one of SEQ ID NOs:

1 to 8.

30. The method of any one of claims 13 to 29, wherein the rAAV is administered by intrathecally, intracerebrally, intraventricularly or intravenously.

14. A method of inhibiting C9orf72 expression in the central nervous system (CNS) of a subject, the method comprising:

administering to the CNS of the subject an inhibitory nucleic acid that targets an RNA encoded by the C9orf72 gene, wherein the inhibitory nucleic acid is a microRNA.

17. The method of any one of claims 14 to 16, wherein the step of administering the inhibitory nucleic acid to the subject comprises administering to the subject a recombinant adeno-associated virus (rAAV) harboring a nucleic acid that is engineered to express the inhibitory nucleic acid in a cell of the subject.

19. The method of claim 17 or 18, wherein the rAAV targets CNS tissue.

20. The method of claim 17, 18 or 19, wherein the rAAV comprises an AAV.Rh10 or AAV9 capsid protein.

15. A method of inhibiting C9orf72 expression in the central nervous system (CNS) of a subject, the method comprising:

administering to the CNS of the subject an inhibitory nucleic acid that targets both pre-mRNA and mRNA encoded by a C9orf72 gene.

16. The method of claim 15, wherein the inhibitory nucleic acid is a microRNA.

18. A method of treating a subject having or suspected of having FTD or ALS, the method comprising:

administering to the subject an effective amount of a recombinant adeno-associated virus (rAAV) harboring a nucleic acid that is engineered to express, in a cell of the subject, an inhibitory nucleic acid that targets both pre-mRNA and mRNA encoded by a C9orf72 gene.

31. A method of inhibiting SOD1 expression in a cell, the method comprising:

delivering to the cell an miRNA that targets SOD1 mRNA, wherein the miRNA comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence as set forth in;

32. A method of treating a subject having or suspected of having ALS, the method comprising:

administering to the subject an effective amount of a recombinant adeno-associated virus (rAAV) harboring a nucleic acid that is engineered to express, in a cell of the subject, an miRNA that targets RNA encoded by a SOD1 gene.

33. The method of claim 32, wherein the miRNA comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 consecutive nucleotides of a sequence as set forth in:

34. The method of claim 32 or 33, wherein the rAAV targets CNS tissue.

35. The method of claim 34, wherein the rAAV comprises an AAV.Rh10 or AAV9 capsid protein.

36. A synthetic microRNA comprising a sequence as set forth in any one of SEQ ID NOs:

1 to 8.

38. The synthetic microRNA of claim 36 or 37 further comprising flanking regions of miR-155.

39. A recombinant nucleic acid encoding the microRNA of any one of claims 36 to 38 and comprising an inverted terminal repeats (ITR) of an AAV serotype.

40. The recombinant nucleic acid of claim 39, wherein the AAV serotype is selected from the group consisting of:

AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVRh10, AAV11 and variants thereof.

41. The recombinant nucleic acid of claim 40, further comprising a promoter operably linked with a region(s) encoding the microRNA.

42. The recombinant nucleic acid of claim 41, wherein the promoter is a tissue-specific promoter.

43. The recombinant nucleic acid of claim 42, wherein the promoter is a polymerase II promoter, such as a β

-actin promoter.

44. The recombinant nucleic acid of claim 42, wherein the promoter is a polymerase III promoter, such as a U6 promoter.

45. A composition comprising the recombinant nucleic acid of any one of claims 39 to 44.

46. A recombinant Adeno-Associated Virus (AAV) harboring a recombinant nucleic acid of any one of claims of claims 39 to 44.

47. The recombinant AAV of claim 46, further comprising one or more capsid proteins of one or more AAV serotypes selected from the group consisting of:

AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11 and variants thereof.

48. A composition comprising the recombinant AAV of claim 46 or 47.

49. The composition of claim 48, further comprising a pharmaceutically acceptable carrier.

50. A kit comprising a container housing the composition of claim 48 or 49.

37. A synthetic microRNA comprising a sequence as set forth as SEQ ID NO:

15;