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1. (WO2019043234) PROMOTEUR RÉTINIEN ET SES UTILISATIONS
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CLAIMS

1. An isolated nucleic acid molecule having promoter activity for use in the treatment of ocular disease, wherein said nucleic acid molecule comprises at least Neurofilament heavy gene promoter conserved region A; and optionally one or more of Neurofilament heavy gene promoter conserved regions D, F, Dl, K, B, C and E;

wherein Neurofilament heavy gene promoter conserved region A is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 1, or a functional variant thereof; Neurofilament heavy gene promoter conserved region D is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 2, or a functional variant thereof; Neurofilament heavy gene promoter conserved region F is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 3, or a functional variant thereof; Neurofilament heavy gene promoter conserved region Dl is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 4, or a functional variant thereof; Neurofilament heavy gene promoter conserved region K is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 5, or a functional variant thereof; Neurofilament heavy gene promoter conserved region B is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 6, or a functional variant thereof;

Neurofilament heavy gene promoter conserved region C is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 7, or a functional variant thereof; and Neurofilament heavy gene promoter conserved region E is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 8, or a functional variant thereof.

2. The isolated nucleic acid molecule for use according to claim 1, wherein the isolated nucleic acid molecule comprises at least one of Neurofilament heavy gene promoter conserved region D and Neurofilament heavy gene promoter conserved region F.

3. The isolated nucleic acid molecule for use according to claim 1 or claim 2, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved region A, Neurofilament heavy gene promoter conserved region D, and Neurofilament heavy gene promoter conserved region F.

4. The isolated nucleic acid molecule for use according to any one of the preceding claims, wherein the isolated nucleic acid molecule comprises at least one of the conserved regions selected from: Neurofilament heavy gene promoter conserved region Dl and Neurofilament heavy gene promoter conserved region K.

5. The isolated nucleic acid molecule for use according to any one of the preceding claims, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, and K.

6. The isolated nucleic acid molecule for use according to claim 5, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, and K having the nucleic acid sequences shown as SEQ ID NOS: 1, 2, 3, 4, and 5 respectively.

7. The isolated nucleic acid molecule for use according to claim 1, wherein of Neurofilament heavy gene promoter conserved regions D, F, Dl, K, B, C and E, the isolated nucleic acid molecule comprises fewer than three.

8. The isolated nucleic acid molecule for use according to claim 7, wherein, of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, K, B, C and E, said isolated nucleic acid molecule comprises only conserved regions A and F.

9. The isolated nucleic acid molecule for use according to claim 1, wherein, of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, K, B, C and E said isolated nucleic acid molecule comprises only conserved region A.

10. The isolated nucleic acid molecule for use according to any one of claims 2 to 8, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180% of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 21.

11. The isolated nucleic acid molecule for use according to claim 10, wherein the isolated nucleic acid molecule comprises between each recited conserved region and its adjacent recited conserved region in said isolated nucleic acid molecule a spacer sequence of a length in the range 20-180% of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 21.

12. The isolated nucleic acid molecule for use according to any one of claims 2 to 8, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180% of the sequence separating one of said recited conserved regions from one of its adjacent conserved regions in the nucleic acid sequence shown as SEQ ID NO: 21.

13. The isolated nucleic acid molecule for use according to claim 6, wherein the isolated nucleic acid molecule consists of the nucleic acid sequence shown as SEQ ID NO: 21.

14. The isolated nucleic acid molecule for use according to any one of claims 1 to

5, wherein the isolated nucleic acid molecule comprises at least one of the conserved regions selected from: Neurofilament heavy gene promoter conserved region B, Neurofilament heavy gene promoter conserved region C, and Neurofilament heavy gene promoter conserved region E.

15. The isolated nucleic acid molecule for use according to claim 14, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, B, C, and E.

16. The isolated nucleic acid molecule for use according to claim 15, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, B, C, and E having the nucleic acid sequences shown as SEQ ID NOS: 9, 12, 15,

6, 7, and 8 respectively.

17. The isolated nucleic acid molecule for use according to claim 14, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, B, C, and E having the nucleic acid sequences shown as SEQ ID NOS: 10, 13, 16, 18, 19, and 20 respectively.

18. The isolated nucleic acid molecule for use according to claim 14, wherein, of Neurofilament heavy gene promoter conserved regions B, C, and E, the isolated nucleic acid molecule comprises only one or two.

19. The isolated nucleic acid molecule for use according to any one of claims 14 to 18, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180% of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 22.

20. The isolated nucleic acid molecule for use according to claim 19, wherein the isolated nucleic acid molecule comprises between each recited conserved region and its adjacent recited conserved region in said isolated nucleic acid molecule a spacer sequence of a length in the range 20-180%) of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 22.

21. The isolated nucleic acid molecule for use according to any one of claims 14 to 18, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180%) of the sequence separating one of said recited conserved regions from one of its adjacent conserved regions in the nucleic acid sequence shown as SEQ ID NO: 22.

22. The isolated nucleic acid molecule for use according to claim 15 or claim 16, wherein the isolated nucleic acid molecule consists of the nucleic acid sequence shown as SEQ ID NO: 22.

23. An isolated nucleic acid molecule having promoter activity, wherein said nucleic acid molecule comprises Neurofilament heavy gene promoter conserved region A and optionally one or more of Neurofilament heavy gene promoter conserved regions D, F, Dl , K, B, C and E, wherein said nucleic acid molecule comprises no more than three of the group of Neurofilament heavy gene promoter conserved regions consisting of Neurofilament heavy gene promoter conserved regions D, F, Dl, and K and no more than four of the group of Neurofilament heavy gene promoter conserved regions consisting of Neurofilament heavy gene promoter conserved regions D, F, B, C, and E;

wherein Neurofilament heavy gene promoter conserved region A is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 1, or a functional variant thereof; Neurofilament heavy gene promoter conserved region D is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 2, or a functional variant thereof; Neurofilament heavy gene promoter conserved region F is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 3, or a functional variant thereof; Neurofilament heavy gene promoter conserved region Dl is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 4, or a functional variant thereof; Neurofilament heavy gene promoter conserved region K is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 5, or a functional variant thereof; Neurofilament heavy gene promoter conserved region B is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 6, or a functional variant thereof;

Neurofilament heavy gene promoter conserved region C is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 7, or a functional variant thereof; and Neurofilament heavy gene promoter conserved region E is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 8, or a functional variant thereof.

24. The isolated nucleic acid molecule according to claim 23, wherein of Neurofilament heavy gene promoter conserved regions D, F, Dl, K, B, C and E, the isolated nucleic acid molecule comprises fewer than three.

25. The isolated nucleic acid molecule according to claim 23, wherein, of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, K, B, C and E said isolated nucleic acid molecule comprises only conserved region A.

26. The isolated nucleic acid molecule according to claim 25, wherein, of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, K, B, C and E, said isolated nucleic acid molecule comprises only conserved regions A and F.

27. The isolated nucleic acid molecule according to claim 23, wherein the isolated nucleic acid molecule comprises at least one of Neurofilament heavy gene promoter conserved region D and Neurofilament heavy gene promoter conserved region F.

28. The isolated nucleic acid molecule according to claim 27, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved region A, Neurofilament heavy gene promoter conserved region D, and Neurofilament heavy gene promoter conserved region F.

29. The isolated nucleic acid molecule according to any one of claims 23, 24, 27 and 28, wherein the isolated nucleic acid molecule comprises at least one of the conserved regions selected from: Neurofilament heavy gene promoter conserved region Dl and

Neurofilament heavy gene promoter conserved region K;

30. The isolated nucleic acid molecule according to any one of claims 23-24 or claims 27-29, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180% of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 21.

31. The isolated nucleic acid molecule according to claim 30, wherein the isolated nucleic acid molecule comprises between each recited conserved region and its adjacent recited conserved region in said isolated nucleic acid molecule a spacer sequence of a length in the range 20-180%) of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 21.

32. The isolated nucleic acid molecule according to any one of claims 23-24 or claims 27-29, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180%) of the sequence separating one of said recited conserved regions from one of its adjacent conserved regions in the nucleic acid sequence shown as SEQ ID NO: 21.

33. The isolated nucleic acid molecule according to any one of claim 23 or claims 27-32, wherein the isolated nucleic acid molecule comprises at least one of the conserved regions selected from: Neurofilament heavy gene promoter conserved region B, Neurofilament heavy gene promoter conserved region C, and Neurofilament heavy gene promoter conserved region E;

34. The isolated nucleic acid molecule for use according to claim 33, wherein, of Neurofilament heavy gene promoter conserved regions B, C, and E, the isolated nucleic acid molecule comprises only one or two.

35. The isolated nucleic acid molecule for use according to claim 33 or claim 34, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20 -180% of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 22.

36. The isolated nucleic acid molecule for use according to claim 35, wherein the isolated nucleic acid molecule comprises between each recited conserved region and its adjacent recited conserved region in said isolated nucleic acid molecule a spacer sequence of a length in the range 20-180% of the sequence separating said recited conserved regions in the nucleic acid sequence shown as SEQ ID NO: 22.

37. The isolated nucleic acid molecule for use according to any to claim 33 or claim 34, wherein the isolated nucleic acid molecule comprises between two recited conserved regions a spacer sequence of a length in the range 20-180%) of the sequence separating one of said recited conserved regions from one of its adjacent conserved regions in the nucleic acid sequence shown as SEQ ID NO: 22.

38. A method of treatment of ocular disease, wherein said method comprises administering to an eye an isolated nucleic acid molecule having promoter activity, wherein said nucleic acid molecule comprises at least Neurofilament heavy gene promoter conserved region A and optionally one or more of Neurofilament heavy gene promoter conserved regions D, F, Dl, K, B, C and E;

wherein Neurofilament heavy gene promoter conserved region A is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 1, or a functional variant thereof; Neurofilament heavy gene promoter conserved region D is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 2, or a functional variant thereof; Neurofilament heavy gene promoter conserved region F is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 3, or a functional variant thereof; Neurofilament heavy gene promoter conserved region D 1 is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 4, or a functional variant thereof; Neurofilament heavy gene promoter conserved region K is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 5, or a functional variant thereof; Neurofilament heavy gene promoter conserved region B is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 6, or a functional variant thereof;

Neurofilament heavy gene promoter conserved region C is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 7, or a functional variant thereof; and Neurofilament heavy gene promoter conserved region E is a nucleotide sequence having the nucleotide sequence shown as SEQ ID NO: 8, or a functional variant thereof.

39. The method according to claim 38, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved region A, Neurofilament heavy gene promoter conserved region D, and Neurofilament heavy gene promoter conserved region F.

40. The method according to claims 38 or claim 39, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, and K.

41. The method according to claim 40, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, Dl, and K having the nucleic acid sequences shown as SEQ ID NOS: 1, 2, 3, 4, and 5 respectively.

42. The method according to claim 40 or claim 41, wherein the isolated nucleic acid molecule consists of the nucleic acid sequence shown as SEQ ID NO: 21.

43. The method according to claim 38, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, B, C, and E.

44. The method according to claim 43, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, B, C, and E having the nucleic acid sequences shown as SEQ ID NOS: 9, 12, 15, 6, 7, and 8 respectively.

45. The method according to claim 43 or claim 44, wherein the isolated nucleic acid molecule consists of the nucleic acid sequence shown as SEQ ID NO: 22

46. The method according to claim 43, wherein the isolated nucleic acid molecule comprises each of Neurofilament heavy gene promoter conserved regions A, D, F, B, C, and E having the nucleic acid sequences shown as SEQ ID NOS: 10, 13, 16, 18, 19, and 20

respectively.

47. The method according to claim 38, wherein the isolated nucleic acid molecule is the isolated nucleic acid molecule according to any one of claims 23 to 37.

48. An expression cassette comprising the isolated nucleic acid molecule according to any one of claims 23 to 37 and one or more heterologous polynucleotide sequences with which the nucleic acid molecule is operably linked.

49. The isolated nucleic acid molecule according to any one of claims 23 to 37 or the expression cassette according to claim 48, wherein said nucleic acid molecule having promoter activity provides preferential expression of said one or more heterologous

polynucleotide sequences in the ganglion cell layer of the eye.

50. A vector comprising the isolated nucleic acid according to any one of claims 23 to 37 or the expression cassette according to claim 48 or claim 49.

The vector according to claim 50, wherein the vector is a viral vector.

The vector according to claim 51 , wherein the vector is an AAV vector.

53. The vector according to any one of claims 50-52, wherein the vector comprises a sequence encoding a neurotrophic or neuroprotective factor.

54. A cell comprising the isolated nucleic acid molecule according to any one of claims 23 to 37, the expression cassette according to claim 48 or claim 49, or the vector according to any one of claims 50-53.

55. A therapeutic composition comprising the isolated nucleic acid molecule according to any one of claims 23 to 37, the expression cassette according to claim 48 or claim 49, the vector according to any one of claims 50-53, or the cell according to claim 54.

56. The isolated nucleic acid molecule according to any one of claims 23 to 37, the expression cassette according to claim 48 or claim 49, the vector according to any one of claims 50-53 for use in medicine.

57. The isolated nucleic acid molecule for use according to any one of claims 1 to 22 or the method according to any one of claims 38 to 47, wherein the ocular disease is Leber Hereditary Optic Neuropathy (LHON), dominant optic atophy (DOA), or glaucoma.