Modified vector

Abstract

This disclosure generally relates to viral vectors useful for gene therapy applications. In particular, this disclosure relates to lentiviral vectors, and more specifically to lentiviral vectors comprising modified insulator sequences.

Claims

1. A nucleic acid construct comprising a modified HS4 insulator or a fragment thereof, wherein the modified HS4 insulator comprises a splice acceptor site (SA4) that is deactivated relative to an unmodified HS4-650 insulator. SA4 is located at nucleotide positions 352–353 in the unmodified HS4-650 insulator in the numbering for SEQ ID NO: 2, SEQ ID NO: 2 is the reverse complementary sequence of the unmodified HS4-650 insulator shown in SEQ ID NO: 1, and / or SA4 is a nucleic acid construct containing the sequence ATCTCTCCAG^GCAAGCTCTT (SEQ ID NO: 49), where ^ represents the splice position.

2. The nucleic acid construct according to claim 1, wherein the modified HS4 insulator comprises a mutation that inactivates SA4 compared to the unmodified HS4-650 insulator.

3. The nucleic acid construct according to claim 2, wherein the mutation is a mutation from A to T at position 351 in the numbering of sequence number 2.

4. The nucleic acid construct according to any one of claims 1 to 3, wherein the modified HS4 insulator is an HS4-650 insulator.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein the modified HS4 insulator comprises a sequence indicated by any one of sequence numbers 3, 11, 19, 27, 35, and 43.

6. A nucleic acid construct comprising a modified HS4 insulator, wherein the modified HS4 insulator comprises a splice acceptor site (SA5) that is deactivated relative to the unmodified HS4-650 insulator. SA5 is located at nucleotide positions 311-312 in the unmodified HS4-650 insulator in the numbering for SEQ ID NO: 2, SEQ ID NO: 2 is the reverse complementary sequence of the unmodified HS4-650 insulator shown in SEQ ID NO: 1, and / or SA5 is a nucleic acid construct containing the sequence AATTTCTCCAG^CTGCCTGTCC (SEQ ID NO: 50), where ^ represents the splice position.

7. The nucleic acid construct according to claim 6, wherein the modified HS4 insulator comprises a mutation that inactivates SA5 compared to the unmodified HS4-650 insulator.

8. The nucleic acid construct according to claim 7, wherein the mutation is a mutation from A to T at position 310 in the numbering of sequence number 2.

9. The nucleic acid construct according to any one of claims 6 to 8, wherein the modified HS4 insulator is an HS4-650 insulator.

10. The nucleic acid construct according to any one of claims 6 to 9, wherein the modified HS4 insulator comprises a sequence indicated by any one of sequence numbers 4, 12, 20, 28, 36, and 44.

11. The nucleic acid construct according to any one of claims 1 to 8, wherein the modified HS4 insulator further comprises one or more additional deactivated splice acceptor sites compared to the unmodified HS4-650 insulator.

12. One or more additional deactivated splice acceptor sites, (i) an inactivated splice acceptor site (SA1) located at positions 385-386 of the nucleotide in the numbering for SEQ ID NO: 2 in an unmodified HS4-650 insulator, and / or comprising the sequence TTGCATCCAG^ACACCATCAA (SEQ ID NO: 51), wherein SA1 may be inactivated via an A-to-T mutation at position 384, (ii) an inactivated splice acceptor site (SA2) located at positions 446–447 of the nucleotide in the numbering for SEQ ID NO: 2 in an unmodified HS4-650 insulator, and / or comprising the sequence ATCCCCCCAG^GTGTCTGCAG (SEQ ID NO: 52), wherein SA2 may be inactivated via an A-to-T mutation at position 445, and / or (iii) An inactivated splice acceptor site (SA2) located at positions 456-457 of the nucleotide in the numbering for SEQ ID NO: 2 in an unmodified HS4-650 insulator, and / or comprising the sequence GTGTCTGCAG^GCTCAAAGAG (SEQ ID NO: 53), wherein SA3 may be inactivated via an A-to-T mutation at position 455. Selected from, The nucleic acid construct according to claim 11, wherein ^ represents the splice position.

13. The nucleic acid construct according to any one of claims 1 to 5 or 11, wherein the modified HS4 insulator comprises deactivated splice acceptor sites SA4, SA2, and SA3.

14. The nucleic acid construct according to claim 13, wherein the modified HS4 insulator comprises a sequence indicated by any one of sequence numbers 5, 13, 21, 29, 37, and 45.

15. The nucleic acid construct according to any one of claims 1 to 5 or 11, wherein the modified HS4 insulator comprises deactivated splice acceptor sites SA4, SA1, SA2, and SA3.

16. The nucleic acid construct according to claim 15, wherein the modified HS4 insulator comprises a sequence indicated by any one of sequence numbers 6, 14, 22, 30, 38, and 46.

17. The nucleic acid construct according to any one of claims 1 to 11, wherein the modified HS4 insulator comprises deactivated splice acceptor sites SA4, SA5, SA1, SA2, and SA3.

18. The nucleic acid construct according to claim 17, wherein the modified HS4 insulator comprises a sequence indicated by any one of sequence numbers 7, 15, 23, 31, 39, and 47.

19. The nucleic acid construct according to any one of claims 1 to 11, wherein the modified HS4 insulator comprises deactivated splice acceptor sites SA4, SA5, SA2, and SA3.

20. The nucleic acid construct according to claim 19, wherein the modified HS4 insulator comprises a sequence indicated by any one of sequence numbers 8, 16, 24, 32, 40, and 48.

21. The nucleic acid construct according to any one of claims 1 to 20, further comprising a first promoter operably linked to a first polynucleotide, wherein the first polynucleotide may contain a xenotransgene.

22. The nucleic acid construct according to claim 21, wherein the first polynucleotide encodes a Wiscott-Aldrich syndrome protein.

23. The nucleic acid construct according to claim 22, wherein the Wiscott-Aldrich syndrome protein comprises the amino acid sequence shown in Sequence ID No. 59 or a sequence having at least 95% sequence identity thereto.

24. The nucleic acid construct according to claim 22 or 23, wherein the first polynucleotide comprises the sequence shown by any one of SEQ ID NOs. 60 to 62 or a sequence having at least 95% sequence identity thereto.

25. The nucleic acid construct according to any one of claims 21 to 24, wherein the modified HS4 insulator is oriented opposite to the first polynucleotide.

26. The nucleic acid construct according to claim 25, wherein the first polynucleotide is oriented forward within the nucleic acid construct and the modified HS4 insulator is oriented backward.

27. The nucleic acid construct according to any one of claims 21 to 24, wherein the modified HS4 insulator is oriented in the same orientation as the first polynucleotide.

28. The nucleic acid construct according to claim 27, wherein the first polynucleotide and the modified HS4 insulator are forward-oriented within the nucleic acid construct.

29. The nucleic acid construct according to any one of claims 21 to 28, wherein the modified HS4 insulator is located downstream of the first polynucleotide.

30. The nucleic acid construct according to any one of claims 21 to 29, further comprising a woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE) between the first polynucleotide and a modified HS4 insulator.

31. The nucleic acid construct according to claim 30, wherein WPRE comprises a nucleic acid sequence represented by either SEQ ID NO: 63 or 64 or a sequence having at least 95% sequence identity thereto.

32. The nucleic acid construct according to any one of claims 21 to 31, wherein the first promoter is an MND promoter.

33. The nucleic acid construct according to claim 32, wherein the MND promoter includes the nucleic acid sequence shown in Sequence ID No. 65 or a sequence having at least 95% sequence identity therewith.

34. The nucleic acid construct according to any one of claims 21 to 33, further comprising a second promoter operably linked to a second polynucleotide.

35. The nucleic acid construct according to claim 34, wherein the second polynucleotide encodes a nucleic acid that inhibits HPRT expression.

36. The nucleic acid construct according to claim 35, wherein the nucleic acid that inhibits HPRT expression is an shRNA which may contain the hairpin loop sequence shown in SEQ ID NO:

66.

37. The nucleic acid construct according to claim 36, wherein the shRNA comprises the nucleic acid sequence shown in SEQ ID NO: 67 or 68 or a sequence having at least 95% sequence identity thereto.

38. The nucleic acid construct according to any one of claims 34 to 37, wherein the second promoter comprises a Pol III promoter or a Pol II promoter.

39. The nucleic acid construct according to claim 38, wherein the Pol III promoter comprises 7sk which may include a nucleic acid sequence represented by any one of sequence numbers 69 to 71 or a sequence having at least 95% sequence identity therewith.

40. The nucleic acid construct according to any one of claims 34 to 39, wherein the second promoter and the operably linked second polynucleotide are in reverse orientation and upstream of the first promoter and the operably linked first polynucleotide.

41. The nucleic acid construct according to any one of claims 21 to 40, further comprising a polyadenylation signal downstream of a first polynucleotide and a modified HS4 insulator.

42. A nucleic acid construct according to any one of claims 1 to 41, comprising a sequence represented as nucleotides 2396 to 4301 of any one of sequence numbers 76 to 79, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

43. A nucleic acid construct according to any one of claims 1 to 42, which is present in a vector.

44. The nucleic acid construct according to claim 43, wherein the vector is a plasmid or a viral vector.

45. The nucleic acid construct according to claim 44, wherein the vector is a retroviral vector.

46. The nucleic acid construct according to claim 45, wherein the vector is a lentiviral vector.

47. A vector comprising the nucleic acid construct according to any one of claims 1 to 42.

48. The vector according to claim 47, which is a viral vector.

49. The vector according to claim 48, which is a retroviral vector.

50. The vector according to claim 49, which is a lentiviral vector.

51. The vector according to any one of claims 47 to 50, comprising a sequence represented as nucleotides 2396 to 4301 of any one of sequence numbers 76 to 79, or a sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

52. A host cell comprising the nucleic acid construct according to any one of claims 1 to 46.

53. A host cell comprising the vector according to any one of claims 47 to 50, or transduced by thereof.

54. A host cell according to claim 52 or 53, which is a hematopoietic stem cell.

55. A host cell according to any one of claims 52 to 54, which is HPRT deficient.

56. A method for treating a subject having Wiscott-Aldrich syndrome, comprising administering to a host cell according to any one of claims 52 to 55, wherein the first polynucleotide encodes Wiscott-Aldrich syndrome.

57. The method according to claim 56, further comprising administering a purine analog to a target to increase the engraftment of host cells.

58. The method according to claim 57, wherein the purine analog is selected from the group consisting of 6-thioguanine ("6TG"), 6-mercaptopurine ("6MP"), or azathiopurine ("AZA").

59. The method according to any one of claims 56 to 58, further comprising preconditioning the subject with a purine analog prior to administration of host cells.

60. Use of host cells according to any one of claims 52 to 55 in the preparation of a medicament for the treatment of Wiscott-Aldrich syndrome.

Images