Globin gene therapy for treating hemoglobinopathies
The presently disclosed subject matter provides for expression cassettes that allow for expression of a globin gene or a functional portion thereof, vectors comprising thereof, and cells transduced with such expression cassettes and vectors. The presently disclosed subject matter further provides methods for treating a hemoglobinopathy in a subject comprising administering an effective amount of such transduced cells to the subject.
1. A viral vector comprising an expression cassette comprising an insulator that comprises the nucleotide sequence set forth in SEQ ID NO:1, SEQ ID NO: 24, or SEQ ID NO: 25, and a globin gene operably linked to a β-globin locus control region (LCR) region.
2. The viral vector of claim 1 , wherein the β-globin LCR region does not comprise a HS1 region.
3. The viral vector of claim 2 , wherein the β-globin LCR region does not comprise a core sequence of the HS1.
4. The viral vector of claim 3 , wherein the core sequence of the HS1 consists of the nucleotide sequence set forth in SEQ ID NO:22 or SEQ ID NO: 23.
5. The viral vector of claim 2 , wherein the HS1 region sustains the HS1 function.
6. The viral vector of claim 2 , wherein the β-globin LCR region comprises a HS3 region and a HS4 region.
7. The viral vector of claim 6 , wherein the HS3 region is positioned between the globin gene and the HS4 region.
8. The viral vector of claim 2 , wherein the β-globin LCR region comprises a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO:5 and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO:6, and the (β-globin LCR region does not comprise a HS2 region.
9. The viral vector of claim 1 , wherein the β-globin LCR region does not comprise a Dnase I hypersensitive site-2 (HS2) region.
10. The viral vector of claim 9 , wherein the β-globin LCR region does not comprise a core sequence of HS2.
11. The viral vector of claim 10 , wherein the core sequence of HS2-consists of the nucleotide sequence set forth in SEQ ID NO:20 or SEQ ID NO: 21.
12. The viral vector of claim 9 , wherein the β-globin LCR region comprises a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO:2, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO:5, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO:6.
13. The viral vector of claim 9 , wherein the β-globin LCR region comprises a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO:3, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO:5, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO:8.
14. The viral vector of claim 9 , wherein the β-globin LCR region comprises a HS1 region consisting of the nucleotide sequence set forth in SEQ ID NO:4, a HS3 region consisting of the nucleotide sequence set forth in SEQ ID NO:5, and a HS4 region consisting of the nucleotide sequence set forth in SEQ ID NO:8.
15. The viral vector of claim 1 , wherein the β-globin LCR region does not comprise a HS2 region that sustains the enhancer activity of HS2.
16. The viral vector of claim 1 , wherein the β-globin LCR region comprises a Dnase I hypersensitive site-1 (HS1) region, a Dnase I hypersensitive site-3 (HS3) region, and a Dnase I hypersensitive site-4 (HS4) region.
17. The viral vector of claim 16 , wherein the HS3 region is positioned between the HS1 region and the HS4 region.
18. The viral vector of claim 16 , wherein the HS1 region is about 1.1 kb in length.
19. The viral vector of claim 18 , wherein the HS1 region consists of the nucleotide sequence set forth in SEQ ID NO:2.
20. The viral vector of claim 16 , wherein the HS1 region is about 600 bp in length.
21. The viral vector of claim 20 , wherein the HS1 region consists of the nucleotide sequence set forth in SEQ ID NO:3.
22. The viral vector of claim 16 , wherein the HS1 region is about 490 bp in length.
23. The viral vector of claim 22 , wherein the HS1 region consists of the nucleotide sequence set forth in SEQ ID NO:4.
24. The viral vector of claim 16 , wherein the HS3 region is about 1300 bp in length.
25. The viral vector of claim 24 , wherein the HS3 region consists of the nucleotide sequence set forth in SEQ ID NO:5.
26. The viral vector of claim 16 , wherein the HS4 region is about 1.1 kb in length.
27. The viral vector of claim 26 , wherein the HS4 region consists of the nucleotide sequence set forth in SEQ ID NO:6.
28. The viral vector of claim 26 , wherein the HS4 region consists of the nucleotide sequence set forth in SEQ ID NO:7.
29. The viral vector of claim 16 , wherein the HS4 region is about 450 bp in length.
30. The viral vector of claim 29 , wherein the HS4 region consists of the nucleotide sequence set forth in SEQ ID NO:8.
31. The viral vector of claim 1 , wherein the β-globin LCR region comprises a HS2 region, a HS3 region, and a HS4 region.
32. The viral vector of claim 31 , wherein the HS2 region is about 860 bp in length.
33. The viral vector of claim 32 , wherein the HS2 region consists of the nucleotide sequence set forth in SEQ ID NO:9.
34. The viral vector of claim 31 , wherein the HS3 region is about 1300 bp in length.
35. The viral vector of claim 34 , wherein the HS3 region consists of the nucleotide sequence set forth in SEQ ID NO:5.
36. The viral vector of claim 31 , wherein the HS4 region is about 1.1 kb in length.
37. The viral vector of claim 36 , wherein the HS4 region consists of the nucleotide sequence set forth in SEQ ID NO:7.
38. The viral vector of claim 31 , wherein the HS2 region consists of the nucleotide sequence set forth in SEQ ID NO:9, the HS3 region consists of the nucleotide sequence set forth in SEQ ID NO:5, and the HS4 region consists of the nucleotide sequence set forth in SEQ ID NO:7.
39. The viral vector of claim 31 , wherein the β-globin LCR region further comprises a HS1 region.
40. The viral vector of claim 1 , further comprising a β-globin promoter.
41. The viral vector of claim 40 , wherein the β-globin promoter is positioned between the globin gene and the β-globin LCR region.
42. The viral vector of claim 40 , wherein the β-globin promoter is a human β-globin promoter that is about 613 bp in length.
43. The viral vector of claim 42 , wherein the human β-globin promoter consists of the nucleotide sequence set forth in SEQ ID NO:10.
44. The viral vector of claim 40 , wherein the β-globin promoter is a human β-globin promoter that is about 265 bp in length.
45. The viral vector of claim 44 , wherein the human β-globin promoter consists of the nucleotide sequence set forth in SEQ ID NO:11.
46. The viral vector of claim 1 , wherein the globin gene is selected from the group consisting of β-globin gene, γ-globin gene, and δ-globin gene.
47. The viral vector of claim 46 , wherein the globin gene is human β-globin gene.
48. The viral vector of claim 47 , wherein the human β-globin gene is selected from the group consisting of a wild-type human β-globin gene, a human β-globin gene comprising one or more deletions of intron sequences, and a mutated human β-globin gene encoding at least one anti-sickling amino acid residue.
49. The viral vector of claim 48 , wherein the human β-globin gene is human β A -globin gene encoding a threonine to glutamine mutation at codon 87 (β A-T87Q ).
50. The viral vector of claim 1 , wherein the expression cassette consists of one of the insulator or two of the insulators.
51. The viral vector of claim 1 , further comprising a human β-globin 3′ enhancer.
52. The viral vector of claim 51 , wherein the human β-globin 3′ enhancer is positioned in the upstream of the globin gene.
53. The viral vector of claim 51 , wherein the human β-globin 3′ enhancer is about 879 bp in length.
54. The viral vector of claim 53 , wherein the human β-globin 3′ enhancer consists of the nucleotide sequence set forth in SEQ ID NO:12.
55. The viral vector of claim 1 , further comprising one or both of a Woodchuck hepatitis post-regulatory element (WPRE) and a bovine growth hormone polyadenylation signal in the 3′ long terminal repeat (LTR) of the vector.
56. The viral vector of claim 1 , wherein the viral vector is a retroviral vector.
57. The viral vector of claim 56 , wherein the retroviral vector is a lentiviral vector.
58. An in vitro cell transduced with the viral vector of claim 1 .
59. The cell of claim 58 , wherein the hematopoietic stem cell is a CD34 + hematopoietic stem cell.
60. A pharmaceutical composition comprising an effective amount of the cell of claim 58 and a pharmaceutically acceptable carrier.
61. A kit for treating a hemoglobinopathy comprising the cell of claim 58 .