Compositions and methods for hemoglobin production
Methods and compositions for producing hemoglobin and treating alpha-thalassemia are disclosed.
1. A viral vector comprising a nucleic acid molecule comprising:
i) a 5′ long terminal repeat (LTR) and a 3′ LTR, wherein one of said LTRs is self-inactivating;
ii) at least one promoter;
iii) a beta globin gene locus control region (LCR), wherein said LCR comprises HS1, HS2, HS3, and HS4, wherein said HS1 is in sense orientation and said HS2, HS3, and HS4 are in antisense orientation;
iv) an ankyrin insulator element (Ank);
v) a beta globin 3′ enhancer; and
vi) a sequence encoding human alpha globin.
2. The viral vector of claim 1 , further comprising a Woodchuck Post-Regulatory Element (WPRE).
3. The viral vector of claim 2 , wherein said WPRE is not between the 5′ LTR and 3′ LTR.
4. The viral vector of claim 1 , further comprising a Rev response element (RRE).
5. The viral vector of claim 4 , wherein said RRE is located between the 5′ LTR and the beta globin 3′ enhancer.
6. The viral vector of claim 1 , wherein said promoter is the beta globin promoter.
7. The viral vector of claim 1 , wherein said sequence encoding human alpha globin is the HBA1 or HBA2 gene.
8. The viral vector of claim 7 , wherein said sequence encoding human alpha globin comprises exons 1, 2, and 3, introns 1 and 2, 5′ UTR, and 3′ UTR.
9. The viral vector of claim 7 , wherein said sequence encoding human alpha globin is codon optimized.
10. The viral vector of claim 1 , wherein the beta globin 3′ enhancer, the promoter, the sequence encoding human alpha globin, and the beta globin gene locus control region are in antisense orientation.
11. The viral vector of claim 1 , wherein the viral vector is a lentiviral vector.
12. Isolated CD34+ cells comprising the viral vector of claim 1 .
13. The isolated CD34+ cells of claim 12 , wherein the CD34+ cells have been isolated from an individual who has alpha-thalassemia.
14. A composition comprising the viral vector of claim 1 and a pharmaceutically acceptable carrier.
15. A composition comprising viral particles, wherein the viral particles are synthesized from the viral vector of claim 1 .
16. A method for inducing expression of human alpha globin in cells comprising introducing into said cells a viral vector of claim 1 .
17. The method of claim 16 , wherein said method is in vitro.
18. A method of inhibiting and/or treating alpha-thalassemia in a subject in need thereof, said method comprising introducing the viral vector of claim 1 into cells and delivering the cells to said subject.
19. The method of claim 18 , wherein the cells are isolated from the subject to be treated.
20. A viral vector comprising a nucleic acid molecule comprising:
i) a 5′ long terminal repeat (LTR) and a 3′ LTR, wherein one of said LTRs is self-inactivating;
ii) at least one promoter;
iii) a beta globin gene locus control region (LCR);
iv) an ankyrin insulator element (Ank);
v) a beta globin 3′ enhancer; and
vi) a sequence encoding human alpha globin, wherein said viral vector is selected from the group consisting of Alpha 1/AA305, ALS19α, and ALS20α.
21. Isolated CD34+ cells comprising the viral vector of claim 20 .
22. The isolated CD34+ cells of claim 21 , wherein the CD34+ cells have been isolated from an individual who has alpha-thalassemia.
23. A composition comprising the viral vector of claim 20 and a pharmaceutically acceptable carrier.
24. A composition comprising viral particles, wherein the viral particles are synthesized from the viral vector of claim 20 .
25. A method for inducing expression of human alpha globin in cells comprising introducing into said cells a viral vector of claim 20 .
26. A method of inhibiting and/or treating alpha-thalassemia in a subject in need thereof, said method comprising introducing the viral vector of claim 20 into cells and delivering the cells to said subject.
27. The method of claim 26 , wherein the cells are isolated from the subject to be treated.
28. A viral vector comprising a nucleic acid molecule comprising:
i) a 5′ long terminal repeat (LTR) and a 3′ LTR, wherein one of said LTRs is self-inactivating;
ii) at least one promoter;
iii) a beta globin gene locus control region (LCR);
iv) an ankyrin insulator element (Ank);
v) a beta globin 3′ enhancer; and
vi) a sequence encoding human alpha globin, wherein said nucleic acid molecule has a nucleotide sequence having at least 95% identity with SEQ ID NO: 1 or SEQ ID NO: 2.
29. The viral vector of claim 28 , wherein said nucleic acid molecule comprises SEQ ID NO: 1.
30. The viral vector of claim 28 , wherein said nucleic acid molecule has a nucleotide sequence having at least 95% identity with SEQ ID NO: 2.
31. The viral vector of claim 30 , wherein said nucleic acid molecule comprises SEQ ID NO: 2.
32. The viral vector of claim 28 , wherein said nucleic acid molecule has a nucleotide sequence having at least 95% identity with SEQ ID NO: 1.
33. Isolated CD34+ cells comprising the viral vector of claim 28 .
34. The isolated CD34+ cells of claim 33 , wherein the CD34+ cells have been isolated from an individual who has alpha-thalassemia.
35. A composition comprising the viral vector of claim 28 and a pharmaceutically acceptable carrier.
36. A composition comprising viral particles, wherein the viral particles are synthesized from the viral vector of claim 28 .
37. A method for inducing expression of human alpha globin in cells comprising introducing into said cells a viral vector of claim 28 .
38. A method of inhibiting and/or treating alpha-thalassemia in a subject in need thereof, said method comprising introducing the viral vector of claim 28 into cells and delivering the cells to said subject.
39. The method of claim 38 , wherein the cells are isolated from the subject to be treated.