Vector encoding human globin gene and use thereof in treatment of hemoglobinopathies

• US 20030022303A1
• Filed: 07/01/2002
• Published: 01/30/2003
• Est. Priority Date: 06/29/2001
• Status: Active Grant

• Current Assignee: Sloan-Kettering Institute For Cancer Research (Memorial Sloan-Kettering Cancer Center)
• Original Assignee: Sloan-Kettering Institute For Cancer Research (Memorial Sloan-Kettering Cancer Center)
• Inventors: May, Chad, Bertino, Joseph, Sadelain, Michel, Rivella, Stefano
• Granted Patent: US 7,541,179 B2
• Time in Patent Office: Days
• US Class Current: 435/69.1
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0066 Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/805 Haemoglobins; Myoglobins
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2740/15043 viral genome or elements thereof as genetic vector

Abstract

Recombinant lentiviral vectors having a region encoding a functional β-globin gene; and large portions of the β-globin locus control regions which include DNase I hypersensitive sites HS2, HS3 and HS4 provides expression of β-globin when introduced into a mammal, for example a human, in vivo. Optionally, the vector further includes a region encoding a dihydrofolate reductase. The vector may be used in treatment of hemoglobinopathies, including β-thalessemia and sickle-cell disease. For example, hematopoietic progenitor or stem cells may be transformed ex vivo and then restored to the patient. Selection processes may be used to increase the percentage of transformed cells in the returned population. For example, a selection marker which makes transformed cells more drug resistant than un-transformed cells allows selection by treatment of the cells with the corresponding drug.

Claims

1. A recombinant lentiviral vector comprising:

(a) a region comprising a functional globin gene; and

(b) large portions of the β

-globin locus control region, which include DNase I hypersensitive sites HS2, HS3 and HS4, said vector providing expression of the globin gene when introduced into a mammal in vivo.

2. The vector of claim 1, further comprising a region encoding a dihydrofolate reductase.

3. The vector of claim 2, further comprising a mouse PGK promoter, wherein the mouse PGK promoter controls the expression of the region encoding a dihydroflate reductase.

4. The vector of claim 3, wherein the dihydrofolate reductase is a human dihdrofolate reductase.

5. The vector of claim 4, wherein the human dihydrofolate reductase is a mutant form having increased resistance to antifolates as compared to wild-type human dihydrofolate reductase, said mutant form differing in amino acid sequence from wild-type human dihydrofolate reductase as a result of a set of mutations.

6. The vector of claim 5, wherein the set of mutations comprises a mutation at an amino acid corresponding to amino acid 22 of the wild-type sequence and a mutation at an amino acid corresponding to amino acid 31 of the wild type sequence.

7. The vector of claim 2, wherein the dihydrofolate reductase is a human dihdrofolate reductase.

8. The vector of claim 7, wherein the human dihydrofolate reductase is a mutant form having increased resistance to antifolates as compared to wild-type human dihydrofolate reductase, said mutant form differing in amino acid sequence from wild-type human dihydrofolate reductase as a result of a set of mutations.

9. The vector of claim 8, wherein the set of mutations comprises a mutation at an amino acid corresponding to amino acid 22 of the wild-type sequence and a mutation at an amino acid corresponding to amino acid 31 of the wild type sequence.

10. The vector of claim 1, wherein the functional globin gene encodes human β

-globin.

11. The vector of claim 10, further comprising a region encoding a dihydrofolate reductase.

12. The vector of claim 11, further comprising a mouse PGK promoter, wherein the mouse PGK promoter controls the expression of the region encoding a dihydroflate reductase.

13. The vector of claim 12, wherein the dihydrofolate reductase is a human dihydrofolate reductase.

14. The vector of claim 13, wherein the human dihydrofolate reductase is a mutant form having increased resistance to antifolates as compared to wild-type human dihydrofolate reductase, said mutant form differing in amino acid sequence from wild-type human dihydrofolate reductase as a result of a set of mutations.

15. The vector of claim 14, wherein the set of mutations comprises a mutation at an amino acid corresponding to amino acid 22 of the wild-type sequence and a mutation at an amino acid corresponding to amino acid 31 of the wild type sequence.

16. The vector of claim 11, wherein the dihydrofolate reductase is a human dihdrofolate reductase.

17. The vector of claim 16, wherein the human dihydrofolate reductase is a mutant form having increased resistance to antifolates as compared to wild-type human dihydrofolate reductase, said mutant form differing in amino acid sequence from wild-type human dihydrofolate reductase as a result of a set of mutations.

18. The vector of claim 17, wherein the set of mutations comprises a mutation at an amino acid corresponding to amino acid 22 of the wild-type sequence and a mutation at an amino acid corresponding to amino acid 31 of the wild type sequence.

19. A method for treatment of hemoglobinopathy in a mammalian individual suffering from a hemoglobinopathy comprising the steps of:

introducing to the mammalian individual a recombinant lentiviral vector comprising;

(a) a region comprising a functional globin gene; and

(b) large portions of the β

-globin locus control region, which include DNase I hypersensitive sites HS2, HS3 and HS4, said vector providing expression of β

-globin when introduced into a mammal in vivo; and

expressing the functional globin gene in the mammal, thereby providing a therapeutic benefit to the mammalian individual.

20. The method of claim 19, wherein the vector further comprises a region encoding a dihydrofolate reductase.

21. The method of claim 20, wherein the vector further comprises a mouse PGK promoter, wherein the mouse PGK promoter controls the expression of the region encoding a dihydroflate reductase.

22. The method of claim 21, wherein the dihydrofolate reductase is a human dihdrofolate reductase.

23. The method of claim 22, wherein the human dihydrofolate reductase is a mutant form having increased resistance to antifolates as compared to wild-type human dihydrofolate reductase, said mutant form differing in amino acid sequence from wild-type human dihydrofolate reductase as a result of a set of mutations.

24. The method of claim 23, wherein the set of mutations comprises a mutation at an amino acid corresponding to amino acid 22 of the wild-type sequence and a mutation at an amino acid corresponding to amino acid 31 of the wild type sequence.

25. The method of claim 19, wherein the step of introducing the recombinant vector is performed by transforming hematopoietic progenitor cells or stem cells with the recombinant vector ex vivo and then restoring the transformed cells to the mammalian individual.

26. The method of claim 25, wherein the transformed cells are subjected to a selection process to increase the percentage of transformed cells prior to restoring the cells to the mammalian individual.

27. The method of claim 26, wherein the vector further comprises a region encoding a dihydrofolate reductase, and wherein the selection process comprises exposure of the cells to an antifolate,

28. The method of claim 27, wherein the antifolate is methotrexate.

29. The method of claim 19, wherein the globin gene encodes human β

-globin.

30. A mammalian hematopoietic progenitor or stem cell transduced with a recombinant lentivector comprising:

(a) a region comprising a functional globin gene; and

(b) large portions of the β

-globin locus control regions, which include DNase I hypersensitive sites HS2, HS3 and HS4, wherein said cells express the functional β

-globin gene.

31. The transduced cell of claim 30, wherein the mammalian hematopoietic progenitor or stem cell is a human cell.

32. The transduced cell of claim 31, wherein the vector further comprises a region encoding a dihydrofolate reductase.

33. The transduced cell of claim 32, wherein the vector further comprises a mouse PGK promoter, wherein the mouse PGK promoter controls the expression of the region encoding a dihydroflate reductase.

34. The transduced cell of claim 33, wherein the dihydrofolate reductase is a human dihdrofolate reductase.

35. The transduced cell of claim 34, wherein the human dihydrofolate reductase is a mutant form having increased resistance to antifolates as compared to wild-type human dihydrofolate reductase, said mutant form differing in amino acid sequence from wild-type human dihydrofolate reductase as a result of a set of mutations.

36. The transduced cell of claim 35, wherein the set of mutations comprises a mutation at an amino acid corresponding to amino acid 22 of the wild-type sequence and a mutation at an amino acid corresponding to amino acid 31 of the wild type sequence.

37. The transduced cell of claim 30, wherein the dihydrofolate reductase is a human dihdrofolate reductase.

38. The transduced cell of claim 30, wherein the globin gene encodes human β

-globin.

39. A method for making a therapeutic composition for treatment of hemoglobinopathy in a mammalian individual, comprising the steps of preparing a recombinant lentiviral vector comprising:

(a) a region comprising a functional globin gene; and

(b) large portions of the β

-globin locus control region, which include DNase I hypersensitive sites HS2, HS3 and HS4, said vector providing expression of the globin gene when introduced into a mammal in vivo., obtaining hematopoietic progenitor or stem cells from the mammalian individual, and transducing the cells with the recombinant vector.

40. The method of claim 39, further comprising the step of performing an ex vivo selection using an antifolate.

41. The method of claim 39, wherein the globin gene encodes human β

-globin.