Hematopoietic stem cells and methods of treatment of neovascular eye diseases therewith

• US 20050063961A1
• Filed: 04/28/2004
• Published: 03/24/2005
• Est. Priority Date: 07/25/2002
• Status: Active Grant

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Da Silva, Karen, Otani, Atsushi, Friedlander, Martin, Moreno, Stacey (Hanekamp)
• Granted Patent: US 7,838,290 B2
• Time in Patent Office: Days
• US Class Current: 424/93.210
• 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: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 5/0647 Haematopoietic stem cells; Uncommitted or multipotent progenitors
• CPC Class Codes: C12N 5/0692 Stem cells; Progenitor cells; Precursor cells

Abstract

Isolated, mammalian, adult bone marrow-derived, lineage negative hematopoietic stem cell populations (Lin⁻ HSCs) contain endothelial progenitor cells (EPCs) capable of rescuing retinal blood vessels and neuronal networks in the eye. Preferably at least about 20% of the cells in the isolated Lin⁻ HSCs express the cell surface antigen CD31. The isolated Lin⁻ HSC populations are useful for treatment of ocular vascular diseases. In a preferred embodiment, the Lin⁻ HSCs are isolated by extracting bone marrow from an adult mammal; separating a plurality of monocytes from the bone marrow; labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens; removing of monocytes that are positive for the lineage surface antigens from the plurality of monocytes, and recovering a Lin⁻ HSC population containing EPCs. Isolated Lin⁻ HSCs that have been transfected with therapeutically useful genes are also provided, and are useful for delivering genes to the eye for cell-based gene therapy. Methods of preparing isolated stem cell populations of the invention, and methods of treating ocular diseases and injury are also described.

Claims

1. An isolated, mammalian lineage negative hematopoietic stem cell population, which comprises hematopoietic stem cells and endothelial progenitor cells.

2. The isolated stem cell population of claim 1 wherein at least about 20% of the cells express the surface antigen CD31.

3. The isolated stem cell population of claim 1 wherein at least about 50% of the cells express the surface antigen CD31.

4. The isolated stem cell population of claim 1 wherein at least about 75% of the cells express the surface antigen CD31.

5. The isolated stem cell population of claim 1 wherein at least about 50% of the cells express the surface antigen for integrin α

6.

6. The isolated stem cell population of claim 1 wherein the cells are derived from adult bone marrow.

7. The isolated stem cell population of claim 1 wherein the cells are murine cells.

8. The isolated stem cell population of claim 7 wherein at least about 50% of the cells express the surface antigen CD31 and at least about 50% of the cells express the surface antigen CD117.

9. The isolated stem cell population of claim 7 wherein at least about 65% of the cells express the surface antigen CD117.

10. The isolated stem cell population of claim 7 wherein at least about 80% of the cells express the surface antigen CD31 and at least about 70% of the cells express the surface antigen CD 117.

11. The isolated stem cell population of claim 1 wherein the cells are human cells.

12. The isolated stem cell population of claim 11 wherein the cells are CD133 negative, at least about 50% of the cells express the surface antigen for integrin α

6, and at least about 50% of the cells express the surface antigen CD31.

13. The isolated stem cell population of claim 11 wherein the cells are CD133 positive, less than about 30% of the cells express the surface antigen for integrin α

6, and less than about 30% of the cells express the surface antigen CD31.

14. The isolated stem cell population of claim 1 further including a cell culture medium.

31. A method of enhancing retinal neovascularization in a mammal comprising intravitreally injecting lineage negative hematopoietic stem cell population of claim 1 into the eye of a mammal in need of retinal neovascularization wherein the stem cells are derived from bone marrow of the same species of mammal as the species into whose eye the cells are injected.

32. The method of claim 31 wherein the stem cells are derived from the bone marrow of the same individual mammal into whose eye the stem cells are injected.

33. The method of claim 31 wherein the mammal is a mouse.

34. The method of claim 31 wherein the mammal is a human.

52. A transfected lineage negative hematopoietic stem cell population comprising a stem cell population of claim 1 transfected with a gene that operably encodes a therapeutically useful peptide.

53. The transfected stem cell population of claim 52 wherein the therapeutically useful peptide is an anti-angiogenic peptide.

54. The transfected stem cell population of claim 52 wherein the anti-angiogenic peptide is a protein fragment.

55. The transfected stem cell population of claim 54 wherein the protein fragment is an anti-angiogenic fragment of TrpRS.

57. The transfected stem cell population of claim 52 wherein the therapeutically useful peptide is a neurotrophic agent.

58. The transfected stem cell population of claim 57 wherein the neurotrophic agent is selected form the group consisting of nerve growth factor, neurotrophin-3, neurotrophin-4, neurotrophin-5, ciliary neurotrophic factor, retinal pigmented epithelium-derived neurotrophic factor, insulin-like growth factor, glial cell line-derived neurotrophic factor, and brain-derived neurotrophic factor.

59. A method of inhibiting retinal angiogenesis in the eye of a mammal comprising intravitreally injecting a transfected stem cell population according to claim 53 into the eye of the mammal.

60. A method of inhibiting retinal neuronal degeneration in the eye of a mammal comprising intravitreally injecting a transfected stem cell population according to claim 57 into the eye of the mammal.

64. A method of delivering transgenes to the retinal vasculature of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of claim 52 into the eye of the mammal.

66. A method of rescuing neuronal networks in the eye of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of claim 1 into the eye of the mammal.

67. A method of rescuing blood vessels in the eye of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of claim 1 into the eye of the mammal.

68. A method of stimulating upregulation of anti-apoptotic genes in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of claim 1 into the eye of the mammal.

69. A method of repairing ischemic tissue in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of claim 1 into the eye of the mammal.

70. A method of targeted delivery of stem cells to astrocytes in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of claim 1 into the eye of the mammal.

71. A method of targeted delivery of transgenes to astrocytes in the eye of a mammal comprising intravitreally injecting a lineage negative hematopoietic stem cell population of claim 52 into the eye of the mammal.

15. A method of isolating an adult bone marrow-derived, lineage negative hematopoietic stem population including endothelial progenitor cells comprising the steps of:

(a) extracting bone marrow from an adult mammal;

(b) separating a plurality of monocytes from the bone marrow;

(c) labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;

(d) removing monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells.

16. The method of claim 15 wherein the mammal is a mouse.

17. The method of claim 15 wherein the mammal is a mouse and the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD3, CD11, CD45, Ly-6G, and TER-119.

18. The method of claim 15 wherein the mammal is a human.

19. The method of claim 15 wherein the mammal is a human and the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, and CD235a.

20. The method of claim 18 wherein the mammal is a human and the method includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody and recovering a population of CD133 positive, lineage negative hematopoietic stem cells.

21. The method of claim 18 wherein the mammal is a human and the method includes the additional steps of labeling the monocytes with a biotin-conjugated CD133 antibody, removing CD133 positive cells, and recovering a population of CD133 negative, lineage negative hematopoietic stem cells.

22. An isolated, mammalian, adult bone marrow-derived lineage negative hematopoietic stem cell population containing endothelial progenitor cells produced by:

(a) extracting bone marrow from an adult mammal;

(b) separating a plurality of monocytes from the bone marrow;

(c) labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;

(d) removing monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells;

wherein at least about 20% of the cells express the surface antigen CD31.

23. The isolated stem cell population of claim 22 wherein at least about 50% of the cells express the surface antigen CD31.

24. The isolated stem cell population of claim 22 wherein at least about 50% of the cells express the surface antigen for integrin α

6.

25. The isolated stem cell population of claim 22 wherein the mammal is a mouse and at least about 80% of the cells express the surface antigen CD31 cell marker and at least about 70% of the cells express the surface antigen CD117.

26. The isolated stem cell population of claim 22 wherein the mammal is a mouse, at least about 50% to about 85% of the cells express the surface antigen CD31, and about 70% to about 75% of the cells express the surface antigen CD117.

27. The isolated stem cell population of claim 22 wherein the mammal is a human, the cells are CD 133 negative, at least about 50% of the cells express the surface antigen for integrin α

6, and at least about 50% of the cells express the surface antigen CD31.

28. The isolated stem cell population of claim 22 wherein the mammal is a human, the cells are CD133 positive, less than about 30% of the cells express the surface antigen for integrin α

6, and less than about 30% of the cells express the surface antigen CD31.

29. The isolated stem cell population of claim 22 wherein the cells are murine cells and the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD3, CD 11, CD45, Ly-6G, and TER-119.

30. The isolated stem cell population of claim 22 wherein the cells are human cells and the monocytes are labeled in step (c) with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11a, Mac-1, CD14, CD16, CD19, CD33, CD38, CD45RA, CD64, CD68, CD86, and CD235a.

35. A method of treating an ocular disease in a mammal comprising isolating from the bone marrow of the mammal a lineage negative hematopoietic stem cell population that includes endothelial progenitor cells by:

(a) extracting bone marrow from a mammal suffering from an ocular disease;

(b) separating a plurality of monocytes from the bone marrow;

(c) labeling the monocytes with biotin-conjugated lineage panel antibodies to one or more lineage surface antigens selected from the group consisting of CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;

(d) separating monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells;

and subsequently intravitreally injecting the isolated stem cells into an eye of the mammal in a number sufficient to ameliorate the effects of the disease.

36. The method of claim 35 wherein the number of stem cells is effective for repairing retinal damage of the mammal'"'"'s eye.

37. The method of claim 35 wherein the number of stem cells is effective for stabilizing retinal neovasculature of the mammal'"'"'s eye.

38. The method of claim 35 wherein the number of stem cells is effective for maturing retinal neovasculature of the mammal'"'"'s eye.

39. The method of claim 35 wherein the disease is a retinal degenerative disease.

40. The method of claim 35 wherein the disease is a retinal vascular degenerative disease.

41. The method of claim 35 wherein the disease is an ischemic retinopathy.

42. The method of claim 35 wherein the disease is a vascular hemorrhage.

43. The method of claim 35 wherein the disease is a vascular leakage.

44. The method of claim 35 wherein the disease is a choroidopathy.

45. The method of claim 35 wherein the disease is age related macular degeneration.

46. The method of claim 35 wherein the disease is diabetic retinopathy.

47. The method of claim 35 wherein the disease is presumed ocular histoplasmosis.

48. The method of claim 35 wherein the mammal is a neonatal mammal.

49. The method of claim 48 wherein the disease is retinopathy of prematurity.

50. The method of claim 35 wherein the disease is sickle cell anemia.

51. The method of claim 35 wherein the disease is retinitis pigmentosa.

56. The transfected stem cell population of claim 45 wherein the fragment of TrpRS is T2-TrpRS.

61. A transfected lineage negative hematopoietic stem cell population prepared by:

(a) extracting bone marrow from an adult mammal;

(b) separating a plurality of monocytes from the bone marrow;

(c) labeling the plurality of monocytes with biotin-conjugated lineage panel antibodies to CD2, CD3, CD4, CD11, CD11a, Mac-1, CD14, CD16, CD19, CD24, CD33, CD36, CD38, CD45, Ly-6G, TER-119, CD45RA, CD56, CD64, CD68, CD86, CD66b, HLA-DR, and CD235a;

(d) separating monocytes that are positive for said one or more lineage surface antigens from the plurality of monocytes and recovering a population of lineage negative hematopoietic stem cells containing endothelial progenitor cells; and

(e) transfecting the lineage negative hematopoietic stem cells recovered in step (d) with a polynucleotide that operably encodes a therapeutically useful peptide.

62. The transfected stem cell population of claim 61 wherein the therapeutically useful peptide is an anti-angiogenic peptide.

63. The transfected stem cell population of claim 61 wherein the therapeutically useful peptide is a neurotrophic agent.

65. A method of delivering transgenes to the retinal vasculature of a mammal comprising intravitreally injecting a transfected lineage negative hematopoietic stem cell population of claim 61 into the eye of the mammal.