Anti-angiogenic methods and compositions

• US 20060210539A1
• Filed: 02/28/2006
• Published: 09/21/2006
• Est. Priority Date: 02/28/2005
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Zhang, H. Steve, Gregory, Philip D.
• Granted Patent: US 7,358,085 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 14/4705 stimulating, promoting or activating activity
• CPC Class Codes: C07K 14/811 Serine protease (E.C. 3.4.21) inhibitors
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 2799/022 where the vector is derived from an adenovirus
• CPC Class Codes: C12N 2799/025 where the vector is derived from a parvovirus
• CPC Class Codes: C12N 2799/028 where the vector is derived from a herpesvirus

Abstract

Disclosed herein are methods and compositions for treatment of conditions requiring inhibition of angiogenesis. Such conditions include those characterized by neovascularization, such as retinopathies, macular degeneration and various malignancies.

Claims

1. An engineered zinc finger protein that binds to and regulates expression of the gene encoding pigment epithelium-derived factor (PEDF) wherein the protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

2. An engineered zinc finger protein according to claim 1, further comprising a functional domain.

3. An engineered zinc finger protein according to claim 2, wherein the functional domain is an activation domain.

4. An engineered zinc finger protein according to claim 3, wherein the activation domain is selected from the group consisting of the VP16 activation domain, the VP64 activation domain and the p65 activation domain.

5. An engineered zinc finger protein according to claim 4, comprising two p65 activation domains.

6. An engineered zinc finger protein according to claim 2, wherein the functional domain is a repression domain.

7. A cell comprising an engineered zinc finger protein according to claim 1.

8. A polynucleotide encoding an engineered zinc finger protein according to claim 1.

9. A cell comprising a polynucleotide according to claim 8.

10. A method for modulating angiogenesis in an organism by regulating expression of the endogenous PEDF gene.

11. The method of claim 10, wherein expression of the endogenous PEDF gene is regulated by an engineered zinc finger protein.

12. The method of claim 11 wherein the engineered zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

13. The method of claim 10, wherein expression of the PEDF gene is activated.

14. The method of claim 12, wherein the engineered zinc finger protein further comprises an activation domain selected from the group consisting of the VP 16 activation domain, the VP64 activation domain and the p65 activation domain.

15. The method of claim 14, wherein the zinc finger protein comprises two p65 activation domains.

16. The method of claim 10, wherein expression of the PEDF gene is repressed.

17. The method of claim 10, wherein the organism is a mammal.

18. The method of claim 17, wherein the mammal is a human.

19. A method for the treatment of ocular neovascularization in an organism, wherein the method comprises activating expression of an endogenous PEDF gene in one or more cells of the organism.

20. The method of claim 19, wherein expression of the endogenous PEDF gene is activated by binding of a first engineered zinc finger protein to a target site in the endogenous PEDF gene.

21. The method of claim 20, wherein the first engineered zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

22. The method of claim 21, wherein the first engineered zinc finger protein further comprises an activation domain selected from the group consisting of the VP16 activation domain, the VP64 activation domain and the p65 activation domain.

23. The method of claim 22, wherein the first engineered zinc finger protein comprises two p65 activation domains.

24. The method of claim 19, wherein the method further comprises inhibiting the expression of an endogenous gene encoding an angiogenic factor in one or more cells of the organism.

25. The method of claim 24, wherein the angiogenic factor is a vascular endothelial growth factor (VEGF).

26. The method of claim 25, wherein the VEGF is vascular endothelial growth factor A (VEGF-A).

27. The method of claim 26, wherein expression of the endogenous VEGF-A gene is inhibited by binding of a second engineered zinc finger protein to a target site in the endogenous VEGF-A gene.

28. The method of claim 27, wherein the second engineered zinc finger protein comprises three zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

29. The method of claim 28, wherein the second zinc finger protein further comprises a repression domain.

30. The method of claim 29,wherein the repression domain is selected from the group consisting of the v-erbA repression domain and the KOX repression domain.

31. The method of claim 19, wherein the ocular neovascularization occurs in a disease selected from the group consisting of age-related macular degeneration (AMD, diabetic retinopathy (DR) and retinopathy of prematurity.

32. A polynucleotide encoding two engineered zinc finger proteins, wherein the first zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

33. The polynucleotide of claim 32, further comprising an internal ribosome entry site (IRES) disposed between the sequences encoding the first and second zinc finger proteins.

34. The polynucleotide of claim 32, further comprising a sequence encoding a 2A peptide disposed between the sequences encoding the first and second zinc finger proteins.

35. A method for the treatment of ocular neovascularization in an organism, wherein the method comprises introducing the polynucleotide of claim 32 into one or more cells of the organism.

36. The method of claim 35, in which the polynucleotide is introduced into one or more retinal epithelial cells.

37. The method of claim 35, in which the polynucleotide is encapsidated in a viral vector selected from the group consisting of adeno-associated virus (AAV), Adenovirus and lentivirus.

38. The method of claim 37, in which the viral vector is an adeno-associated virus (AAV).

39. The method of claim 38, in which the viral vector is AAV Type 2 or AAV Type 4.

40. The method of claim 35, wherein the organism is a mammal.

41. The method of claim 40, wherein the mammal is a human.

42. A method for the treatment of a malignancy in an organism, wherein the method comprises activating expression of the endogenous PEDF gene in one or more cells of the organism.

43. The method of claim 42, wherein expression of the endogenous PEDF gene is activated by binding of a first engineered zinc finger protein to a target site in the endogenous PEDF gene.

44. The method of claim 43 wherein the first engineered zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

45. The method of claim 44 wherein the first engineered zinc finger protein further comprises an activation domain selected from the group consisting of the VP16 activation domain, the VP64 activation domain and the p65 activation domain.

46. The method of claim 45 wherein the first engineered zinc finger protein comprises two p65 activation domains.

47. The method of claim 42, wherein the method further comprises activating the expression of an endogenous gene encoding a cytokine in one or more cells of the organism.

48. The method of claim 47, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF).

49. The method of claim 48, wherein expression of the endogenous GM-CSF gene is activated by binding of a second engineered zinc finger protein to a target site in the endogenous GM-CSF gene.

50. The method of claim 49, wherein the second zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

51. The method of claim 50, wherein the second zinc finger protein further comprises an activation domain.

52. The method of claim 51, wherein the activation domain is selected from the group consisting of the p65 activation domain, the VP64 activation domain and the VP 16 activation domain.

53. The method of claim 42, wherein the malignancy occurs in a disease selected from the group consisting of head and neck cancer, glioblastoma, prostate cancer and pancreatic cancer.

54. A method for the treatment of a malignancy in an organism, wherein the method comprises activating expression of an endogenous gene encoding a cytokine in one or more cells of the organism.

55. The method of claim 54, wherein the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF).

56. The method of claim 55, wherein expression of the endogenous GM-CSF gene is activated by binding of an engineered zinc finger protein to a target site in the endogenous GM-CSF gene.

57. The method of claim 56, wherein the engineered zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

58. The method of claim 57, wherein the engineered zinc finger protein further comprises an activation domain.

59. The method of claim 58, wherein the activation domain is selected from the group consisting of the p65 activation domain, the VP64 activation domain and the VP 16 activation domain.

60. The method of claim 54, wherein the malignancy occurs in a disease selected from the group consisting of head and neck cancer, glioblastoma, prostate cancer and pancreatic cancer.

61. The method of claim 56, wherein a polynucleotide encoding the engineered zinc finger protein is introduced into one or more cells of the organism.

62. The method of claim 61, in which the polynucleotide is encapsidated in a viral delivery vehicle selected from the group consisting of adeno-associated virus (AAV), Adenovirus and lentivirus.

63. The method of claim 62, in which the viral delivery vehicle is an adenovirus.

64. The method of claim 63, in which the viral delivery vehicle is Adenovirus Type 5 or Adenovirus Type 35 or a hybrid Adenovirus Type 5/35.

65. The method of claim 63, in which the delivery vehicle is a virus that selectively replicates in tumor cells.

66. The method of claim 54, wherein the organism is a mammal.

67. The method of claim 66, wherein the mammal is a human.

68. A polynucleotide encoding two engineered zinc finger proteins, wherein the first zinc finger protein comprises six zinc fingers and the amino acid sequence of the recognition region of each of the zinc fingers is as follows:

69. The polynucleotide of claim 68, further comprising an internal ribosome entry site (IRES) disposed between the sequences encoding the first and second zinc finger proteins.

70. The polynucleotide of claim 68, further comprising a sequence encoding a 2A peptide disposed between the sequences encoding the first and second zinc finger proteins.

71. The polynucleotide of claim 68, wherein sequences encoding the first zinc finger protein are operably linked to a tumor-specific promoter.

72. The polynucleotide of claim 68, wherein sequences encoding the second zinc finger protein are operably linked to a tumor-specific promoter.

73. The polynucleotide of claim 68, wherein sequences encoding the first and second zinc finger proteins are operably linked to a tumor-specific promoter.

74. The polynucleotide of any of claims 71, 72 or 73 wherein the tumor specific promoter is selected from the group consisting of the E2F promoter, the survivin promoter, the human telomerase reverse transcriptase (hTERT) promoter, the COX-2 promoter, the EGD-2 promoter and the ELF-1 promoter.

75. The polynucleotide of claim 68, wherein sequences encoding the first zinc finger protein are operably linked to a hypoxia-specific promoter.

76. The polynucleotide of claim 68, wherein sequences encoding the second zinc finger protein are operably linked to a hypoxia-specific promoter.

77. The polynucleotide of claim 68, wherein sequences encoding the first zinc finger protein are operably linked to a tissue-specific promoter.

78. The polynucleotide of claim 68, wherein sequences encoding the second zinc finger protein are operably linked to a tissue-specific promoter.

79. A method for the treatment of a malignancy in an organism, wherein the method comprises introducing the polynucleotide of claim 68 into one or more cells of the organism.

80. A method for the treatment of a malignancy in an organism, wherein the method comprises introducing the polynucleotide of claim 74 into one or more cells of the organism.

81. The method of claim 79, in which the polynucleotide is introduced into a tumor.

82. The method of claim 79, in which the polynucleotide is introduced into an endothelial or mesenchymal stem cell and the stem cell is subsequently introduced into the organism.

83. The method of claim 79, in which the polynucleotide is encapsidated in a viral delivery vehicle selected from the group consisting of adeno-associated virus (AAV), Adenovirus and Herpes simplex virus.

84. The method of claim 83, in which the viral delivery vehicle is an Adenovirus.

85. The method of claim 84, wherein the adenovirus replicates preferentially in tumor cells.

86. The method of claim 84, in which the adenovirus vector is a non-replicating adenovirus vector.

87. The method of claim 86, in which the viral vector is Adenovirus Type 5.

88. The method of claim 79, wherein the organism is a human.

89. The method of claim 79, wherein the malignancy occurs in a disease selected from the group consisting of head and neck cancer, glioblastoma, prostate cancer and pancreatic cancer.