Promoters exhibiting endothelial cell specificity and methods of using same for regulation of angiogenesis

• US 20060204478A1
• Filed: 05/23/2006
• Published: 09/14/2006
• Est. Priority Date: 11/17/2000
• Status: Active Grant

• Current Assignee: Vascular Biogenics Ltd.
• Original Assignee: Vascular Biogenics Ltd.
• Inventors: Harats, Dror, Greenberger, Shoshana
• Granted Patent: US 8,039,261 B2
• Time in Patent Office: Days
• US Class Current: 424/93.200
• CPC Class Codes: A01K 2267/0331 Animal model for proliferative diseases
• CPC Class Codes: A01K 2267/0393 Animal model comprising a reporter system for screening tests
• CPC Class Codes: A61K 31/198 Alpha-amino acids, e.g. alanine or edetic acid [EDTA] betaine A61K31/205; proline A61K31/401; tryptophan A61K31/405; histidine A61K31/4172; peptides not degraded to individual amino acids A61K38/00
• CPC Class Codes: A61K 31/573 substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
• CPC Class Codes: A61K 38/1858 Platelet-derived growth factor [PDGF]
• CPC Class Codes: A61K 38/1866 Vascular endothelial growth factor [VEGF]
• 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 48/0058 Nucleic acids adapted for tissue specific expression, e.g. having tissue specific promoters as part of a contruct
• 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 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• 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: A61P 9/00 Drugs for disorders of the cardiovascular system
• 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 15/85 for animal cells
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2710/10343 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2830/002 inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2840/203 : having an IRES

Abstract

Isolated polynucleotide sequences exhibiting endothelial cell specific promoter activity, novel cis regulatory elements and methods of use thereof enabling treatment of diseases characterized by aberrant neovascularization or cell growth are disclosed.

Claims

1. A method of regulating angiogenesis in a tissue, the method comprising expressing in the tissue a nucleic acid construct including:

(a) an endothelial cell specific promoter;

(b) at least one copy of a hypoxia response element set forth in SEQ ID NO;

5; and

(c) a nucleic acid sequence encoding an angiogenesis regulator, said nucleic acid sequence being under regulatory control of said promoter and said hypoxia response element.

2. The method of claim 1, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

3. The method of claim 2, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing expression of said angiogenesis regulator.

4. The method of claim 3, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

5. The method of claim 1, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said endothelial specific promoter in a synergic manner.

6. The method of claim 5, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

7. The method of claim 6, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

8. The method of claim 6, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

9. The method of claim 8, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

10. The method of claim 6, wherein said endothelin receptor antagonist is an endothelin receptor antagonist excluding Bosentan.

11. A method of regulating angiogenesis in a tissue, the method comprising expressing in the tissue a nucleic acid construct including:

a nucleic acid sequence encoding an angiogenesis regulator, said nucleic acid sequence being under regulatory control of a cis regulatory element including at least a portion of the sequence set forth in SEQ ID NO;

15 covalently linked to at least a portion of the sequence set forth in SEQ ID NO;

16, thereby regulating angiogenesis in the tissue.

12. The method of claim 11, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

13. The method of claim 12, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

14. The method of claim 13, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

15. The method of claim 11, wherein said nucleic acid sequence encodes a proangiogenic factor and wherein said regulating angiogenesis is upregulating angiogenesis.

16. The method of claim 11, wherein said nucleic acid sequence encodes an inhibitor of angiogenesis and wherein said regulating angiogenesis is downregulating angiogenesis.

17. The method of claim 11, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

18. The method of claim 17, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

19. The method of claim 18, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

20. The method of claim 18, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

21. The method of claim 20, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

22. The method of claim 18, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.

23. A method of downregulating angiogenesis in a tissue of a subject, the method comprising administering to the subject a nucleic acid construct designed and configured for generating cytotoxicity in a sub-population of angiogenic cells, said nucleic acid construct including:

(a) a first polynucleotide region encoding a chimeric polypeptide including a ligand binding domain fused to an effector domain of a cytotoxic molecule; and

(b) a second polynucleotide region encoding a cis regulatory element being for directing expression of said chimeric polypeptide in said sub-population of angiogenic cells;

wherein said ligand binding domain is selected such that it is capable of binding a ligand present in, or provided to, said sub-population of angiogenic cells, whereas binding of said ligand to said ligand binding domain activates said effector domain of said cytotoxic molecule, thereby down-regulating angiogenesis in the tissue.

24. The method of claim 23, further comprising administering to said subject at least one additional therapeutic modality, said additional therapeutic modality selected capable of further potentiating said cytotoxicity in a synergic manner.

25. The method of claim 24, wherein said at least one additional therapeutic modality is selected from the group comprising chemotherapy, radiotherapy, phototherapy and photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachiotherapy, proton beam therapy, immunotherapy, cellular therapy and photon beam radiosurgical therapy.

26. The method of claim 23, further comprising administering to said subject at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

27. The method of claim 26, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

28. The method of claim 27, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

29. The method of claim 27, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

30. The method of claim 29, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

31. The method of claim 27, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.

32. A method of down-regulating angiogenesis in a tissue of a subject, the method comprising:

(a) expressing in the tissue of the subject a nucleic acid construct designed and configured for generating cytotoxicity in a sub-population of angiogenic cells, said nucleic acid construct including;

(i) a first polynucleotide region encoding a chimeric polypeptide including a ligand binding domain fused to an effector domain of a cytotoxic molecule, wherein said effector domain is selected such that it is activated following binding of a ligand to said ligand binding domain; and

(ii) a second polynucleotide region encoding a cis acting regulatory element being for directing expression of said chimeric polypeptide in said sub-population of angiogenic cells; and

(b) administering to the subject said ligand, thereby down-regulating angiogenesis in the tissue.

33. The method of claim 32, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

34. The method of claim 33, further comprising administering to said subject at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

35. The method of claim 34, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

36. The method of claim 32, further comprising administering to said subject at least one modulator of angiogenesis selected capable of further potentiating activity of said cis acting regulatory element in a synergic manner.

37. The method of claim 36, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

38. The method of claim 37, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

39. The method of claim 37, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

40. The method of claim 39, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

41. The method of claim 37, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.

42. A method of treating a disease or condition associated with excessive neo-vascularization, the method comprising administering a therapeutically effective amount of a nucleic acid construct designed and configured for generating cytotoxicity in a sub-population of angiogenic cells, said nucleic acid construct including:

(i) a first polynucleotide region encoding a chimeric polypeptide including a ligand binding domain fused to an effector domain of a cytotoxic molecule; and

(ii) a second polynucleotide region encoding a cis acting regulatory element being for directing expression of said chimeric polypeptide in said sub-population of angiogenic cells;

wherein said ligand binding domain is selected such that it is capable of binding a ligand present in, or provided to, said sub-population of angiogenic cells, whereas binding of said ligand to said ligand binding domain activates said effector domain of said cytotoxic molecule, thereby down-regulating angiogenesis in the tissue and treating the disease or condition associated with excessive neo-vascularization.

43. The method of claim 42, wherein said cis acting regulatory element is an endothelial cell-specific or periendothelial cell-specific promoter selected from the group consisting of the PPE-1 promoter, the PPE-1-3x promoter, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willebrand promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.

44. The method of claim 42, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

45. The method of claim 44, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

46. The method of claim 45, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

47. The method of claim 42, further comprising administering to said tissue at least one angiogenic modulator selected capable of further potentiating activity of said cis acting regulatory element in a synergic manner.

48. The method of claim 47, wherein said angiogenic modulator is an endothelin receptor antagonist.

49. The method of claim 48, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

50. The method of claim 48, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

51. The method of claim 50, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

52. The method of claim 48, wherein said endothelin receptor antagonist is an endothelin receptor antagonist, excluding Bosentan.

53. A method of treating a tumor in a subject, the method comprising administering a therapeutically effective amount of a nucleic acid construct designed and configured for generating cytotoxicity in cells of the tumor, said nucleic acid construct including:

(i) a first polynucleotide region encoding a chimeric polypeptide including a ligand binding domain fused to an effector domain of a cytotoxic molecule; and

(ii) a second polynucleotide region encoding a cis regulatory element being for directing expression of said chimeric polypeptide in said cells of the tumor;

wherein said ligand binding domain is selected such that it is capable of binding a ligand present in, or provided to, said cells of the tumor, whereas binding of said ligand to said ligand binding domain activates said effector domain of said cytotoxic molecule to thereby direct cytotoxicity in said cells of the tumor.

54. The method of claim 53, wherein said cis regulatory element is an endothelial cell-specific or periendothelial cell-specific promoter selected from the group consisting of the PPE-1 promoter, the PPE-1-3x promoter, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willebrand promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.

55. The method of claim 53, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

56. The method of claim 53, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

57. The method of claim 56, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

58. The method of claim 54, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

59. The method of claim 58, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

60. The method of claim 59, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

61. The method of claim 59, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

62. The method of claim 61, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

63. The method of claim 59, wherein said endothelin receptor antagonist is an endothelin receptor antagonist, excluding Bosentan.

64. A method of treating a disease or condition associated with ischemia, the method comprising administering a therapeutically effective amount of a nucleic acid construct designed and configured for generating angiogenesis in a sub-population of angiogenic cells, said nucleic acid construct including:

(i) a first polynucleotide region encoding a proangiogenic factor; and

(ii) a second polynucleotide region encoding a cis regulatory element being for directing expression of said proangiogenic factor in said sub-population of angiogenic cells;

thereby up-regulating angiogenesis in the tissue and treating the disease or condition associated with ischemia.

65. The method of claim 64, wherein said cis regulatory element is an endothelial cell-specific or periendothelial cell-specific promoter selected from the group consisting of the PPE-1 promoter, the PPE-1-3x promoter, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willebrand promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.

66. The method of claim 64, wherein said disease or condition associated with ischemia is selected from the group consisting of wound healing, ischemic stroke, ischemic heart disease and gastrointestinal lesions.

67. The method of claim 64, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

68. The method of claim 67, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

69. The method of claim 68, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

70. The method of claim 65, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

71. The method of claim 70, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

72. The method of claim 71, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

73. The method of claim 71, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

74. The method of claim 73, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

75. The method of claim 71, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.

76. A method of down-regulating angiogenesis in a tissue of a subject, the method comprising:

(a) expressing in the tissue a nucleic acid construct designed and configured for cytotoxicity in angiogenic cells, said nucleic acid construct including;

(i) a first polynucleotide region encoding a suicide gene and

(ii) a second polynucleotide region encoding a cis acting regulatory element capable of directing expression of said suicide gene in said angiogenic cells; and

(b) administering to the subject a therapeutic amount of a prodrug sufficient to cause apoptosis of said tissue when said prodrug is converted to a toxic compound by said suicide gene

thereby down-regulating angiogenesis in the tissue.

77. The method of claim 76, wherein said cis regulatory element is an endothelial cell-specific or periendothelial cell-specific promoter selected from the group consisting of the PPE-1 promoter, the PPE-1-3x promoter, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willebrand promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.

78. The method of claim 76, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

79. The method of claim 78, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

80. The method of claim 79, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

81. The method of claim 76, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

82. The method of claim 81, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

83. The method of claim 82, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

84. The method of claim 82, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

85. The method of claim 84, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

86. The method of claim 82, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.

87. The method of claim 76, further comprising administering to said subject at least one additional therapeutic modality, said additional therapeutic modality selected capable of further potentiating cytotoxicity in a synergic manner.

88. The method of claim 87, wherein said at least one additional therapeutic modality is selected from the group comprising chemotherapy, radiotherapy, phototherapy and photodynamic therapy, surgery, nutritional therapy, ablative therapy, combined radiotherapy and chemotherapy, brachiotherapy, proton beam therapy, immunotherapy, cellular therapy and photon beam radiosurgical therapy.

89. A method of down-regulating angiogenesis in a tissue of a subject, the method comprising administering to the subject a nucleic acid construct designed and configured for generating cytotoxicity in angiogenic cells, said nucleic acid construct including:

(a) a first polynucleotide region encoding a suicide gene; and

(b) a second polynucleotide region encoding a cis regulatory element capable of directing expression of said suicide gene in said angiogenic cells;

wherein said suicide gene is selected capable of converting a prodrug to a toxic compound capable of causing cytotoxicity, thereby down-regulating angiogenesis in the tissue.

90. The method of claim 89, wherein said cis acting regulatory element is an endothelial cell-specific or periendothelial cell-specific promoter selected from the group consisting of the PPE-1 promoter, the PPE-1-3x promoter, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willebrand promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.

91. The method of claim 89, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

92. The method of claim 91, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

93. The method of claim 92, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

94. The method of claim 90, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

95. The method of claim 94, wherein modulator of angiogenesis is an endothelin receptor antagonist.

96. The method of claim 95, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

97. The method of claim 95, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

98. The method of claim 97, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

99. The method of claim 95, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.

100. A method of treating a disease or condition associated with excessive neo-vascularization, the method comprising administering a therapeutically effective amount of a nucleic acid construct designed and configured for cytotoxicity in angiogenic cells, said nucleic acid construct including:

(i) a first polynucleotide region encoding a suicide gene; and

(ii) a second polynucleotide region encoding a cis acting regulatory element capable of directing expression of said suicide gene in said angiogenic cells;

wherein said suicide gene is selected capable of converting a prodrug to a toxic compound capable of causing cytotoxicity, thereby down-regulating angiogenesis in the tissue and treating the disease or condition associated with excessive neo-vascularization.

101. The method of claim 100, wherein said cis regulatory element is an endothelial cell-specific or periendothelial cell-specific promoter selected from the group consisting of the PPE-1 promoter, the PPE-1-3x promoter, the TIE-1 promoter, the TIE-2 promoter, the Endoglin promoter, the von Willebrand promoter, the KDR/flk-1 promoter, The FLT-1 promoter, the Egr-1 promoter, the ICAM-1 promoter, the VCAM-1 promoter, the PECAM-1 promoter and the aortic carboxypeptidase-like protein (ACLP) promoter.

102. The method of claim 100, wherein said nucleic acid construct further comprises a conditionally replicating adenovirus.

103. The method of claim 102, further comprising administering to said tissue at least one compound selected capable of enhancing copy number of said adenovirus and/or enhancing transgene expression.

104. The method of claim 103, wherein said compound is a corticosteroid and/or N-acetyl cysteine.

105. The method of claim 101, further comprising administering to said tissue at least one modulator of angiogenesis selected capable of further potentiating activity of said cis regulatory element in a synergic manner.

106. The method of claim 105, wherein said modulator of angiogenesis is an endothelin receptor antagonist.

107. The method of claim 106, wherein said endothelin receptor antagonist is a dual A-form and B-form endothelin receptor antagonist.

108. The method of claim 106, wherein said endothelin receptor antagonist is a B-form specific endothelin receptor antagonist.

109. The method of claim 108, wherein said B-form specific endothelin receptor antagonist is selected from the group consisting of A192,621;

BQ788;

Res 701-1 and Ro 46-8443.

110. The method of claim 106, wherein said endothelin receptor antagonist is any endothelin receptor antagonist excluding Bosentan.