Regulation of endogenous gene expression in cells using zinc finger proteins

• US 6,534,261 B1
• Filed: 01/12/1999
• Issued: 03/18/2003
• Est. Priority Date: 01/12/1999
• Status: Expired due to Term

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Spratt, Sharon Kaye, Jarvis, Eric Edward, Cox, George Norbert III, Case, Casey Christopher, Eisenberg, Stephen P.
• Primary Examiner(s): Brusca, John S.
• Application Number:
  US09/229,037
• Time in Patent Office: 1,526 Days
• Field of Search: 514/44, 514/725, 435/29, 435/6, 530/387.1, 536/23.1, 536/23.5, 536/24.1
• US Class Current: 435/6.12
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 67/0275 Genetically modified vertebrates, e.g. transgenic
• 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 48/005 characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 17/06 Antipsoriatics
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 21/04 for myasthenia gravis
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/18 for HIV
• CPC Class Codes: A61P 33/02 Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 : Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• 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: 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: C07K 14/47 : from mammals
• CPC Class Codes: C07K 14/52 : Cytokines; Lymphokines; Interferons
• CPC Class Codes: C07K 2319/00 : Fusion polypeptide
• 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 15/63 : Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 15/67 : General methods for enhancing the expression
• CPC Class Codes: C12N 15/8216 : Methods for controlling, regulating or enhancing expression of transgenes in plant cells
• CPC Class Codes: C12N 15/85 : for animal cells
• CPC Class Codes: C12N 2830/002 : inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
• CPC Class Codes: C12N 2830/005 : repressible enhancer/promoter combination, e.g. KRAB
• CPC Class Codes: C12N 2830/008 : cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/85 : mammalian
• CPC Class Codes: C12Q 1/66 : involving luciferase
• CPC Class Codes: C12Q 1/6897 : involving reporter genes operably linked to promoters
• CPC Class Codes: G01N 33/5091 : for testing the pathological state of an organism

Abstract

The present invention provides methods for modulating expression of endogenous cellular genes using recombinant zinc finger proteins.

Claims

1. A method of inhibiting transcription of an endogenous cellular gene in an isolated cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first zinc finger protein, wherein said first zinc finger protein is engineered, wherein said polynucleotide sequence is operably linked to a promoter, and wherein the nucleic acid molecule expresses the first zinc finger protein in the cell; and

contacting a first target site in the endogenous cellular gene with the first zinc finger protein, wherein the K_d of the zinc finger protein is less than about 25 nM;

thereby inhibiting transcription of the endogenous cellular gene by at least about 20%.

2. The method of claim 1, wherein the step of administering further comprises administering a second zinc-finger protein-encoding nucleic acid operably linked to a promoter that expresses a second zinc finger protein in the cell, and wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with the second zinc finger protein.

3. The method of claim 2, wherein the first and second target sites are adjacent.

4. The method of claim 3, wherein the first and second zinc finger proteins are covalently linked, forming a fusion protein.

5. The method of claim 1, wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.

6. The method of claim 5, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

7. The method of claim 2, wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.

8. The method of claim 7, wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

10. The method of claim 1, wherein the cell is selected from the group consisting of animal cell, a plant cell, a bacterial cell, a protozoal cell, or a fungal cell.

11. The method of claim 10, wherein the cell is a mammalian cell.

12. The method of claim 11, wherein the cell is a human cell.

13. The method of claim 1, wherein expression of the endogenous cellular gene is inhibited by at least about 75%-100%.

14. The method of claim 1, wherein the endogenous cellular gene is a selected from the group consisting of VEGF, ERα

, IGF-I, c-myc, c-myb, ICAM, and Her2/Neu.

15. The method of claim 1, wherein the endogenous cellular gene is VEGF.

16. The method of claim 1, wherein the inhibition of gene expression prevents gene activation.

17. The method of claim 5 or 7, wherein the regulatory domain is selected from the group consisting of a transcriptional repressor, an endonuclease, a methyl transferase, and a histone deacetylase.

18. The method of claim 1, wherein the step of administering the nucleic acid molecule to the cell comprises administering the nucleic acid molecule in a lipid:

nucleic acid complex or as naked nucleic acid.

19. The method of claim 1, wherein the nucleic acid molecule is an expression vector comprising a zinc finger protein encoding-nucleic acid operably linked to a promoter.

20. The method of claim 19, wherein the expression vector is a viral expression vector.

21. The method of claim 20, wherein the expression vector is a retroviral expression vector, an adenoviral expression vector, or an AAV expression vector.

22. The method of claim 1, wherein the promoter to which the zinc finger encoding nucleic acid is operably linked is an inducible promoter.

23. The method of claim 1, wherein the promoter to which the zinc finger encoding nucleic acid is operably linked is a weak promoter.

24. The method of claim 1, wherein the cell comprises less than about 1.5×

10⁶ copies of the zinc finger protein.

25. The method of claim 1, wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.

26. The method of claim 1, wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.

27. The method of claim 1, wherein the target site is adjacent to an RNA polymerase pause site wherein the RNA polymerase pause site is downstream of a transcription initiation site of the endogenous cellular gene.

28. The method of claim 1, wherein the zinc finger protein comprises an SP-1 backbone.

29. The method of claim 28, wherein the zinc finger protein comprises a regulatory domain and is humanized.

9. A method of inhibiting transcription of an endogenous cellular gene in an isolated cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a fusion zinc finger protein encoding nucleic acid operably linked to a promoter, wherein the nucleic acid molecule expresses a fusion zinc finger protein in the cell, and wherein the fusion zinc finger protein comprises six fingers and a regulatory domain; and

contacting a target site in the endogenous cellular gene with the fusion zinc finger protein, wherein the K_d of the fusion zinc finger protein is less than about 25 nM;

thereby inhibiting transcription of the endogenous cellular gene by at least about 20%.

30. A method of activating transcription of an endogenous cellular gene in an isolated cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first zinc finger protein, wherein said first zinc finger protein is engineered, wherein said polynucleotide sequence is operably linked to a promoter, and wherein the nucleic acid molecule expresses the zinc finger protein in the cell; and

contacting a first target site in the endogenous cellular gene with the first zinc finger protein, wherein the K_d of the zinc finger protein is less than about 25 nM;

thereby activating transcription of the endogenous cellular gene to at least about 150%.

31. The method of claim 30, wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with a second zinc finger protein, wherein the second zinc finger protein is encoded by a second zinc finger protein-encoding nucleic acid operably linked to a promoter.

32. The method of claim 31, wherein the first and second target sites are adjacent.

33. The method of claim 32, wherein the first and second zinc finger proteins are covalently linked.

34. The method of claim 30, wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.

35. The method of claim 34, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

36. The method of claim 31, wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.

37. The method of claim 36, wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

39. The method of claim 30, wherein the cell is selected from the group consisting of animal cell, a plant cell, a bacterial cell, a protozoal cell, or a fungal cell.

40. The method of claim 39, wherein the cell is a mammalian cell.

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

42. The method of claim 30, wherein expression of the endogenous cellular gene is activated to at least about 200%-500%.

43. The method of claim 30, wherein the endogenous cellular gene is a selected from the group consisting of FAD2-1, EPO, GM-CSF, GDNF, VEGF, and LDL-R, and Her2/Neu.

44. The method of claim 30, wherein the endogenous cellular gene is VEGF.

45. The method of claim 30, wherein the activation of gene expression prevents repression of gene expression.

46. The method of claim 34 or 36, wherein the regulatory domain is selected from the group consisting of a transcriptional activator, or a histone acetyltransferase.

47. The method of claim 30, wherein the step of administering the nucleic acid molecule to the cell comprises administering the nucleic acid molecule in a lipid:

nucleic acid complex or as naked nucleic acid.

48. The method of claim 30, wherein the nucleic acid molecule is an expression vector comprising a zinc finger protein-encoding nucleic acid operably linked to a promoter.

49. The method of claim 48, wherein the expression vector is a viral expression vector.

50. The method of claim 49, wherein the expression vector is a retroviral expression vector, an adenoviral expression vector, or an AAV expression vector.

51. The method of claim 30, wherein the promoter is an inducible promoter.

52. The method of claim 30, wherein the promoter is a weak promoter.

53. The method of claim 30, wherein the cell comprises less than about 1.5×

10⁶ copies of the zinc finger protein.

54. The method of claim 30, wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.

55. The method of claim 30, wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.

56. The method of claim 30, wherein the target site is adjacent to an RNA polymerase pause site downstream of a transcription initiation site of the endogenous cellular gene.

57. The method of claim 30, wherein the zinc finger protein comprises an SP-1 backbone.

58. The method of claim 57, wherein the zinc finger protein comprises a regulatory domain and is humanized.

38. A method of activating transcription of an endogenous cellular gene in an isolated cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a fusion zinc finger protein-encoding nucleic acid operably linked to a promoter, wherein the nucleic acid molecule expresses a fusion zinc finger protein in the cell, and wherein the fusion zinc finger protein comprises six fingers and a regulatory domain; and

contacting a target site in the endogenous cellular gene with the fusion zinc finger protein, wherein the K_d of the fusion zinc finger protein is less than about 25 nM;

thereby activating transcription of the endogenous cellular gene to at least about 150%.

59. A method of modulating transcription of an endogenous cellular gene in an isolated cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first zinc finger protein, wherein said first zinc finger protein is engineered, wherein said polynucleotide sequence is operably linked to a promoter, and wherein the nucleic acid molecule expresses the zinc finger protein in the cell; and

contacting a first target site in the endogenous cellular gene with the zinc finger protein, thereby modulating transcription of the endogenous cellular gene.

60. The method of claim 59, wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with a second zinc finger protein, wherein the second zinc finger protein is encoded by a second zinc finger protein-encoding nucleic acid operably linked to a promoter.

61. The method of claim 60, wherein the first and second target sites are adjacent.

62. The method of claim 61, wherein the first and second zinc finger proteins are covalently linked.

63. The method of claim 59, wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.

64. The method of claim 63, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

65. The method of claim 60, wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.

66. The method of claim 65, wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

68. The method of claim 59, wherein the cell is selected from the group consisting of animal cell, a plant cell, a bacteria cell, a protozoal cell, or a fungal cell.

69. The method of claim 68, wherein the cell is a mammalian cell.

70. The method of claim 69, wherein the cell is a human cell.

71. The method of claim 59, wherein the endogenous cellular gene is a selected from the group consisting of VEGF, ERα

, IGF-I, c-myc, c-myb, ICAM, Her2Neu, FAD2-1, EPO, GM-CSF, GDNF, and LDL-R.

72. The method of claim 59, wherein the endogenous cellular gene is VEGF.

73. The method of claim 63 or 65, wherein the regulatory domain is selected from the group consisting of a transcriptional repressor, a transcriptional activator, an endonuclease, a methyl transferase, a histone acetyltransferase, and a histone deacetylase.

74. The method of claim 59, wherein the step of administering the nucleic acid molecule to the cell comprises administering the nucleic acid molecule in a lipid:

nucleic acid complex or as naked nucleic acid.

75. The method of claim 59, wherein the nucleic acid molecule is an expression vector comprising a zinc finger protein encoding-nucleic acid operably linked to a promoter.

76. The method of claim 59, wherein the expression vector is a viral expression vector.

77. The method of claim 76, where in the expression vector is a retroviral expression vector, an adenoviral expression vector, or an AAV expression vector.

78. The method of claim 59, wherein the promoter is an inducible promoter.

79. The method of claim 59, wherein the promoter is a weak promoter.

80. The method of claim 59, wherein the cell comprises less than about 1.5×

10⁶ copies of the zinc finger protein.

81. The method of claim 59, wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.

82. The method of claim 59, wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.

83. The method of claim 59, wherein the target site is adjacent to an RNA polymerase pause site downstream of a transcription initiation site of the endogenous cellular gene.

84. The method of claim 59, wherein the zinc finger protein comprises an SP-1 backbone.

85. The method of claim 84, wherein the zinc finger protein comprises a regulatory domain and is humanized.

67. A method of modulating transcription of an endogenous cellular gene in an isolated cell, the method comprising the steps of:

administering to the cell a nucleic acid molecule comprising a fusion zinc finger protein-encoding nucleic acid operably linked to a promoter, wherein the nucleic acid molecule expresses a fusion zinc finger protein in the cell, and wherein the fusion zinc finger protein comprises six fingers and a regulatory domain; and

contacting a target site in the endogenous cellular gene with the fusion zinc finger protein;

thereby modulating transcription of the endogenous cellular gene.