Regulation of endogenous gene expression in cells using zinc finger proteins

• US 6,979,539 B2
• Filed: 07/02/2001
• Issued: 12/27/2005
• Est. Priority Date: 01/12/1999
• Status: Expired due to Term

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, 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:
  US09/897,844
• Publication Number:
  US 20030087817A1
• Time in Patent Office: 1,639 Days
• Field of Search: 435/6, 435/69.1, 435/320.1, 536/23.1, 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 expression of an endogenous cellular pine in a cell, the method comprising the step of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first engineered zinc finger protein, wherein

(i) said polynucleotide sequence is operably linked to a promoter,

(ii) the nucleic acid molecule expresses the zinc finger protein is less cell;

(iii) the zinc finger protein contacts a first target site in the endogenous cellular gene; and

(iv) the K_d of the zinc finger protein is less than about 25 nM;

thereby inhibiting expression of the endogenous cellular gene.

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

8. The method of claim 7, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

9. 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.

10. The method of claim 9, wherein the cell is a mammalian cell.

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

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

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

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

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

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

16. The method of claim 1, wherein the step of administering the nucleic acid molecule to the cell comprises administering acid molecule in a lipidnucleic acid complex or as nucleic acid.

17. 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.

18. The method of claim 1, wherein the expression vector is a viral expression vector.

19. The method of claim 18, wherein the expression vector is a retroviral expression vector, an aderoviral expression vector, or an AAV expression vector.

20. The method of claim 18 wherein the promoter to which the zinc finger-encoding nucleic acid is operably linked is an inductable promoter.

21. The method of claim 18, wherein the promoter to which the zinc finger-encoding nucleic acid is operably linked is a weak promoter.

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

10⁶ copies of the zinc finger protein.

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

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

25. 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.

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

27. The method of claim 26, wherein the zinc finger protein comprises a regulatory domain and is humanized.

2. The method of claim 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 stop of contacting father comprises contacting a second target site in endogenous cellular gene with the second zinc finger protein.

3. The method claim 2, wherein the flat wad second target sites are adjacent.

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

5. The method of claim 2, wherein the first and second zinc finger proteins are fusion proteins, each computing a regularly domain.

6. The method of claim 5, wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

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

29. A method of inhibiting expression of an endogenous cellular pine in a cell, the method comprising the step of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first engineered zinc finger protein, wherein

(i) said polynucleotide sequence is operably linked to a promoter;

(ii) the nucleic acid molecule expresses the zinc finger protein in the cell;

(iii) the fusion zinc finger protein comprises six fingers and a regulatory domain;

(iv) the fusion zinc finger protein contacts a target site in the endogenous cellular gene and;

(v) the K_d of the zinc finger protein is less than about 25 nM;

thereby inhibiting expression of the endogenous cellular gene.

30. A method of activating expression of an endogenous cellular gene in a cell the method comprising the step of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first engineered zinc finger protein, wherein

(i) said polynucleotide sequence is operably linked to a promoter,

(ii) the nucleic add molecule expresses a the zinc finger protein in the cell;

(iii) the zinc finger protein contacts a first target site in the endogenous cellular gene; and

(iv) the K_d of the zinc finger protein is less than about 25 nm;

thereby activating expression of the endogenous cellular gene.

31. The method of claim 30, wherein the step of administering father comprises administering a second zinc finger protein-encoding nucleic add 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 ix the endogenous cellular gene with the second zinc finger protein.

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, forming a fusion protein.

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

35. The method of claim 34, wherein the first and the second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

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

37. The method of claim 36, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

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

39. The method of claim 38, wherein the cell is a mammalian cell.

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

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

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

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

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

45. 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 as marked nucleic acid.

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

47. The method of claim 46, wherein the expression vector is a viral expression vector.

48. The method of claim 47, wherein the expression vector is a retroviral expression vector, an adenoviral expression vector, or an AAV expression vector.

49. The method of claim 47, wherein the promoter to which the zinc finger protein encoding by a nucleic acid operably linked to an inducible promoter.

50. The method of claim 47, wherein the promoter to which the zinc finger protein encoding by a nucleic acid operably linked is a weak promoter.

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

10⁶ copies of the zinc finger protein.

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

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

54. The method of claim 31, 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.

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

56. The method of claim 55, wherein the zinc finger protein comprises a regulatory domain and is humanized.

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

58. A method of activating expression of an endogenous cellular gene in a cell the method comprising the step of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes an engineered fusion zinc finger protein, wherein

(i) said polynucleotide sequence is operably linked to a promoter;

(ii) the nucleic acid molecule expresses the fusion zinc linger protein in the cell;

(iii) the fusion zinc finger protein comprise six fingers and a regulatory domain;

(iv) the fusion zinc finger protein contacts a target site in the endogenous cellular gene and;

(v) the K_d of the fusion zinc finger protein is less than about 25 nm;

thereby activating expression of the endogenous cellular gene.

59. A method of modulating expression of an endogenous cellular gene in a cell, the method comprising the step of:

administering to the cell a nucleic acid molecule comprising a polynucleotide sequence which encodes a first engineered zinc finger protein, wherein

(i) said polynucleotide sequence is operably linked to a promoter,

(ii) the nucleic acid molecule expresses the zinc finger protein in the cell, and

(iii) the zinc finger protein contacts a first target site in the endogenous cellular gene,

thereby inhibiting expression of the endogenous cellular gene.

60. The method of claim 59, 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.

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 forming a fusion.

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

64. The method of claim 63, wherein the first and second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

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

66. The method of claim 65, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

67. The method of claim 59, 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.

68. The method of claim 67, wherein the cell is a mammalian cell.

69. The method of claim 59, wherein the cell is a human cell.

70. 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, Her2/Neu, FAD2-1, EPO, GM-CSF, GDNF, and LDL-R.

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

72. 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.

73. 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.

74. The method of claim 73, wherein the expression vector is a viral expression vector.

75. The method of claim 74, wherein the expression vector is a retroviral expression vector, an adenoviral expression vector or an AAV expression vector.

76. The method of claim 74, wherein the promoter to which the zinc finger encoding nucleic acid is operably linked is an inducible promoter.

77. The method of claim 74 wherein the promoter to which the zinc finger encoding nucleic acid is operably linked is a weak promoter.

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

10⁶ copies of the zinc finger protein.

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

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

81. The method of claim 59, 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.

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

83. The method of claim 82, wherein the zinc finger protein comprises a regulatory domain and is humanized.

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

85. A method of modulating expression of an endogenous cellular gene in a cell, the method comprising the step of:

administering to the cell a nucleic acid molecule comprising a polynucleic sequence which encodes an engineered fusion zinc finger protein, wherein

(i) said polynucleotide sequence is operably linked to a promoter,

(ii) the nucleic acid molecule, expresses the fusion zinc finger protein in the cell,

(iii) the fusion zinc finger protein comprises six fingers and a regulatory domain, and

(iv) the fusion zinc finger protein contacts a target site in the endogenous cellular gene;

thereby modulating expression of the endogenous cellular gene.