Regulation of endogenous gene expression in cells using zinc finger proteins

• US 6,824,978 B1
• Filed: 11/03/2000
• Issued: 11/30/2004
• 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:
  US09/706,243
• Time in Patent Office: 1,488 Days
• Field of Search: 435/6, 435/69.1, 435/455, 435/468, 435/320.1, 530/350, 536/23.4, 536/23.1
• 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 gene in a cell, the method comprising the step of:

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

thereby inhibiting expression of the endogenous cellular gene.

2. The method of claim 1, wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with a 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 are covalently linked to form a fusion protein.

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

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

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

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

10. The method of claim 8, wherein the regulatory domain is selected from the group consisting of a transcriptional repressor, an endonuclease, a methyl transferase, and a histone deacetylase.

11. The method of claim 1, wherein expression of the endogenous cellular gene is inhibited by at least about 20%.

12. The method of claim 1, wherein the endogenous cellular gene is selected from the group consisting of VEGF, ER*, IGF-1, c-myc, c-myb, ICAM, and Her2/Neu.

13. The method of claim 12, wherein the endogenous cellular gene is VEGF.

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

15. The method of claim 1, wherein the method further comprises the step of first administering to the cell a delivery vehicle comprising the zinc finger protein, wherein the delivery vehicle comprises a liposome or a membrane translocation polypeptide.

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

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

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

19. The method of claim 18, wherein the zinc finger protein comprises a regulatory domain and is humanized.

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

21. The method of claim 20, wherein the cell is a mammalian cell.

22. The method of claim 21, wherein the cell is a human cell.

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

contacting a target site in the endogenous cellular gene with an engine fusion zinc finger protein comprising six fingers and a regulatory domain, wherein the Kd of the zinc finger protein is less than about 25 nM;

thereby inhibiting expression of the endogenous cellular gene.

24. A method of activating expression of an endogenous cellular gene, the method comprising the step of:

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

thereby activating expression of the endogenous cellular gene.

25. The method of claim 24, wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with a second zinc finger protein.

26. The method of claim 25, wherein the first and second target sites adjacent.

27. The method of claim 26, wherein the first and second zinc finger proteins are covalently linked to form a fusion protein.

28. The method of claim 25, wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.

29. The method of claim 28, wherein the first and the second zinc finger protein are fusion proteins, each comprising at least two regulatory domains.

30. The method of claim 28, wherein the regulatory domain is selected from the group consisting of a transcriptional activator and a histone acetyltransferase.

31. The method of claim 24, wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.

32. The method of claim 31, wherein the first zinc finger protein is a fusion protein comprising at least two regulatory domains.

33. The method of claim 31, wherein the regulatory domain is selected from the group consisting of a transcriptional activator and a histone acetyltransferase.

34. The method of claim 24, wherein the cell is selected from the group consisting of an animal cell, a plant cell, a bacterial cell, a protozoal cell, and a fungal cell.

35. The method of claim 34, wherein the cell is a mammalian cell.

36. The method of claim 35, wherein the cell is a human cell.

37. The method of claim 24, wherein expression of the endogenous cellular gene is activated to at least about 150%.

38. The method of claim 24, wherein the endogenous cellular gene is selected from the group consisting of FAD2-1, EPO, GM-CSF, GDNF, VEGF, and LDL-R.

39. The method of claim 38, wherein the endogenous cellular gene is VEGF.

40. The method of claim 24, wherein the activation of gene expression prevents repression of gene expression.

41. The method of claim 24, wherein the target site is upstream of a transcription initiation site of the endogenous cellular gene.

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

43. The method of claim 24, wherein the zinc finger protein comprises an SP-1 backbone.

44. The method of claim 43, wherein the zinc finger protein comprises a regulatory domain and is humanized.

45. A method of activating expression of an endogenous cellular gene, the method comprising the step of:

contacting a target site in the endogenous cellular gene with an engineered fusion zinc finger protein comprising six fingers and a regulatory domain, wherein the Kd of the zinc finger protein is less than about 25 nM;

thereby activating expression of the endogenous cellular gene.

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

contacting a first target site in the endogenous cellular gene with a first zinc finger protein, wherein said first zinc finger protein is engineered;

thereby modulating expression of the endogenous cellular gene.

47. The method of claim 46, wherein the step of contacting further comprises contacting a second target site in the endogenous cellular gene with a second zinc finger protein.

48. The method of claim 47, wherein the first and second target sites are adjacent.

49. The method of claim 48, wherein the first and second zinc finger proteins are covalently linked to form a fusion protein.

50. The method of claim 47, wherein the first and second zinc finger proteins are fusion proteins, each comprising a regulatory domain.

51. The method of claim 50, wherein the first and second zinc finger protein are fusion proteins, each consisting at least two regulatory domains.

52. The method of claim 50, 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.

53. The method of claim 46, wherein the first zinc finger protein is a fusion protein comprising a regulatory domain.

54. The method of claim 53, wherein the first zinc finger protein is a fusion in comprising at least two regulatory domains.

55. The method of claim 53, 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.

56. The method of claim 46, wherein the cell is selected from the group consisting of an animal cell, a plant cell, a bacterial cell, a protozoal cell, and a fungal cell.

57. The method of claim 56, wherein the cell is a mammalian cell.

58. The method of claim 57, wherein the cell is a human cell.

59. The method of claim 46, wherein the endogenous cellular gene is selected from the group consisting of VEGF, ER*, IGF-1, c-myc, c-myb, ICAM, Her2/Neu, FAD2-1, EPO, GM-CSF, GDNF, and LDL-R.

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

61. The method of claim 46, wherein the method further comprises the step of first administering to the cell a delivery vehicle comprising the zinc finger protein, wherein the delivery vehicle comprises a liposome or a membrane translocation polypeptide.

62. The method of claim 46, wherein the target site is upstream of transcription initiation site of the endogenous cellular gene.

63. The method of claim 46, wherein the target site is adjacent to a transcription initiation site of the endogenous cellular gene.

64. The method of claim 46, wherein the zinc finger protein comprises an SP-1 backbone.

65. The method of claim 64, wherein the zinc finger protein comprises a regulatory domain and is humanized.

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

contacting a target site in the endogenous cellular gene with an engineered fusion zinc finger protein comprising six fingers and a regulatory domain;

thereby modulating expression of the endogenous cellular gene.