Zinc finger protein derivatives and methods therefor

• US 6,242,568 B1
• Filed: 12/30/1996
• Issued: 06/05/2001
• Est. Priority Date: 01/18/1994
• Status: Expired due to Term

• Current Assignee: The Scripps Research Institute
• Original Assignee: The Scripps Research Institute
• Inventors: Gottesfeld, Joel M., Barbas, Carlos F. III, Wright, Peter E.
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Moore, William W.
• Application Number:
  US08/676,318
• Time in Patent Office: 1,618 Days
• Field of Search: 530/350, 530/400, 514/6, 424/450, 435/69.1, 435/252.3, 435/320.1, 435/471, 536/23.5, 536/23.6, 536/23.72
• US Class Current: 530/350
• 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: 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: A61P 1/00 Drugs for disorders of the alimentary tract or the digestive system
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 15/00 Drugs for genital or sexual disorders for disorders of sex hormones A61P5/24; Contraceptives
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/46 from vertebrates
• CPC Class Codes: C07K 14/4702 Regulators; Modulating activity
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/02 containing a signal sequence
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C07K 2319/24 containing a MBP (maltose binding protein)-tag
• CPC Class Codes: C07K 2319/42 containing a HA(hemagglutinin)-tag
• CPC Class Codes: C07K 2319/71 containing domain for transcriptional activaation, e.g. VP16
• CPC Class Codes: C07K 2319/73 containing coiled-coiled motif (leucine zippers)
• CPC Class Codes: C07K 2319/735 containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
• CPC Class Codes: C07K 2319/81 : containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/62 : DNA sequences coding for fusion proteins
• CPC Class Codes: C12Q 1/68 : involving nucleic acids
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/5011 : for testing antineoplastic activity
• CPC Class Codes: G01N 33/502 : for testing non-proliferative effects
• CPC Class Codes: G01N 33/56988 : HIV or HTLV
• CPC Class Codes: G01N 33/68 : involving proteins, peptides or amino acids involving lipoproteins G01N33/92

Abstract

The present invention provides zinc finger nucleotide binding polypeptide variants that have at least two zinc finger modules that bind to a target cellular nucleotide sequence and modulate the transcriptional function of the cellular nucleotide sequence. Also provided are methods of use of such zinc finger nucleotide binding polypeptide variants and methods for isolating the same using expression libraries encoding the polypeptide variants containing randomized substitutions of amino acids. Exemplary zinc finger nucleotide binding polypeptide variants of the invention include two cysteines and two histidines whereby both cysteines are amino proximal to both histidines.

Claims

1. A method for isolating a zinc finger-nucleotide binding polypeptide variant which binds to a cellular nucleotide sequence comprising:

a) identifying the amino acids in a zinc finger-nucleotide binding polypeptide that bind to a first cellular nucleotide sequence and modulate the function of a nucleotide sequence;

b) creating an expression library encoding the polypeptide variant containing randomized substitution of the amino acids identified in step a) above;

c) expressing the library in a suitable host cell; and

d) isolating a clone that produces a polypeptide variant that binds to a second cellular nucleotide sequence and modulates the function of the second nucleotide sequence;

wherein the variant is comprised of at least two zinc finger modules and wherein the amino acid sequence of each module that binds the second nucleotide sequence comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of two modules of said variant has at least one amino acid sequence modification.

2. The method of claim 1, wherein the library is expressed in a phage surface expression system.

3. The method of claim 1, wherein the phage expression system includes a reducing reagent which allows folding of expression products on the phage surface.

4. The method of claim 3, wherein the reducing reagent is dithiothreitol.

5. The method of claim 1, wherein the library is randomized by PCR using primers containing degenerate triplet codons at sequence locations corresponding to the determined amino acids.

6. The method of claim 1, wherein the modulation of function is enhancement of transcription of a gene operatively linked to the cellular nucleotide sequence.

7. The method of claim 1, wherein the modulation of function is suppression of transcription of a gene operatively linked to the cellular nucleotide sequence.

8. The method of claim 1, wherein the cellular nucleotide sequence is DNA.

9. The method of claim 1, wherein the cellular nucleotide sequence is RNA.

10. The method of claim 1, wherein the variant is derived from a zinc finger-nucleotide binding polypeptide selected from the group consisting of zif 268 and TFIIIA.

11. The method of claim 1, wherein the variant derived from the zinc finger-nucleotide binding polypeptide zif268 is modified at any of residues 1, 3, 4, 5, 6 or 7 as set forth in SEQ ID NO:

14.

12. The method of claim 1, wherein the variant derived from the zinc finger-nucleotide binding polypeptide zif268 is modified at any of residues 1, 2, 3, 4, 5 or 6 as set forth in SEQ ID NO:

15.

13. The method of claim 1, wherein the variant derived from the zinc finger-nucleotide binding polypeptide zif268 is modified at any of residues 1, 3, 4, 5, 6 or 7 as set forth in SEQ ID NO:

5.

27. The method of claims 1, 14, or 22 wherein the variant is derived from a zinc finger-nucleotide binding polypeptide selected from the group consisting of zif268 and TFIIIA.

34. The variant of claim 1, wherein the polypeptide contains a linker region between zinc fingers comprising the amino acid sequence TGEKP.

14. A method for identifying a zinc finger-nucleotide binding polypeptide variant comprised of at least two zinc finger modules, which modulates the transcriptional function of cellular nucleotide sequence and binds to a zinc finger-nucleotide binding motif, wherein the amino acid sequence of each module that binds to a zinc finger nucleotide binding motif comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of two modules of said variant has at least one amino acid sequence modification, said method comprising:

a) incubating the components comprising a nucleotide sequence encoding the putative modulating variant operably linked to a first inducible promoter, and a reporter gene operably linked to a second inducible promoter and a zinc finger-nucleotide binding motif, wherein the incubating is carried out under conditions sufficient to allow the components to interact; and

b) measuring the effect of the putative modulating variant on the expression of the reporter gene.

15. The method of claim 14, wherein the modulation is inhibition of gene expression.

16. The method of claim 14, wherein the modulation is enhancement of gene expression.

17. The method of claim 14, wherein the first inducible promoter is the arabinase promoter.

18. The method of claim 14, wherein the second inducible promoter is the lactose promoter.

19. The method of claim 14, wherein the incubating is performed in vitro.

20. The method of claim 14, wherein the incubating is performed in vivo.

21. The method of claim 14, wherein the reporter gene is β

-galactosidase.

22. A method of modulating transcription of a cellular nucleotide sequence associated with a zinc finger-nucleotide binding motif, comprising contacting the zinc finger-nucleotide in cells in vitro with an effective amount of a zinc finger-nucleotide binding polypeptide variant comprised of at least two zinc finger modules that binds to the zinc finger-nucleotide binding motif, wherein the amino acid sequence of each module that binds the motif comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of two modules of said variant has at least one amino acid sequence modification, thereby modulating transcriptional activity of the cellular nucleotide sequence.

23. The method of claim 22, wherein an expression vector comprising a polynucleotide sequence encoding a zinc finger-nucleotide binding polypeptide variant is introduced into the cells.

24. The method of claim 23, wherein the expression vector is a virus.

25. The method of claim 22, wherein the modulation is enhancement of transcription of a gene operatively linked to the cellular nucleotide sequence.

26. The method of claim 22, wherein the modulation is suppression of transcription of a gene operatively linked to the cellular nucleotide sequence.

50. The method of claim 26, wherein the zinc finger binding polypeptide variant is a truncated zinc finger protein.

28. An isolated zinc finger-nucleotide binding polypeptide variant comprising at least two zinc finger modules that bind to a target cellular nucleotide sequence and modulate the transcriptional function of the cellular nucleotide sequence, wherein the amino acid sequence of each zinc finger module that binds a target cellular nucleotide comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of two modules of said variant has at least one amino acid sequence modification.

29. The variant of claim 28, wherein the modulation is enhancement of transcription of a gene operatively linked to the cellular nucleotide sequence.

30. The variant of claim 28, wherein the modulation is suppression of transcription of a gene operatively linked to the cellular nucleotide sequence.

31. The variant of claim 28, which is derived from a zinc finger-nucleotide binding polypeptide selected from the group consisting of zif 268 and TFIIIA.

32. The variant of claim 28, wherein the cellular nucleotide sequence is DNA.

33. The variant of claim 28, wherein the cellular nucleotide sequence is RNA.

35. The variant of claim 28, wherein the cellular nucleotide sequence is a structural gene nucleotide sequence.

36. The variant of claim 28, wherein the cellular nucleotide sequence is a promoter nucleotide sequence.

37. The variant of claim 36, wherein the promoter is an onco-promoter.

38. The variant of claim 37, wherein the promoter is a viral promoter.

39. The variant of claim 28, wherein the cellular nucleotide sequence is a retroviral nucleotide sequence.

40. The variant of claim 39, wherein the retrovirus is a human T-cell lymphotrophic virus (HTLV).

41. The variant of claim 40, wherein the retrovirus is HTLV-1 or HTLV-2.

42. The variant of claim 39, wherein the retrovirus is a human immunodeficiency virus (HIV).

43. The variant of claim 42, wherein the retrovirus is HIV-1 or HIV-2.

44. The variant of claim 28, wherein the cellular nucleotide sequence is an oncogene nucleotide sequence.

45. The variant of claim 28, wherein the cellular nucleotide sequence is a plant cellular nucleotide sequence.

46. A zinc finger-nucleotide binding polypeptide variant produced by the method of claim 36.

47. An isolated nucleotide sequence encoding a zinc finger-nucleotide binding polypeptide variant of claim 28.

48. A recombinant expression vector containing a nucleotide sequence of claim 47.

49. A method for inhibiting a transcriptional function of a target cellular nucleotide sequence comprising a zinc finger-nucleotide binding motif, the method comprising contacting the motif with an effective amount of a zinc finger-nucleotide binding polypeptide variant comprised of at least two zinc finger molecules, wherein the amino acid sequence of each zinc finger module that binds the motif comprises two cysteines and two histidines whereby both cysteines are amino proximal to both histidines and wherein each of two modules has at least one amino acid sequence modification.

51. The method of claim 49, wherein the cellular nucleotide sequence is DNA.

52. The method of claim 49, wherein the cellular nucleotide sequence is RNA.

53. The method of claim 49, wherein the cellular nucleotide sequence is a structural gene nucleotide sequence.

54. The method of claim 49, wherein the cellular nucleotide sequence is a promoter nucleotide sequence.

55. The method of claim 49, wherein the cellular nucleotide sequence is an oncogene nucleotide sequence.

56. The method of claim 49, wherein the cellular nucleotide sequence is a plant cellular nucleotide sequence.