Methods and compositions for inactivation of dihydrofolate reductase

• US 20080015164A1
• Filed: 05/17/2007
• Published: 01/17/2008
• Est. Priority Date: 05/19/2006
• Status: Abandoned Application

• Current Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Original Assignee: Sangamo Biosciences, Inc. (Sangamo Therapeutics, Inc.)
• Inventors: Collingwood, Trevor
• Application Number:
  US11/804,222
• Publication Number:
  US 20080015164A1
• Time in Patent Office: Days
• US Class Current: 514/44
• 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 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for inactivating dihydrofolate reductase genes, using fusion proteins comprising a zinc finger protein and a cleavage domain or cleavage half-domain. Polynucleotides encoding said fusion proteins are also provided, as are cells comprising said polynucleotides and fusion proteins.

Claims

1. A protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:

3. The protein according to claim 1, further comprising a cleavage domain.

5. The protein of claim 3, wherein the cleavage domain is a cleavage half-domain.

7. The protein of claim 5, wherein the cleavage half-domain is a wild-type or engineered FokI cleavage half-domain.

9. A polynucleotide encoding the protein of claim 1.

11. An isolated cell comprising the protein of claim 1.

13. An isolated cell comprising the polynucleotide of claim 9.

2. A protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:

4. The protein according to claim 2, further comprising a cleavage domain.

6. The protein of claim 4, wherein the cleavage domain is a cleavage half-domain.

8. The protein of claim 6, wherein the cleavage half-domain is a wild-type or engineered FokI cleavage half-domain.

10. A polynucleotide encoding the protein of claim 2.

12. An isolated cell comprising the protein of claim 2.

14. An isolated cell comprising the polynucleotide of claim 10.

15. A method for inactivating an endogenous dhfr gene in a cell, the method comprising:

(a) introducing, into a cell, a first nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises;

(i) a zinc finger DNA-binding domain that is engineered to bind to a first target site in an endogenous dhfr gene; and

(ii) a cleavage domain;

such that the polypeptide is expressed in the cell, whereby the polypeptide binds to the target site and cleaves the dhfr.

16. The method of claim 15, wherein the nucleic acid comprises a polynucleotide encoding a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:

17. The method of claim 15, wherein the nucleic acid comprises a polynucleotide encoding a protein comprising an engineered zinc finger protein DNA-binding domain, wherein the DNA-binding domain comprises four zinc finger recognition regions ordered F1 to F4 from N-terminus to C-terminus, and wherein F1, F2, F3, and F4 comprise the following amino acid sequences:

18. The method of claim 15, wherein the first nucleic acid further encodes a second polypeptide, wherein the second polypeptide comprises:

(i) a zinc finger DNA-binding domain that is engineered to bind to a second target site in the dhfr gene; and

(ii) a cleavage domain;

such that the second polypeptide is expressed in the cell, whereby the first and second polypeptides bind to their respective target sites and cleave the dhfr gene.

19. A method of producing a recombinant protein of interest in a host cell, the method comprising the steps of:

(a) providing a host cell comprising an endogenous dhfr gene;

(b) inactivating the endogenous dhfr gene of the host cell by the method of claim 15;

(c) introducing an expression vector comprising transgene, the transgene comprising a dhfr gene and a sequence encoding a protein of interest into the host cell; and

(d) selecting cells in which the transgene is stably integrated into and expressed by the host cell, thereby producing the recombinant protein.

20. The method of claim 19, further comprising the step of exposing the host cell comprising the integrated transgene to a DHFR inhibitor.

21. The method of claim 19, wherein the host cell is a CHO cell.

22. A method of treating a subject having a cell proliferative disorder, the method comprising the step of inactivating a dhfr gene according to the method of claim 15 in one or more cells of the subject.

23. The method of claim 22, wherein the cell proliferative disorder is a cancer.