Methods and compositions for generation of Bax-and Bak-deficient cell lines

• US 8,597,912 B2
• Filed: 06/10/2009
• Issued: 12/03/2013
• Est. Priority Date: 06/10/2008
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Collingwood, Trevor, Gregory, Philip D.
• Primary Examiner(s): LEAVITT, MARIA GOMEZ
• Application Number:
  US12/456,043
• Publication Number:
  US 20100003756A1
• Time in Patent Office: 1,637 Days
• Field of Search: 435/69.1, 435/252.3, 435/6.1, 435/440, 435/455, 530/350
• US Class Current: 435/69.7
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 2501/48 Regulators of apoptosis
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 5/0018 Culture media for cell or tissue culture media for specific animal cell type C12N5/06
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Disclosed herein are methods and compositions for generation of Bak- and/or Bax-deficient cell lines using engineered nucleases.

Claims

1. A protein comprising an engineered zinc finger protein DNA-binding domain that specifically binds to a target site in a Bak or Bax gene, wherein the DNA-binding domain comprises four, five or six zinc finger recognition regions, ordered F1 to F4 for proteins with four zinc finger domains, ordered F1 to F5 for proteins with five zinc finger domains and ordered F1 to F6 for proteins with six zinc finger domains from N-terminus to C-terminus, and further wherein the zinc finger proteins is selected from the group consisting of a protein comprising the following amino acid sequences in the zinc finger recognition regions:

(i) F1;

RSDHLTT (SEQ ID NO;

9);

F2;

RSDHLSE (SEQ ID NO;

10);

F3;

RNDNRKT (SEQ ID NO;

11);

F4;

QSGHLQR (SEQ ID NO;

12); and

F5;

QSGHLSR (SEQ ID NO;

13) and wherein the protein binds to the target sequence shown in SEQ ID NO;

23;

(ii) F1;

RSDSLSA (SEQ ID NO;

14);

F2;

DRSSRTK (SEQ ID NO;

15);

F3;

RSDDLTR (SEQ ID NO;

16); and

F4;

RSDALAR (SEQ ID NO;

17) and wherein the protein binds to the target sequence shown in SEQ ID NO;

24;

(iii) F1;

DRSHLSR (SEQ ID NO;

34);

F2;

TSGSLTR (SEQ ID NO;

35);

F3;

RSDSLSA (SEQ ID NO;

14);

F4;

DRSSRTK (SEQ ID NO;

15);

F5;

RSDNLSE (SEQ ID NO;

36); and

F6;

HSNARKT (SEQ ID NO;

37); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

33;

(iv) F1;

RSDNLSV (SEQ ID NO;

39);

F2;

DRSNLTR (SEQ ID NO;

40);

F3;

RSDTLSE (SEQ ID NO;

41); and

F4;

RSQTRKT (SEQ ID NO;

42); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

38;

(v) F1;

QNAHRKT (SEQ ID NO;

50);

F2;

QSGDLTR (SEQ ID NO;

     53);

F3;

QSGDLTR (SEQ ID NO;

53);

F4;

QSSNLAR (SEQ ID NO;

54); and

F5;

TSSNRKT (SEQ ID NO;

     55); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

52;

(vi) F1;

RSDNLAR (SEQ ID NO;

57);

F2;

QSGDLTR (SEQ ID NO;

     53);

F3;

DNRQLIT (SEQ ID NO;

58);

F4;

TSSNLSR (SEQ ID NO;

8);

F5;

RSDTLSR (SEQ ID NO;

59); and

F6;

RNDDRIT (SEQ ID NO;

60); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

56;

(vii) F1;

RSDNLTT (SEQ ID NO;

62);

F2;

RSDHLSR (SEQ ID NO;

63);

F3;

QSSDLRR (SEQ ID NO;

64);

F4;

QSSHLTR (SEQ ID NO;

3); and

F5;

RSDHLTQ (SEQ ID NO;

65); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

61;

(viii) F1;

QSGSLTR (SEQ ID NO;

67);

F2;

TSSNRKT (SEQ ID NO;

55);

F3;

DRSHLTR (SEQ ID NO;

68);

F4;

RSDDRKT (SEQ ID NO;

69);

F5;

NRTDLIR (SEQ ID NO;

7); and

F6;

TSSNLSR (SEQ ID NO;

8); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

66;

(ix) F1;

RSDHLST (SEQ ID NO;

1);

F2;

DRSHLAR (SEQ ID NO;

2);

F3;

QSSHLTR (SEQ ID NO;

3); and

F4;

RSDNLRE (SEQ ID NO;

4); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

1;

(x) F1;

RSANLSV (SEQ ID NO;

5);

F2;

DRANLSR (SEQ ID NO;

6);

F3;

NRTDLIR (SEQ ID NO;

7); and

F4;

TSSNLSR (SEQ ID NO;

8); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

22;

(xi) F1;

DRSDLSR (SEQ ID NO;

45);

F2;

NRTDLIR (SEQ ID NO;

7);

F3;

TSSNLSR (SEQ ID NO;

8);

F4;

RSDTLSQ (SEQ ID NO;

46); and

F5;

DRSARTR (SEQ ID NO;

47); and

wherein the protein binds to the target sequence shown in SEQ ID NO;

44; and

(xii) F1;

RSDALSV (SEQ ID NO;

48);

F2;

DSSHRTR (SEQ ID NO;

49);

F3;

QNAHRKT (SEQ ID NO;

50);

F4;

RSDHLST (SEQ ID NO;

1); and

F5;

TSGHLSR (SEQ ID NO;

51) and wherein the protein binds to the target sequence shown in SEQ ID NO;

44.

2. A fusion protein comprising a zinc finger DNA-binding domain according to claim 1 and a FokI cleavage half-domain.

3. The fusion protein of claim 2, wherein the FokI cleavage half-domain is a wild-type Fold cleavage half-domain.

4. The fusion protein of claim 2, wherein the FokI cleavage half-domain is an engineered Fold cleavage half-domain.

5. A polynucleotide encoding the zinc finger DNA-binding domain according to claim 1.

6. An isolated cell comprising a protein according to claim 1.

7. A method of inactivating an endogenous cellular BAK or BAX gene in a cell, the method comprising:

(a) introducing, into a cell, a first nucleic acid encoding a first fusion protein according to claim 2,

(b) introducing, into the cell, a second nucleic acid encoding a second fusion protein according to claim 2, whereby the first and second fusion proteins bind to and dimerize to cleave at least one of the BAK and BAX genes.

8. The method of claim 7, wherein the first and second fusion proteins are encoded by the same nucleic acid.

9. The method of claim 7, wherein the first and second fusion proteins are encoded by different nucleic acids.

10. 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 endogenous BAX and BAK genes;

(b) inactivating one or both of the endogenous BAX and BAK genes of the host cell by the method of claim 7; and

(c) introducing an expression vector comprising a transgene, the transgene comprising a sequence encoding a protein of interest into the host cell, thereby producing the recombinant protein.

11. The method of claim 10, wherein the protein of interest comprises an antigen.

12. The method of claim 11, wherein the antigen comprises an influenza antigen.

13. A cell line in which at least one of Bak and Bax are partially or fully inactivated, wherein the cell line is produced by

(a) inactivating at least one of Bak and Bax in a cell according to the method of claim 7;

and

(b) culturing the cell under conditions suitable for generating a cell line in which at least one of Bak and Bax is partially or fully inactivated.

14. The cell line of claim 13, wherein the cell is a mammalian cell selected from the group consisting of a COS cell, a CHO cell, a VERO cell, a MDCK cell, a WI38 cell, a V79 cell, a B14AF28-G3 cell, a BHK cell, a HaK cell, a NS0 cell, a SP2/0-Ag14 cell, a HeLa cell, an HEK293 cell, and a perC6 cell.

15. The method of claim 7, wherein the first and second fusion proteins form a homodimer.

16. The method of claim 7, wherein the first and second fusion proteins form a heterodimer.