Methods and compositions for modification of the;locus

• US 8,895,264 B2
• Filed: 10/25/2012
• Issued: 11/25/2014
• Est. Priority Date: 10/27/2011
• Status: Active Grant

• Current Assignee: Sangamo Biosciences, Inc., Regents of the University of California (University of California)
• Original Assignee: Sangamo Biosciences, Inc., Regents of the University of California (University of California)
• Inventors: Cost, Gregory J., Holmes, Michael C., Kasahara, Noriyuki, Laganiere, Josee, Miller, Jeffrey C., Paschon, David, Rebar, Edward J., Urnov, Fyodor, Zhang, Lei
• Primary Examiner(s): Steadman, David J
• Assistant Examiner(s): HOLLAND, PAUL J
• Application Number:
  US13/660,843
• Publication Number:
  US 20130122591A1
• Time in Patent Office: 761 Days
• Field of Search: None
• US Class Current: 435/69.1
• CPC Class Codes: C07K 2319/81 containing a Zn-finger domain for DNA binding
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/1077 Pentosyltransferases (2.4.2)
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Y 204/02008 Hypoxanthine phosphoribosyltransferase (2.4.2.8)
• CPC Class Codes: C12Y 301/21004 Type II site-specific deoxyribonuclease (3.1.21.4)

Abstract

Nucleases and methods of using these nucleases for modification of an HPRT locus and for increasing the frequency of gene modification at a targeted locus and clones and for generating animals.

Claims

1. A non-naturally occurring fusion protein comprising a transcription activator like effector domain DNA binding protein (TALE) that binds to a target site in an endogenous hypoxanthine guanine phosphoribosyltransferase (HPRT) gene comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs:

69-78 and a nuclease cleavage domain, wherein the TALE domain comprises a repeat variable diresidue (RVD) sequence in the order presented and selected from a single row of Table 4.

2. A polynucleotide encoding the non-naturally occurring fusion proteins of claim 1.

3. An isolated cell comprising the fusion proteins of claim 1.

4. An isolated cell comprising the polynucleotides of claim 2.

5. The isolated cell of claim 3, wherein the cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.

6. The isolated cell of claim 5, wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell and a hepatic stem cell.

7. The isolated cell of claim 4, wherein the cell is selected from the group consisting of a T-cell, a B-cell, or a stem cell.

8. The isolated cell of claim 7, wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell, and a hepatic stem cell.

9. A kit comprising the fusion protein of claim 1.

10. A kit comprising the polynucleotide of claim 2.

11. A method of cleaving an endogenous HPRT gene in an isolated cell, the method comprising:

transforming or transfecting said isolated cell comprising said HRPT gene with the polynucleotide of claim 2, culturing said transformed or transfected isolated cell under conditions such that said fusion proteins is expressed and the HPRT gene is cleaved.

12. The method of claim 11, wherein the cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.

13. The method of claim 12, wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell, and a hepatic stem cell.

14. A method of enriching for cells modified by a nuclease at an endogenous HPRT locus, the method comprising:

cleaving said endogenous HPRT gene in said isolated cell according to the method of claim 11;

introducing into the cell, said polynucleotide encoding said fusion protein for modification of a second locus of the endogenous HPRT gene;

subjecting the cells to selection with 6-thioguanine, thereby enriching the cells for those in which the endogenous locus has been modified.

15. The method of claim 14, wherein the endogenous locus is inactivated.

16. The method of claim 14, wherein the isolated cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.

17. The method of claim 16, wherein the stem cell is selected from the group consisting of an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a CD34+ hematopoietic stem cell and a hepatic stem cell.