METHODS AND COMPOSITIONS FOR MODIFICATION OF THE HPRT LOCUS

• US 20130122591A1
• Filed: 10/25/2012
• Published: 05/16/2013
• 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., Laganiere, Josee, Miller, Jeffrey C., Paschon, David, Rebar, Edward J., Urnov, Fyodor, Zhang, Lei, Kasahara, Noriyuki
• Granted Patent: US 8,895,264 B2
• Time in Patent Office: Days
• US Class Current: 435/456
• 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 TAL effector domain DNA binding protein (TALE) that binds to an endogenous hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene and a cleavage domain, wherein the fusion protein modifies the endogenous HPRT gene.

3. A polynucleotide encoding one or more fusion proteins of claim 1.

4. An isolated cell comprising one or more fusion proteins according to claim 1.

5. An isolated cell comprising one or more polynucleotides according to claim 3.

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

7. The cell of claim 6, 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.

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

9. The cell of claim 8, 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.

10. A kit comprising a fusion protein according to claim 1.

11. A kit comprising a polynucleotide according to claim 3.

12. A method of cleaving an endogenous HPRT gene in a cell, the method comprising:

introducing, into the cell, one or more polynucleotides according to claim 3, under conditions such that the one or more fusion proteins are expressed and the HPRT gene is cleaved.

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

14. The cell of claim 13, 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.

15. The method of claim 12, further comprising integrating a transgene into the genome of the cell.

16. The method of claim 15, wherein the transgene is introduced to the cell using a viral vector, as an oligonucleotide or on a plasmid.

17. The method of claim 15, wherein the transgene is integrated into the HPRT locus.

18. The method of claim 15, wherein the transgene is integrated into a CCR5 gene, a CXCR4 gene, an albumin gene, an AAVS1 gene, a Rosa gene or a beta-globin gene.

19. The method of claim 15, wherein the transgene is under the control of an endogenous promoter.

20. The method of claim 15, wherein the transgene is under the control of an exogenous promoter.

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

cleaving an endogenous HPRT gene in a cell according to the method of claim 12;

introducing into the cell, one or more polynucleotides encoding nucleases that cleave the genome of the cell at the endogenous locus;

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

22. The method of claim 21, wherein the endogenous locus is inactivated.

23. The method of claim 21, wherein a transgene is integrated into the endogenous locus following cleavage by the nucleases.

24. The method of claim 21, wherein the endogenous locus is selected from the group consisting of HPRT, AAVS1, albumin, beta-globin and Rosa26.

25. The method of claim 21, wherein the cell is selected from the group consisting of a T-cell, a B-cell or a stem cell.

26. The method of claim 25, 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.

2. The fusion protein of claim 2, wherein the TALE comprises the repeat variable diresidue (RVD) sequences shown in a single row of Table 4.