METHODS AND COMPOSITIONS FOR NUCLEASE-MEDIATED TARGETED INTEGRATION

• US 20150240263A1
• Filed: 02/24/2015
• Published: 08/27/2015
• Est. Priority Date: 02/24/2014
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Biosciences, Inc.
• Inventors: Holmes, Michael C., Wechsler, Thomas
• Granted Patent: US 10,370,680 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2750/14141 Use of virus, viral particle or viral elements as a vector

Abstract

Disclosed herein are methods and compositions for targeted, nuclease-mediated insertion of transgene sequences into the genome of a cell.

Claims

1. A method of integrating an exogenous sequence into a target sequence of an isolated cell, the method comprising sequentially administering (i) one or more nucleases that cleave the target sequence and (ii) one or more donor sequences that are integrated into the target sequence following cleavage of the target sequence by the one or more nucleases, wherein there is a delay of at least 24 hours between the sequential administration.

2. The method of claim 1, wherein the delay between the sequential administrations is between 24 and 72 hours.

3. The method of claim 1, wherein the one or more nucleases are administered prior to the one or more donor sequences.

4. The method of claim 1, wherein the one or more donor sequences are administered prior to the one or more nucleases.

5. The method of claim 1, wherein the one or more nucleases comprise a meganuclease, a zinc finger nuclease (ZFN), a TALE-nuclease (TALEN), or a CRISPR/Cas nuclease system.

6. The method of claim 5, wherein the one or more nucleases are nickases.

7. The method of claim 1, wherein the one or more nucleases are administered using a plasmid, a viral vector or in RNA, mini-circle or linear DNA form.

8. The method of claim 1, wherein the one or more donor sequences are administered using a plasmid, a viral vector, a mini-circle, an oligonucleotide or as linear DNA.

9. The method of claim 3, wherein the one or more nucleases and one or more donor sequences are administered using a viral vector.

10. The method of claim 9, wherein the viral vector is an AAV vector.

11. The method of claim 4, wherein the one or nucleases are administered as RNA.

12. The method of claim 11, wherein the RNA is mRNA.

13. The method of claim 1, wherein the one or more donor sequences express at least one protein product.

14. The method of claim 13, wherein the protein product is a functional version of a protein that is non-functional, deficient or aberrantly expressed in a disorder.

15. The method of claim 1, wherein the target sequence is an endogenous locus.

16. The method of claim 15, wherein the endogenous locus is a safe-harbor locus.

17. A method of providing a functional protein lacking or deficient in a subject, the method comprising integrating one or more donor sequences into an isolated cell according to the method of claim 14 and administering the isolated cell to the subject.

18. The method of claim 17, wherein the cell is a stem cell.

19. A method of providing a functional protein lacking or deficient in a subject, the method comprising integrating one or more donor sequences into a cell of the subject according to the method of claim 14.