Delivery methods and compositions for nuclease-mediated genome engineering

• US 9,957,526 B2
• Filed: 10/16/2014
• Issued: 05/01/2018
• Est. Priority Date: 10/17/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc.
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Holmes, Michael C., Wang, Jianbin
• Primary Examiner(s): Ton, Thaian N
• Application Number:
  US14/516,236
• Publication Number:
  US 20150110762A1
• Time in Patent Office: 1,293 Days
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector

Abstract

The present disclosure is in the field of genome engineering, particularly targeted modification of the genome of a cell.

Claims

1. A method of integrating one or more transgenes into a genome of an isolated population of hematopoietic stem cells or T-cells, the method comprising:

introducing, into the population of hematopoietic stem cells or T-cells, (a) an adeno-associated type 6 (AAV6) donor vector comprising the one or more transgenes and (b) at least one non-naturally occurring nuclease, wherein the at least one nuclease is introduced in mRNA form and cleaves the genome of the population of hematopoietic stem cells or T-cells such that the one or more transgenes are integrated into the genome of the cell, wherein the donor vector is introduced into the cell prior to or after the at least one nuclease and further wherein

(i) if the donor vector is introduced into the population of hematopoietic stem cells or T-cells before the at least one nuclease, the at least one nuclease is introduced into the cell 3 to 48 hours after the donor vector is introduced and;

(ii) if the at least one nuclease is introduced before the donor vector, the donor vector is introduced into the cell within 4 hours after the at least one nuclease is introduced,

such that at least 10% of the one or more transgenes are integrated into the genome of the population of hematopoietic stem cells or T-cells.

2. The method of claim 1, wherein the donor vector is introduced into the cell before the at least one nuclease and the cell is cultured for between 1 and 24 hours after the donor vector is introduced into the cell.

3. The method of claim 1, wherein the AAV6 vector comprises an AAV6 chimeric vector.

4. The method of claim 1, wherein the at least one nuclease is selected from the group consisting of a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), a CRISPR/Cas nuclease system and combinations thereof.

5. The method of claim 1, wherein the nuclease cleaves a safe-harbor gene.

6. The method of claim 5, wherein the safe-harbor gene is selected from the group consisting of a CCR5 gene, an AAVS1 gene, a Rosa gene and an albumin gene.

7. The method of claim 1, wherein the transgene encodes a therapeutic protein.