Delivery methods and compositions for nuclease-mediated genome engineering

• US 10,604,771 B2
• Filed: 05/06/2014
• Issued: 03/31/2020
• Est. Priority Date: 05/10/2013
• Status: Active Grant

• Current Assignee: Sangamo Therapeutics, Inc., University of Southern California
• Original Assignee: Sangamo Therapeutics, Inc.
• Inventors: Cannon, Paula M., Exline, Colin Michael, Holmes, Michael C.
• Primary Examiner(s): Wilson, Michael C
• Application Number:
  US14/271,008
• Publication Number:
  US 20140335063A1
• Time in Patent Office: 2,156 Days
• Field of Search: 435455, 435325, 424 9321
• CPC Class Codes: A01K 2207/12 Animals modified by administration of exogenous cells
• CPC Class Codes: A01K 2207/15 Humanized animals
• CPC Class Codes: A01K 2267/0381 Animal model for diseases of the hematopoietic system
• CPC Class Codes: A61K 35/28 Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• CPC Class Codes: C12N 15/907 in mammalian cells

Abstract

Disclosed herein are methods and compositions for delivery of engineered nucleases and donor molecules into the genome of a cell.

Claims

1. A method of expressing a transgene in an isolated human hematopoietic stem cell (HSC), the method comprising:

a) administering at least one polynucleotide to an isolated human HSC, the polynucleotide encoding a pair of zinc finger nucleases (ZFNs) that generates a double-stranded break in the albumin, CCR5 or AAVS1 gene; and

b) administering a non-viral DNA construct to the human HSC, the DNA construct comprising;

(i) a nucleic acid comprising a transgene encoding a marker protein flanked by nucleic acid sequences homologous to the albumin, CCR5 or AAVS1 gene; and

(ii) a plasmid backbone sequence that is less than 300 base pairs in length and is devoid of bacterial sequences,

wherein the DNA construct does not include an origin of replication, and further wherein the nucleic acid encoding the transgene is integrated into the double-stranded break such that the marker protein is expressed in the isolated human HSC.

2. The method of claim 1, wherein the DNA construct comprises a promoter that drives expression of the marker protein.