Delivery methods and compositions for nuclease-mediated genome engineering
Disclosed herein are methods and compositions for delivery of engineered nucleases and donor molecules into the genome of a cell.
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.