Zinc finger nuclease mediated safe harbor targeted gene transfer in patient iPSCs functionally corrects X-linked chronic granulomatous disease

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NIAID	STEMCELL-7

* FARE Award Winner

Authors

• C Sweeney
• J Zou
• BK Chou
• U Choi
• J Pan
• H Wang
• S Dowey
• L Cheng
• H Malech

Abstract

X-linked chronic granulomatous disease (X-CGD) is a defect of neutrophil microbicidal reactive oxygen species (ROS) production resulting from gp91phox deficiency, caused by a variety of mutations throughout the CYBB gene. We developed induced pluripotent stem cells (iPSCs) from patients with X-CGD, and demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production, reproducing the defining phenotype of X-CGD. Gene therapy of X-CGD using lentiviral vectors carries a risk of mutagenesis due to vector insertion near oncogenes. Homologous recombination allows safe gene delivery to a specific site in the genome, but has very low efficiency. Utilizing zinc finger nucleases to facilitate homologous recombination, we achieved high-efficiency targeted transfer of a therapeutic CYBB minigene into the safe harbor AAVS1 locus in X-CGD iPSCs, without inducing additional detected genetic changes. Upon neutrophil differentiation, corrected clones exhibited functionally restored ROS production (27-42% of cells positive for oxidase by dihydrorhodamine flow cytometry assay, compared to 0.4-0.9% for neutrophils from uncorrected X-CGD iPSCs and 23-29% for neutrophils from normal control iPSCs). Our findings demonstrate that precise safe harbor minigene targeting can be used for the correction of X-CGD using zinc finger nucleases in iPSCs, an approach that might also be applied to other monogenic diseases.

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