We have developed induced pluripotent stem cells (iPSCs) from a patient with X-linked chronic granulomatous disease (X-CGD), a defect of neutrophil microbicidal reactive oxygen species (ROS) generation resulting from gp91phox deficiency. We demonstrated that mature neutrophils differentiated from X-CGD iPSCs lack ROS production, reproducing the pathognomonic CGD cellular phenotype. Targeted gene transfer into iPSCs, with subsequent selection and full characterization to assure no off-target changes, holds promise for correction of monogenic diseases without the insertional mutagenesis caused by multisite integration of viral or plasmid vectors. Zinc finger nuclease mediated gene targeting of a single copy gp91phox therapeutic minigene into one allele of the "safe harbor" AAVS1 locus in X-CGD iPSCs without off-target inserts resulted in sustained expression of gp91phox and substantially restored neutrophil ROS production. Our findings demonstrate how precise gene targeting may be applied to correction of X-CGD using zinc finger nuclease and patient iPSCs.

  • Submitted December 30, 2010.
  • Accepted March 1, 2011.