Introduction Wiskott Aldrich syndrome WAS

Introduction Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from mutations in the WAS gene; WAS encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). WASp is a key regulator of the R 428 cytoskeleton, specifically regulating actin polymerization and formation of immunological synapses. Within the immune system, WASp deficiency results in well-documented functional defects in mature lymphocytes such as reduced antigen-specific proliferation of T cells and significantly reduced cytotoxic activity by natural killer (NK) cells when exposed to tumor cell lines (Orange et al., 2002). Transplantation of hematopoietic stem cells (HSCs) represents a potential therapeutic approach for a variety of hematological disorders. Success in treating WAS via lentiviral-mediated gene delivery has recently been reported (Aiuti et al., 2013; Hacein-Bey Abina et al., 2015). Although no leukemogenic events were reported in up to 3 years following delivery of gene-modified CD34+ cells, it remains difficult to predict whether any of the unique integration sites (e.g., ∼10,000 per treated child in Aiuti et al. [2013]) will result in adverse consequences in the longer term as occurred in the original WAS retroviral gene-therapy trial (Braun et al., 2014). Thus, development of site-specific targeting strategies for treatment of WAS is warranted. In this study, we wished to assess whether targeted gene editing of WASp-deficient induced pluripotent stem cells (iPSCs) would result in functional correction of the derived hematopoietic progeny. WAS can be caused by a diversity of mutations distributed across all 12 exons. To provide a gene correction solution potentially applicable to most, if not all, WAS patient cells, we used zinc finger nuclease (ZFN)-mediated, site-specific, homology-directed repair (HDR) to target the integration of a corrective WAS gene sequence into the endogenous WAS chromosomal locus. We hypothesized that utilizing the endogenous WAS promoter, the natural WAS chromatin environment, and transcription regulatory signals, would provide for a physiologically appropriate WAS transgene expression.
Results
Discussion In this study, we demonstrate the successful, sequence-specific correction of WAS-iPSCs via TI of a WAS transgene into the endogenous WAS locus. We chose to target integration of the WAS2–12 half-gene into intron 1 of the WAS locus with a view toward potentially providing correction for all WAS mutations; for exon 1 mutations, targeted iPSC clones also incorporating the donor WT exon 1 would be utilized. This report provides proof-of-concept data for the potential utility of WASp-deficient iPSCs corrected using site-directed gene editing. Correcting the WAS gene mutations in patient-specific iPSCs versus primary HSCs, has two distinct advantages. The first potential advantage is the ability to comprehensively sequence the corrected iPSC clones to rule out any untoward genetic changes. As a first step toward this end, we compared the WAS and cWAS-iPSCs via whole-exome sequencing and CGH to identify potential consequences of the ZFN-mediated gene editing, Cre-mediated excision, and/or extended iPSC culture. Although some differences were observed (e.g., amplifications or deletions uniquely present in either WAS or cWAS in Table S1, non-synonymous coding variants in Table S2), we did not observe any generation of mutations that we could directly attribute to the ZFN-mediated gene editing. The second potential advantage is that transplantation of corrected iPSC-derived HSCs will result in patients receiving a genetically homogeneous population of corrected cells. Derivation of transplantable HSCs from hESCs/human iPSCs (hiPSCs) remains very inefficient and challenging (Kaufman, 2009; Slukvin, 2013). Although recent studies suggest strategies to improve generation of transplantable HSC from human pluripotent stem cells (Gori et al., 2015), these studies need to be confirmed with a demonstration of long-term, multi-lineage HSC engraftment. Non-random X-inactivation of the WAS gene in female carriers and somatic reversion suggest the possibility of a selective advantage in vivo for corrected blood cells at the level of T cell precursors, common lymphoid progenitor cells, and perhaps even at a more primitive stage (Davis et al., 2008; Wengler et al., 1995). Thus, it may be sufficient to deliver to patients in-vitro-generated T cell precursors/progenitors from corrected iPSC.