Gene editing-based targeted integration for correction of Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a severe X-linked primary immunodeficiency resulting from a diversity of mutations distributed across all 12 exons of the gene. encodes a hematopoietic-specific and developmentally regulated cytoplasmic protein (WASp). The objective of this study was to develop a gene correction strategy potentially applicable to most WAS patients by employing nuclease-mediated, site-specific integration of a corrective gene sequence into the endogenous chromosomal locus. In this study, we demonstrate the ability to target the integration of -containing constructs into intron 1 of the endogenous gene of primary CD34 hematopoietic stem and progenitor cells (HSPCs), as well as WASp-deficient B cell lines and WASp-deficient primary T cells. This intron 1 targeted integration (TI) approach proved to be quite efficient and restored WASp expression in treated cells. Furthermore, TI restored WASp-dependent function to WAS patient T cells. Edited CD34 HSPCs exhibited the capacity for multipotent differentiation to various hematopoietic lineages and in transplanted immunodeficient mice. This methodology offers a potential editing approach for treatment of WAS using patient's CD34 cells.