DNA-PK inhibition enhances gene editing efficiency in HSPCs for CRISPR-based treatment of X-linked hyper IgM syndrome
Cole M. Pugliano, Mason P. Berger, Roslyn M. Ray, Kai Sapkos, Betty Wu, Aidan Laird, Yidian Ye, Daniel Thomson, M. Quinn DeGottardi, Iram Khan, Kristina J. Tatiossian, Brodie A. Miles, Florian Aeschimann, Jérôme Pasquier, Mihee M. Kim, David J. Rawlings
Abstract
Targeted gene editing to restore CD40L expression via homology-directed repair (HDR) in CD34 + hematopoietic stem and progenitor cells (HSPCs) represents a potential long-term therapy for X-linked hyper IgM syndrome. However, clinical translation of HSPC editing is limited by inefficient long-term engraftment of HDR-edited HSPCs. Here, we ameliorate this issue by employing a small-molecule inhibitor of DNA-PKcs, AZD7648, to bias DNA repair mechanisms to facilitate HDR upon CRISPR SpCas9-based gene editing. Using AZD7648 treatment and a clinically relevant HSPC source, mobilized peripheral blood CD34 + cells, we achieve ∼60% HDR efficiency at the CD40LG locus and enhanced engraftment of HDR-edited HSPCs in primary and secondary xenotransplants. Specifically, we observed a 1.6-fold increase of HDR-edited long-term HSPCs in primary transplant recipients without disturbing chimerism levels or differentiation capacity. As CD40L is primarily expressed in T cells, we demonstrate T cell differentiation from HDR-edited HSPCs in vivo and in artificial thymic organoid cultures, and endogenously regulated CD40L expression following activation of in-vivo -derived CD4 + T cells. Our combined findings demonstrate HDR editing at the CD40LG locus at potentially clinically beneficial levels. More broadly, these data support using DNA-PKcs inhibition with AZD7648 as a simple and efficacious addition to HSPC editing platforms.