Editing of ADA2 Point Mutation in Human Hematopoietic Stem Cells

BackgroundThe homozygous ADA2: c.506G>A (p.Arg169Gln; p.R169Q) variant accounts for majority of Deficiency in Adenosine Deaminase 2 (DADA2). This monogenic disorder may be amenable to ex vivo gene therapy by correcting the pathogenic mutation in CD34+ hematopoietic stem and progenitor cells (HSPCs). ObjectiveTo apply CRISPR-Cas9 and homology-directed repair (HDR) as a surrogate strategy to model correction of the pathogenic ADA2 c.506G>A variant in healthy cord blood HSPCs. MethodsHSPCs were electroporated with optimised CRISPR-Cas9 editing reagents, and editing outcomes, including HDR and on-target deletions, were quantified by ddPCR. Cell functionality was assessed through colony-forming unit (CFU) assays and by xenotransplantation into NOD SCID Gamma (NSG) mice. Two HDR enhancement strategies were tested: (1) genetic inhibitors of p53 and non-homologous end joining (NHEJ) pathways, and (2) pharmacological NHEJ inhibition. ResultsSmall-molecule NHEJ inhibitors increased HDR efficiency approximately two-fold (from [~]40 % to [~]80 %). Edited HSPCs retained normal CFU capacity and successfully engrafted in NSG mice. However, up to 8 % of edited cells exhibited on-target chromosome loss, though this declined over time. Up to 40 % of T cells and fibroblasts demonstrated similar losses under NHEJ inhibitors treatment. In contrast, genetically encoded inhibitors did not improve HDR. ConclusionThe ADA2 p. c.506G>A variant can be effectively edited employing surrogate strategy in HSPCs without impairing functionality. Although pharmacological inhibition of NHEJ enhances HDR efficiency, it also increases the risk of on-target chromosome aberrations, highlighting the need for careful consideration of the associated risks and benefits in therapeutic gene editing. Key messages1) The ADA2 p.R169Q variant can be efficiently corrected via HDR, and the edited CD34+ HSPCs retain their engraftment capability in NSG mice. 2) Pharmacological inhibition of NHEJ using small-molecule inhibitors increases HDR efficiency but is associated with significant on-target deletions and chromosomal arm loss, particularly in differentiated cell types, and in a donor-dependent manner. Capsule summaryThe ADA2 p.R169Q variant is a viable target for precision gene editing in hematopoietic stem cells. Although inhibition of NHEJ improves HDR efficiency, it concomitantly increases the risk of large on-target deletions, particularly in differentiated cells.