p38 MAPK inhibition safeguards genome integrity during gene editing of hematopoietic stem and progenitor cells.

Ex vivo genome editing of hematopoietic stem and progenitor cells (HSPCs) holds significant therapeutic potential but remains constrained by genotoxic risks associated with nuclease-induced DNA double-strand breaks, DNA donor template delivery and sensing, and proliferation-induced stress during ex vivo manipulation. These processes can lead to chromosomal instability, large on-target deletions, donor mis-integration, off-target events, and impaired long-term stem cell function, raising safety concerns for clinical translation. Here, we evaluate the impact of transient p38 MAPK inhibition on genomic integrity during clinically relevant CRISPR-Cas9 editing and show that this intervention attenuates ex vivo culture-associated stress without increasing detectable genotoxic outcomes. Comprehensive genotoxicity analyses, including quantification of large on-target deletions, adeno-associated viral vector mis-integration, and CAST-seq mapping of translocations, reveal no measurable differences in gene editing-associated structural alterations upon p38 MAPK inhibition, while micronuclei were significantly reduced. Importantly, long-term xenotransplantation followed by whole-exome sequencing shows that p38i-treated HSPCs display a reduced mutational burden without evidence of increased genomic alterations. Collectively, these findings identify transient p38 MAPK inhibition as a strategy to improve the quality and long-term fitness of gene-edited HSPCs without detectable adverse effects on the genome editing outcomes, supporting its further evaluation as a refinement to clinically relevant CRISPR-Cas9 editing workflows.