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.
CRISPR Gene Editing