In-Depth Characterization of Stem Cell Potency and Genotoxicity for Clinical-Scale Ex Vivo CRISPR/Cas9 Gene Editing

Abstract

Translating CRISPR/Cas9-based homology-directed repair (HDR) strategies into clinical application remains a major challenge due to limited standardization, concerns over safety, and efficacy issues. Here, we present a comprehensive and clinically compliant preclinical framework for the ex vivo correction of Wiskott-Aldrich Syndrome (WAS) using a CRISPR/Cas9-AAV6 platform targeting hematopoietic stem and progenitor cells (HSPCs). In this study, we established a clinical-compatible platform enabling large-scale manufacturing while preserving HSPC viability, stemness, and multilineage functionality. To overcome low HSPC long-term engraftment, we fine-tuned AAV dosing and transiently modulated p53 signalling, achieving significantly improved in vivo correction and repopulation. Importantly, we implemented a multi-tiered genotoxicity assessment strategy, integrating in silico, genome-wide, and orthogonal assays, revealing a largely favorable safety profile with minimal off-target risks and no signs of clonal dominance or transformation. Longitudinal in vivo safety monitoring revealed donor specific rare off-target events and structural variants. This highlights the crucial importance of patient monitoring after transplantation, further emphasized by the identification of a de novo chromosomal rearrangements that could be detected exclusively following cell engraftment in mice. This work offers a robust and adaptable roadmap for future HDR-based gene editing platforms, establishing critical benchmarks for efficacy, safety, and regulatory readiness in the development of advanced therapeutic medicinal products.