In-depth characterization of stem cell potency and genotoxicity for clinical-scale ex vivo CRISPR-Cas9 gene editing

Area of Science:

• Gene therapy and genome editing
• Hematopoietic stem cell biology
• Rare genetic disorders

Background:

• CRISPR-Cas9 homology-directed repair (HDR) faces clinical translation challenges including standardization, safety, and efficacy.
• Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency treatable by hematopoietic stem cell transplantation.

Purpose of the Study:

• To establish a clinically compliant preclinical framework for ex vivo correction of WAS using CRISPR-Cas9-AAV6.
• To optimize hematopoietic stem and progenitor cell (HSPC) editing for improved in vivo engraftment and safety.

Main Methods:

• Developed a clinical-compatible platform for large-scale HSPC manufacturing preserving viability and function.
• Optimized adeno-associated virus (AAV) dosing and modulated the p53BP-dependent DNA damage response pathway.
• Implemented a multi-tiered genotoxicity assessment (in silico, genome-wide, orthogonal assays) and longitudinal in vivo safety monitoring.

Main Results:

• Achieved significantly improved in vivo correction and repopulation of HSPCs.
• Demonstrated a favorable safety profile with minimal off-target risks and no evidence of clonal dominance.
• Identified rare donor-specific off-target events and a de novo chromosomal rearrangement post-engraftment, underscoring the need for patient monitoring.

Conclusions:

• The developed CRISPR-Cas9-AAV6 platform provides a robust roadmap for HDR-based gene editing therapies.
• Established critical benchmarks for efficacy, safety, and regulatory readiness in advanced therapeutic medicinal product development.
• Highlights the importance of rigorous genotoxicity assessment and long-term patient monitoring after gene editing transplantation.