Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells

Gene editing (GE) using homology-directed repair (HDR) in hematopoietic stem and progenitor cells (HSPCs) offers promise for long-range gene correction of inherited genetic disorders. However, cellular responses induced by CRISPR-Cas9/AAV6 engineering impair the long-term repopulating potential of HDR-edited HSPCs, adversely impacting the safety and efficacy of clinical translation. Our study uncovers a durable senescence-like response in genetically engineered HSPCs triggered by p53 and interleukin (IL)-1/nuclear factor KB (NF-kB) activation, which restricts graft size and clonal diversity in long-term transplantation as-says. We show that transient p53 inhibition or blocking inflammatory pathways mitigates senescence-asso-ciated responses, improving the repopulating capacity of edited HSPCs. Importantly, we identify treatment with Anakinra, an IL-1 signaling antagonist, as a promising strategy to enhance polyclonal output in HDR-edi-ted cells while minimizing genotoxicity risks associated with the editing procedure. Overall, our findings pre-sent strategies to overcome key hurdles in HDR-based HSPC gene therapies, providing a framework for enhancing their efficacy and safety in clinical applications.