Senescence and inflammation are unintended adverse consequences of CRISPR-Cas9/AAV6-mediated gene editing in hematopoietic stem cells
Anastasia Conti, Kety Giannetti, Federico Midena, Stefano Beretta, Nicolò Gualandi, Rosaria De Marco, Edoardo Carsana, Angelica Varesi, Teresa Tavella, Laura Alessandrini, Parinaz Zarghamian, Alessandra Weber, Samuele Ferrari, Chiara Brombin, Diego Gilioli, Lucrezia della Volpe, Stephanie Z. Xie, Ivan Merelli, Toni Cathomen, Luigi Naldini, Raffaella Di Micco
Abstract
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 κB (NF-κB) activation, which restricts graft size and clonal diversity in long-term transplantation assays. We show that transient p53 inhibition or blocking inflammatory pathways mitigates senescence-associated 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-edited cells while minimizing genotoxicity risks associated with the editing procedure. Overall, our findings present strategies to overcome key hurdles in HDR-based HSPC gene therapies, providing a framework for enhancing their efficacy and safety in clinical applications.