A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells
Lucrezia della Volpe, Federico Midena, Roberta Vacca, Teresa Tavella, Laura Alessandrini, Giacomo Farina, Chiara Brandas, Elena Lo Furno, Kety Giannetti, Edoardo Carsana, Matteo Maria Naldini, Matteo Barcella, Samuele Ferrari, Stefano Beretta, Antonella Santoro, Simona Porcellini, Angelica Varesi, Diego Gilioli, Anastasia Conti, Ivan Merelli, Bernhard Gentner, Anna Villa, Luigi Naldini, Raffaella Di Micco
Abstract
Ex vivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering ex vivo culture necessary yet detrimental. Mechanistically, ex vivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications. • Shortening HSPC pre-stimulation reduces DDR-mediated responses to gene editing • p38 MAPK-ROS induce fast cell-cycle progression fueling DNA damage and DDR signaling • Preventing p38 activity mitigates proliferative stress and improves HSC fitness • p38 inhibition enhances polyclonal and long-term reconstitution of edited HSPCs Although detrimental, ex vivo activation of hematopoietic stem cells is required for gene editing strategies. Here, della Volpe et al. uncover a p38 MAPK-dependent molecular axis causing functional decline of gene-edited cells. Temporary p38 inhibition enhances the fitness of engineered cells for more effective and safer clinical applications.