Senescence and inflammation are unintended consequences of CRISPR-Cas9 gene-editing in hematopoietic stem cells [WES]

Gene editing (GE) in hematopoietic stem and progenitor cells (HSPCs) represents a promising strategy for site-specific gene correction of several inherited diseases. However, its clinical translation is still hampered by limited efficiency and the emergence ofcellular responses constraining HSPC post-transplant polyclonal hematopoietic reconstitution, specifically for homology-directed repair (HDR) mediated GE. Here we identified the activation of a senescence and interleukin-1 (IL-1)/nuclear factor-kappa beta (NF-kB)-mediated inflammatory programs in HDR-edited HSPCs and their progeny, contributing to their oligoclonal reconstitution upon transplantation. Temporary inhibition of p53 or IL-1-NF-kB axis upon GE increased clonogenicity of edited HSPCs ex vivo, improved their long-term polyclonal reconstitution, and better-preserved self-renewal of HDR-edited HSPC clones. Importantly, senescence modulation did not aggravate the genotoxicity risks of the GE procedure in repopulating HSPCs. Our findings define senescence and inflammatory programs as uncharted barriers to efficient GE paving the way for the development of valuable strategies for more efficient and safer HSPC-based clinical applications.