1:San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Milan, Italy; 2:University Vita-Salute San Raffaele, Milan, Italy; 3:University Center for Statistics in the Biomedical Sciences, Milan, Italy; 4:National research Council, Institute for Biomedical technologies, Milan, Italy

Gene editing (GE) by artificial nucleases hold promise for gene therapy in Hematopoietic Stem and Progenitor Cells (HSPC). Despite rapid advances in GE-based therapies, a few challenges remain to be faced to improve GE efficiency and HSPC repopulating potential. We showed that the combination of nuclease-induced Double Strand Break with DNA repair template for Homology Directed Repair (HDR) delivered by AAV6 caused cumulative activation of the p53-mediated DNA Damage Response (DDR) pathway constraining HSPC proliferation and yield. Protracted DDR signaling leads to the establishment of cellular senescence, a condition of permanent cell cycle arrest. By integrating transcriptional analysis with innovative imaging-based cellular assays we reported induction of cellular senescence markers and pro-inflammatory programs across edited HSPC subtypes and in vivo upon transplantation. Consistently, we found open chromatin at promoters of several senescence-gene categories and inflammatory genes of the IL1 axis and NF-kB pathway especially in HDR-edited cells. Mechanistically, we reported an ATM-p53 dependent activation of inflammatory cytokines in edited HSPC. Temporary inhibition of IL1 and NF-kB pathways at the time of GE increased edited HSPC clonogenicity in-vitro and long-term hematopoietic reconstitution in vivo with a concomitant decrease in senescence markers in edited HSPC. Our in vivo clonal tracking of HDR-edited HSPC revealed that IL1 inhibition improved polyclonal reconstitution preserving self-renewal and multi-potency of individual edited HSPC. Our findings define senescence and inflammatory programs as long-term consequences of CRISPR-Cas9 engineered human HSPC and pave the way for the development of novel strategies to overcome cellular barriers for efficient HSPC-based clinical applications.