Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells

Clinical application of lentiviral vector (LV)‐based hematopoietic stem and progenitor cells (HSPC) gene therapy is rapidly becoming a reality. Nevertheless, LV‐mediated signaling and its potential functional consequences on HSPC biology remain poorly understood. We unravel here a remarkably limited impact of LV on the HSPC transcriptional landscape. LV escaped innate immune sensing that instead led to robust IFN responses upon transduction with a gamma‐retroviral vector. However, reverse‐transcribed LV DNA did trigger p53 signaling, activated also by non‐integrating Adeno‐associated vector, ultimately leading to lower cell recovery ex vivo and engraftment in vivo . These effects were more pronounced in the short‐term repopulating cells while long‐term HSC frequencies remained unaffected. Blocking LV‐induced signaling partially rescued both apoptosis and engraftment, highlighting a novel strategy to further dampen the impact of ex vivo gene transfer on HSPC. Overall, our results shed light on viral vector sensing in HSPC and provide critical insight for the development of more stealth gene therapy strategies. Synopsis Lentiviral (LV) gene therapy vectors escape innate sensing but trigger p53 signaling in human hematopoietic stem and progenitor cells (HSPC), ultimately leading to lower engraftment of short‐term repopulating cells. LV transduction remains remarkably stealth in human HSPC, avoiding innate immune activation but triggering p53 signaling in human HSPC. p53 signaling occurs upon ATM‐dependent nuclear sensing of vector DNA (LV, IDLV, AAV) independently of integration into the host genome. Vector‐mediated activation of p53 leads to cell cycle arrest and apoptosis ex vivo ultimately leading to lower engraftment of short‐term HSPC in vivo . Inhibition of LV‐signaling rescues ex vivo apoptosis and early engraftment of human HSPC. Graphical Abstract Lentiviral (LV) gene therapy vectors escape innate sensing but trigger p53 signaling in human hematopoietic stem and progenitor cells (HSPC), ultimately leading to lower engraftment of short‐term repopulating cells.