Choice of template delivery mitigates the genotoxic risk and adverse impact of editing in human hematopoietic stem cells

• DOI: 10.1016/j.stem.2022.09.001
• Publication Date: 2022-10-06T17:25:08Z
• Authors: Samuele Ferrari; Aurelien Jacob; Daniela Cesana; Marianne Laugel; Stefano Beretta; Angelica Varesi; Giulia Unali; Anastasia Conti; Daniele Canarutto; Luisa Albano; Andrea Calabria; Valentina Vavassori; Carlo Cipriani; Maria Carmina Cas...; Simona Esposito; Chiara Brombin; Federica Cugnata; Oumeya Adjali; Eduard Ayuso; Ivan Merelli; Anna Villa; Raffaella Di Micco; Anna Kajaste-Rudn...; Eugenio Montini; Magalie Penaud-Bu...; Luigi Naldini
• Keywords: Transduction (biophysics); Homology directed repair

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex inverted terminal repeats (ITRs). Accrual cell-cycle-arrested HSPCs led to its frequent integration, predominantly form transcriptionally competent ITRs, at nuclease on- off-target sites. Optimized delivery integrase-defective lentiviral (IDLV) induced lower less persistent DDR, improving clonogenic capacity efficiency long-term repopulating HSPCs. Because insertions fragments are IDLV, choice mitigates adverse impact genotoxic burden HDR should facilitate clinical translation HSPC therapy.