Choice of Template Delivery Mitigates the Genotoxic Risk and Adverse Impact of Editing in Human Hematopoietic Stem Cells (Targeted deep sequencing)

Long-range gene editing by homology-directed repair (HDR) in hematopoietic stem/progenitor cells (HSPCs) often relies on viral transduction with recombinant adeno-associated viral vector (AAV) for template delivery. Here, we uncover unexpected load and prolonged persistence of AAV genomes and their fragments, which trigger sustained p53-mediated DNA damage response (DDR) upon recruiting the MRE11-RAD50-NBS1 (MRN) complex on the AAV inverted terminal repeats (ITRs). Accrual of viral DNA in cell-cycle-arrested HSPCs led to its frequent integration, predominantly in the form of transcriptionally competent ITRs, at nuclease on- and off-target sites. Optimized delivery of integrase-defective lentiviral vector (IDLV) induced lower DNA load and less persistent DDR, improving clonogenic capacity and editing efficiency in long- term repopulating HSPCs. Because insertions of viral DNA fragments are less frequent with IDLV, its choice for template delivery mitigates the adverse impact and genotoxic burden of HDR editing and should facilitate its clinical translation in HSPC gene therapy. The frequency and diversity of insertions/deletions (indels) generated at the target site upon NHEJ-mediated repair of the nuclease-induced DNA double strand break (DSB) can be used as surrogate readout of graft complexity. Moreover, integration of fragments derived from the DNA template can be integrated at the nuclease DNA DSB. Therefore, we performed targeted deep sequencing of the nuclease target site in hematopoietic organs of humanized hematochimeric mice.