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

Summary

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.

Graphical abstract

Highlights

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  AAV DNA activates p53, thereby reducing HSPC engraftment

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  AAV inverted terminal repeats (ITRs) can get trapped at DNA breaks in edited HSPCs

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  AAV ITRs are transcriptionally competent in human HSPCs

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  Integrase-defective lentiviral vector outperforms AAV as DNA template to edit HSPCs

Abstract

Naldini and colleagues describe p53-mediated cellular sensing and frequent integration of transcriptionally competent AAV ITRs at induced DNA breaks as inadvertent consequences of templated gene editing of human HSPCs. To alleviate such potentially adverse effects, they propose an optimized gene editing protocol using integrase-defective lentiviral vectors for template delivery.