期刊
CELL STEM CELL
卷 29, 期 10, 页码 1428-+出版社
CELL PRESS
DOI: 10.1016/j.stem.2022.09.001
关键词
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资金
- Telethon [E5, E4]
- Italian Ministry of Health [PE-2016-02363691]
- Italian Ministry of University and Research [20175XHBPN]
- EU Horizon 2020 Program (UPGRADE)
- Louis-Jeantet Foundation
- EU [819815]
- New York Stem Cell Foundation
- Lady Tata Memorial Trust International Award for Research in Leukemia
- European Research Council (ERC) [819815] Funding Source: European Research Council (ERC)
This study reveals the unexpected load and persistence of AAV genomes and their fragments in gene editing using viral transduction. The accumulation of viral DNA triggers a sustained DNA damage response, leading to frequent integration in cells. Using an integrase-defective lentiviral vector can mitigate the adverse effects and improve gene editing efficiency.
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.
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