San Rafaelle Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy; Vita-Salute San Raffaele University, Milan 20132, Italy.
San Rafaelle Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan 20132, Italy.
Cell Stem Cell. 2022 Oct 6;29(10):1428-1444.e9. doi: 10.1016/j.stem.2022.09.001.
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.
通过同源定向修复 (HDR) 在造血干细胞/祖细胞 (HSPC) 中进行长程基因编辑，通常依赖于使用重组腺相关病毒载体 (AAV) 进行病毒转导以递送模板。在这里，我们揭示了 AAV 基因组及其片段的意外负载和延长的持久性，它们在招募 AAV 反向末端重复序列 (ITR) 上的 MRE11-RAD50-NBS1 (MRN) 复合物后，引发持续的 p53 介导的 DNA 损伤反应 (DDR)。在细胞周期停滞的 HSPC 中积累的病毒 DNA 导致其频繁整合，主要是以转录有效的 ITR 形式，在核酸内切酶的靶内和靶外位点。整合酶缺陷型慢病毒载体 (IDLV) 的优化递送导致较低的 DNA 负载和较少的持续 DDR，从而提高了长期重编程 HSPC 的集落形成能力和编辑效率。由于 IDLV 中病毒 DNA 片段的插入较少，因此选择其作为模板递送可以减轻 HDR 编辑的不利影响和遗传毒性负担，并应有助于其在 HSPC 基因治疗中的临床转化。
