同源重组
生物
氟达拉滨
清脆的
转基因
基因靶向
Cas9
DNA修复
遗传增强
核酸酶
分子生物学
基因
细胞生物学
遗传学
化疗
环磷酰胺
作者
Shinnosuke Tsuji,Calvin J. Stephens,Giulia Bortolussi,Feijie Zhang,Gabriele Baj,Hagoon Jang,Gustavo de Alencastro,Andrés F. Muro,Katja Pekrun,Mark A. Kay
标识
DOI:10.1038/s41587-022-01240-2
摘要
Homologous recombination (HR)-based gene therapy using adeno-associated viruses (AAV-HR) without nucleases has several advantages over classic gene therapy, especially the potential for permanent transgene expression. However, the low efficiency of AAV-HR remains a major limitation. Here, we tested a series of small-molecule compounds and found that ribonucleotide reductase (RNR) inhibitors substantially enhance AAV-HR efficiency in mouse and human liver cell lines approximately threefold. Short-term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease- and CRISPR/Cas9-mediated AAV-HR two- to sevenfold in the murine liver, without causing overt toxicity. Fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Notably, the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice, suggesting that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing AAV-HR efficiency in mice. These results suggest that use of a clinically approved RNR inhibitor can potentiate AAV-HR-based genome-editing therapeutics.
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