美国斯坦福大学Mark A. Kay研究团队发现，氟达拉滨增加小鼠无核酸酶AAV和CRISPR/Cas9介导的同源重组。相关论文于2022年4月7日在线发表在《自然—生物技术》杂志上。

研究人员表示，基于同源重组（HR）的基因治疗，使用不含核酸酶的腺相关病毒（AAV-HR），与传统的基因治疗相比有几个优点，特别是有可能实现永久性的转基因表达。然而，AAV-HR的低效率仍然是一个主要限制。

研究人员测试了一系列的小分子化合物，发现核糖核苷酸还原酶（RNR）抑制剂能大幅提高AAV-HR在小鼠和人类肝脏细胞系中的效率，约为三倍。RNR抑制剂氟达拉滨的短期给药使非核酸酶和CRISPR/Cas9介导的AAV-HR在小鼠肝脏中的体内效率提高了2至7倍，而没有造成明显的毒性。氟达拉滨的施用在增殖期和静止期的肝细胞中都诱发了短暂的DNA损伤信号。

值得注意的是，在氟达拉滨处理的小鼠和对照组小鼠中，大多数AAV-HR事件都发生在非增殖期的肝细胞中，这表明在非增殖期的肝细胞中诱导瞬时DNA修复信号是提高小鼠AAV-HR效率的原因。这些结果表明，使用临床批准的RNR抑制剂可以增强基于AAV-HR的基因组编辑疗法的效果。

附：英文原文

Title: Fludarabine increases nuclease-free AAV- and CRISPR/Cas9-mediated homologous recombination in mice

Author: Tsuji, Shinnosuke, Stephens, Calvin J., Bortolussi, Giulia, Zhang, Feijie, Baj, Gabriele, Jang, Hagoon, de Alencastro, Gustavo, Muro, Andrs F., Pekrun, Katja, Kay, Mark A.

Issue&Volume: 2022-04-07

Abstract: 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. Fludarabine-induced DNA damage pathways enhance the in vivo efficiency of homologous recombination-based gene editing.

DOI: 10.1038/s41587-022-01240-2

Source: https://www.nature.com/articles/s41587-022-01240-2