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

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.

Automated summary

The use of ribonucleotide reductase inhibitors can substantially enhance the efficiency of adeno-associated virus-mediated homologous recombination-based gene therapy. These findings suggest that clinically approved inhibitors, such as fludarabine, can be used to potentiate gene editing therapies.