Recombinant adeno-associated viral (rAAV) vectors continue to gain popularity for in vivo therapeutic gene delivery. Homologous recombination-based gene therapy using rAAV (AAV-HR) without nucleases has several advantages over classical gene therapy, especially when targeting the liver in neonatal/pediatric populations due to its potential for permanent sustained transgene expression. However, the low efficiency of AAV-HR remains a limitation for some clinical applications. Here, we tested

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... s of small molecule compounds with different mechanisms of action in the context of AAV-HR and identified that ribonucleotide reductase (RNR) inhibitors significantly enhanced the AAV-HR efficiency in mouse and human liver cell lines. Furthermore, short term administration of the RNR inhibitor fludarabine increased the in vivo efficiency of both non-nuclease and CRISPR/Cas9-mediated AAV-HR in the murine liver, without causing toxicity. Mechanistic experiments showed that fludarabine administration induced transient DNA damage signaling in both proliferating and quiescent hepatocytes. Surprisingly, in vivo BrdU labeling implicated that the majority of AAV-HR events occurred in non-proliferating hepatocytes in both fludarabine-treated and control mice. These studies suggested that the induction of transient DNA repair signaling in non-dividing hepatocytes was responsible for enhancing the efficiency of AAV-HR in mice during RNR inhibition. In total, we show the use of a clinically approved RNR inhibitor can enhance AAV-HR based genome editing therapeutics.