Efficient in vivo editing of OTC-deficient patient-derived primary human hepatocytes

Abstract Background and aims Genome editing technology has immense therapeutic potential and is likely to rapidly supplant contemporary gene addition approaches. Key advantages include the capacity to directly repair mutant loci with resultant recovery of physiological gene expression and maintenance of durable therapeutic effects in replicating cells. This study aimed to repair a disease-causing point mutation in the ornithine transcarbamylase (OTC) locus in patient-derived primary human hepatocytes in vivo at therapeutically relevant levels. Methods Editing reagents for precise CRISPR/SaCas9-mediated cleavage and homology-directed repair (HDR) of the human OTC locus were first evaluated against an OTC minigene cassette transposed into the mouse liver. The editing efficacy of these reagents were then tested on the native OTC locus in patient-derived primary human hepatocytes xenografted into the FRG (Fah-/-Rag2-/-IL2rg-/-) mouse liver. A highly human hepatotropic capsid (NP59) was used for AAV-mediated gene transfer. Editing events were characterised using next generation sequencing and restoration of OTC expression evaluated using immunofluorescence. Results Following AAV-mediated delivery of editing reagents to patient-derived primary human hepatocytes in vivo, OTC locus-specific cleavage was achieved at efficiencies of ≤72%. Importantly successful editing was observed in up to 29% of OTC alleles at clinically relevant vector doses. No off-target editing events were observed at the top 10 in silico-predicted sites in the genome. Conclusions We report efficient single-nucleotide correction of a disease-causing mutation in the OTC locus in patient-derived primary human hepatocytes in vivo at levels that, if recapitulated in the clinic, would provide therapeutic benefit for even the most therapeutically challenging liver disorders. Key challenges for clinical translation include the cell cycle dependence of classical HDR and mitigation of unintended on and off-target editing events.