Functional editing of the OTC locus by targeted integration with phenotype correction and restoration of endogenous expression patterns.

Here, we report highly efficient functional repair of the ornithine transcarbamylase (OTC) locus in mutant mouse and human hepatocytes in vivo using a dual adeno-associated virus system delivering CRISPR-Cas9 editing reagents and a promoterless donor for targeted integration. The approach was mutation agnostic and targeted intronic sequences to prevent inadvertent inactivation of hypomorphic alleles. Notably, in a murine model, we corrected the metabolic defect and simultaneously achieved liver-wide restoration of physiological metabolic zonation of Otc expression by capturing native cis-acting regulatory elements. The effectiveness of this approach was confirmed using a universally configured therapeutic cassette in patient-derived primary human hepatocytes in vivo. These data provide a powerful template to guide further optimization of this approach and, given the high editing efficacy required for phenotypic effect in OTC deficiency, have broader relevance to other liver disease phenotypes.