Highly Efficient Functional Editing of the Native Ornithine Transcarbamylase Locus by Targeted Integration with Phenotype Correction and Restoration of Physiological Patterns of Gene Expression

Here we report unprecedented efficacy in the functional repair of an exemplar locus, ornithine transcarbamylase (OTC), in mutant primary mouse and human hepatocytes in vivo using a dual AAV vector system configured to deliver CRISPR-Cas9 editing reagents and a promoterless donor for targeted integration by non-homologous end joining. The approach was mutation agnostic and targeted editing events to 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 capture of the native promoter. 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.