481. CRISPR/Cas9-Mediated In Vivo Genome Editing to Correct the OTC spfash Mutation in Newborn Mice

Many genetic liver diseases, including OTC deficiency, present in newborns with repeated, often lethal, metabolic crises. Adeno-associated virus (AAV) neonatal gene therapy in this setting would require multiple vector administrations to maintain the therapeutic effects because the non-integrating genome is lost as developing hepatocytes proliferate. As such, we reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR/Cas9, a powerful genome-editing tool consisting of the Cas9 nuclease and a single-guide RNA (sgRNA). We developed a strategy using an AAV vector with high liver tropism (AAV8) to correct the point mutation in newborn spfash mice using Cas9 enzyme from Staphylococcus aureus (SaCas9). An animal model of OTC deficiency, the male sparse fur ash (spfash) mouse, has a G-to-A point mutation at the donor splice site at the end of exon 4 of the OTC gene, which leads to abnormal splicing and a 20-fold reduction in OTC mRNA and protein.We developed a two-vector approach to incorporate all 3 components of CRISPR/Cas9 into AAV. Vector 1 expresses the SaCas9 gene from a liver-specific TBG promoter, while vector 2 contains both the sgRNA1 sequence expressed from a U6 promoter and the 1.8 kb donor OTC DNA sequence. Spfash pups were injected intravenously on postnatal day 2 with mixtures of vector 1 and vector 2 and subsequently evaluated for indel (insertion and deletion) formation and functional correction of the spfash mutation. Following gene correction (3 and 8 weeks), indels were detected by deep sequencing in 31% of OTC alleles, and HDR-based correction of the G-to-A mutation was observed in 10% of OTC alleles. Liver sections were analyzed by immunohistochemistry for OTC expression, showing 15% OTC-positive cells at 3 weeks and 13% at 8 weeks. Direct measurements of OTC enzyme activity from liver homogenates and OTC mRNA from total cellular RNA from liver revealed similarly high levels of correction in treated animals. We further assessed the impact of gene correction on the clinical manifestations of OTC deficiency by evaluating the tolerance of spfash mice to a one-week course of high-protein diet. Spfash mice treated with the gene-editing vectors had significantly lower plasma ammonia levels and showed a survival improvement as compared with untreated spfash mice. This study provides convincing evidence for efficacy in an authentic animal model of a lethal human metabolic disease following in vivo genome editing.