AAV-Mediated CRISPR/Cas9 Gene Editing in Murine Phenylketonuria.

Phenylketonuria (PKU) due to recessively inherited phenylalanine hydroxylase (PAH) deficiency results in hyperphenylalaninemia, which is toxic to the central nervous system. Restriction of dietary phenylalanine intake remains the standard of PKU care and prevents the major neurologic manifestations of the disease, yet shortcomings of dietary therapy remain, including poor adherence to a difficult and unpalatable diet, an increased incidence of neuropsychiatric illness, and imperfect neurocognitive outcomes. Gene therapy for PKU is a promising novel approach to promote lifelong neurological protection while allowing unrestricted dietary phenylalanine intake. In this study, liver-tropic recombinant AAV2/8 vectors were used to deliver CRISPR/Cas9 machinery and facilitate correction of the allele by homologous recombination. Additionally, a non-homologous end joining (NHEJ) inhibitor, vanillin, was co-administered with the viral drug to promote homology-directed repair (HDR) with the AAV-provided repair template. This combinatorial drug administration allowed for lifelong, permanent correction of the allele in a portion of treated hepatocytes of mice with PKU, yielding partial restoration of liver PAH activity, substantial reduction of blood phenylalanine, and prevention of maternal PKU effects during breeding. This work reveals that CRISPR/Cas9 gene editing is a promising tool for permanent PKU gene editing.