Amelioration of an Inherited Metabolic Liver Disease through Creation of a De Novo Start Codon by Cytidine Base Editing.

Base editing technology efficiently generates nucleotide conversions without inducing excessive double-strand breaks (DSBs), which makes it a promising approach for genetic disease therapy. In this study, we generated a novel hereditary tyrosinemia type 1 (HT1) mouse model, which contains a start codon mutation in the fumarylacetoacetate hydrolase (Fah) gene by using an adenine base editor (ABE7.10). To investigate the feasibility of base editing for recombinant adeno-associated virus (rAAV)-mediated gene therapy, an intein-split cytosine base editor (BE4max) was developed. BE4max efficiently induced C-to-T conversion and restored the start codon to ameliorate HT1 in mice, but an undesired bystander mutation abolished the effect of on-target editing. To solve this problem, an upstream sequence was targeted to generate a de novo in-frame start codon to initiate the translation of FAH. After treatment, almost all C-to-T conversions created a start codon and restored Fah expression, which efficiently ameliorated the disease without inducing off-target mutations. Our study demonstrated that base editing-mediated creation of de novo functional elements would be an applicable new strategy for genetic disease therapy.