Preclinical pharmacology and toxicology study of an AAV8-tATP7B vector for Wilson’s disease

BACKGROUND/AIMS: Wilson's disease (WD) is an autosomal recessive disorder of copper metabolism caused by Atp7b mutations. In this study, a novel gene therapy for WD was developed, and its efficacy and safety were evaluated in relevant animal models. METHODS: Codon-optimized full-length or truncated ATP7B (tATP7B) genes were assembled with liver-specific mini promoters to construct an adeno-associated virus serotype 8 vector (AAV8). The expression and activity of these vectors were evaluated in HepG2 cells. AAV8-tATP7B viral particles were produced using a triple-plasmid cotransfection system under good manufacturing practice conditions. Long-term efficacy was evaluated in Atp7b-/- mice at three doses (5×10¹¹, 5×10¹², and 1×10¹³ vg/kg). Single-dose toxicity was assessed over 13 weeks in Sprague- Dawley rats and cynomolgus macaques. RESULTS: In HepG2 cells, the ability of the tATP7B to export copper was comparable to that of the full-length protein, but the expression efficiency was greater. Alkaline gel electrophoresis confirmed its better compatibility with AAV packaging limits while maintaining genomic integrity, supporting its selection for AAV8-tATP7B production. In a 24- week study in Atp7b-/- mice, AAV8-tATP7B restored copper homeostasis and liver function in a dose-dependent manner and significantly reversed existing liver injury. Toxicity studies in Sprague‒Dawley rats and cynomolgus monkeys revealed no systemic toxicity, whereas reversible liver changes were observed in cynomolgus monkeys at high doses; thus, 6×10¹³ vg/kg was established as the maximum tolerated dose. CONCLUSIONS: These results establish the efficacy and safety profile of AAV8-tATP7B and provide the rationale for its clinical translation in patients with WD.