Abstract
Pathogenic mutations in the Gjb2 gene, encoding connexin 26, are the leading cause of autosomal recessive hereditary deafness. Gene therapy holds significant promise for treating this. Adeno-associated virus (AAV)-mediated therapeutic gene delivery has been shown to be safe and effective in restoring hearing in both animal models and human patients. However, Gjb2 gene therapy has been hindered by the limited specificity and efficiency of the available AAV vectors. In this study, we screened AAV serotypes and found that co-administration of AAV1 and AAV-ie could effectively target Gjb2-expressing cells. However, the ectopic Gjb2 expression in hair cells induced by these AAVs could cause ototoxicity, which was addressed by employing the specific promoter SCpro. Co-injection of AAV1 and AAV-ie carrying exogenous Gjb2 driven by SCpro effectively restored hearing function in Gjb2-deficient mice. Moreover, the combined AAV system can transduce the cochleae of Bama miniature pigs and AAV administration into the inner ear of cynomolgus monkeys did not impair hearing and showed negligible systemic toxicity, indicating the efficiency and safety of this gene therapy in large animals. Thus, this study provides a strategy for Gjb2 gene therapy and lays a foundation for future clinical applications.