Engineering of the AAV-compatible Hair Cell-specific small size Myo15 Promoter for Gene Therapy in the Inner Ear

Adeno-associated virus (AAV) -mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a “multiple vectors in one AAV” strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1611 bp down to 1157 bp, and further down to 956 bp. Under the control of these two promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the two promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these two promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof 26 –/– mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering, and created two truncated Myo15 promoters that not only restored hereditary deafness in animal models, but also show great potential for treating human patients in future.