Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear

Shao Wei Hu; Jun Lv; Zijing Wang; Honghai Tang; Hui Wang; Fang Wang; Daqi Wang; Juan Zhang; Longlong Zhang; Qi Cao; Yuxin Chen; Ziwen Gao; Yu Han; Wuqing Wang; Geng-Lin Li; Yilai Shu; Huawei Li

doi:10.34133/research.0341

Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear

Research (Wash D C). 2024 Apr 25:7:0341. doi: 10.34133/research.0341. eCollection 2024.

Authors

Shao Wei Hu^{1

2

3}, Jun Lv^{1

2

3}, Zijing Wang^{1

2

3}, Honghai Tang^{1

2

3}, Hui Wang^{1

2

3}, Fang Wang^{1

2

3}, Daqi Wang^{1

2

3}, Juan Zhang^{1

2

3}, Longlong Zhang^{1

2

3}, Qi Cao^{1

2

3}, Yuxin Chen^{1

2

3}, Ziwen Gao^{1

2

3}, Yu Han^{1

2

3}, Wuqing Wang^{1

2

3}, Geng-Lin Li^{1

2

3}, Yilai Shu^{1

2

3}, Huawei Li^{1

2

3}

Affiliations

¹ ENT Institute and Department of Otorhinolaryngology, Eye & ENT Hospital, State Key Laboratory of Medical Neurobiology and MOE Frontiers Center for Brain Science, Fudan University, Shanghai, 200031, China.
² Institute of Biomedical Science, Fudan University, Shanghai, 200032, China.
³ NHC Key Laboratory of Hearing Medicine (Fudan University), Shanghai, 200032, China.

Abstract

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 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof ^-/- mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.