In vivo-directed evolution identifies AAV-WM04 as a next-generation vector for potent and sustained hearing restoration in DFNB9.

Abstract

Efficient and cell-specific gene delivery to cochlear inner hair cells (IHCs) remains a major challenge for inner ear gene therapy. Here, we identify and characterize a novel adeno-associated virus (AAV)2-derived capsid, AAV-WM04, that enables highly efficient and selective IHC transduction at low doses. Using an in-vivo-directed evolution strategy, we generated a randomized AAV2 capsid library with 9 amino acid insertions and performed iterative selection in the adult mouse cochlea. Next-generation sequencing revealed enrichment of several variants, among which AAV-WM04 exhibited superior packaging efficiency and pronounced IHC tropism. AAV-WM04 achieved near-complete IHC transduction throughout the cochlear axis in adult mice, outperforming clinically relevant vectors with minimal off-target expression and no detectable ototoxicity. Selective IHC transduction was observed in non-human primates following local delivery, providing proof-of-concept evidence supporting the translational potential of AAV-WM04. Therapeutically, AAV-WM04 enabled efficient dual-AAV trans-splicing delivery of the large OTOF gene, resulting in uniform full-length otoferlin expression in IHCs. In a humanized Otof^Q829X/Q829X mouse model, AAV-WM04 restored auditory function across a broad frequency range at relatively low doses and achieved sustained hearing recovery. These findings establish AAV-WM04 as a next-generation IHC-targeted vector with high efficiency, safety, and cross-species applicability for precision gene therapy of hereditary hearing loss.