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

Tao, Y.; Chu, C.; Cheng, Z.; Sun, Y.; Chen, Y.; Zhang, H.; Bao, S.; yang, B.; Feng, B.; Huang, X.; Lu, Y.; Yang, Q.; Mao, X.; Zhou, Q.; Jin, C.; Duan, Z.; Zhong, G.; Wu, H.

doi:10.64898/2026.03.11.710960

bioRxiv · 10.64898/2026.03.11.710960

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

Tao, Y.·Chu, C.·Cheng, Z.·Sun, Y.·Chen, Y.·Zhang, H.·Bao, S.·yang, B.·Feng, B.·Huang, X.·Lu, Y.·Yang, Q.·Mao, X.·Zhou, Q.·Jin, C.·Duan, Z.·Zhong, G.·Wu, H.

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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 AAV2-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. Robust and exclusive IHC transduction was further validated in non-human primates following round window membrane delivery, underscoring translational potential. 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 durable hearing recovery. Collectively, 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.

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Tao, Y., Chu, C., Cheng, Z., Sun, Y., Chen, Y., Zhang, H., Bao, S., yang, B., Feng, B., Huang, X., Lu, Y., Yang, Q., Mao, X., Zhou, Q., Jin, C., Duan, Z., Zhong, G., Wu, H.. 2026-03-11. In vivo-directed evolution identifies AAV-WM04 as a next-generation vector for potent and durable hearing restoration in DFNB9. https://doi.org/10.64898/2026.03.11.710960

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