Rassf2 overexpression mediated by AAV promotes the supporting cell-to-hair cell transformation in the cochlea

•It is a promising way to promote hair cell regeneration by AAV-mediated foreign genes.•Rassf2 overexpression mediated by AAV-ie could enhance organoids production and supporting cells trans-differentiation into hair cells in the organoids cultured with matrigel-based hydrogel scaffolds in vitro.•The regenerated hair cells due to Rassf2 overexpression in supporting cells were derived from Lgr5+ progenitors by using a lineage tracing approach in vivo. Sensory hair cells are responsible for detecting and transmitting sound in the inner ear, and damage to HCs leads to hearing loss. HCs do not regenerate spontaneously in adult mammals, which makes the hearing loss permanent. However, hair cells and supporting cells have the same precursors in the inner ear, and in newborn mice, the adjacent SCs can be activated by gene manipulation to differentiate into newly regenerated hair cells. Here, we demonstrate the role of the Ras association domain family member 2 (Rassf2) in supporting cell to hair cell trans-differentiation in the inner ear. Using the AAV vector (AAV-ie) to upregulate Rassf2 expression promoted supporting cell division and hair cell production in cultured cochlear organoids. Also, AAV-Rassf2 enhanced the regenerative ability of Lgr5+ SCs in the postnatal cochlea without impairing hearing, and this might due to the modulation of the Wnt, Hedgehog and Notch signaling pathways. Furthermore, AAV-Rassf2 enhances cochlear supporting cell division and hair cell production in the neomycin injury model. In summary, our results suggest that Rassf2 is a key component in HC regenerative repair, and gene modulation mediated by adeno-associated virus may be a promising gene therapy for hearing repair. [Display omitted]