AAV-Net1 facilitates the trans-differentiation of supporting cells into hair cells in the murine cochlea

Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFP^CreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFP^CreERT2/Rosa26-tdTomato^loxp/loxp mice. We further found that the up-regulation of Wnt/β-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.