The study investigates the potential of AAV-mediated gene cocktails to enhance the regeneration of hair cells (HCs) in the mammalian cochlea. HCs are essential for hearing, and their damage leads to severe hearing impairment. The research focuses on the transdifferentiation of supporting cells (SCs) into HCs using key transcription factors Atoh1, Gfi1, Pou4f3, and Six1 (GPAS). AAV-ie, a safe and efficient AAV variant, was used to deliver these genes to SCs in the cochlea. The results show that AAV-ie-mediated co-regulation of multiple genes significantly enhances the regeneration of mature HCs. Single-cell nuclear sequencing and lineage tracing revealed that GPAS-induced HCs are more advanced and functionally mature compared to those induced by Atoh1 alone. Electrophysiological experiments confirmed that the regenerated HCs exhibit similar electrophysiological characteristics to native HCs. Additionally, GPAS was shown to induce HC regeneration in a mouse model with HC damage. The study suggests that AAV-mediated co-regulation of GPAS could be an effective therapeutic approach for functional HC regeneration in the mouse cochlea.The study investigates the potential of AAV-mediated gene cocktails to enhance the regeneration of hair cells (HCs) in the mammalian cochlea. HCs are essential for hearing, and their damage leads to severe hearing impairment. The research focuses on the transdifferentiation of supporting cells (SCs) into HCs using key transcription factors Atoh1, Gfi1, Pou4f3, and Six1 (GPAS). AAV-ie, a safe and efficient AAV variant, was used to deliver these genes to SCs in the cochlea. The results show that AAV-ie-mediated co-regulation of multiple genes significantly enhances the regeneration of mature HCs. Single-cell nuclear sequencing and lineage tracing revealed that GPAS-induced HCs are more advanced and functionally mature compared to those induced by Atoh1 alone. Electrophysiological experiments confirmed that the regenerated HCs exhibit similar electrophysiological characteristics to native HCs. Additionally, GPAS was shown to induce HC regeneration in a mouse model with HC damage. The study suggests that AAV-mediated co-regulation of GPAS could be an effective therapeutic approach for functional HC regeneration in the mouse cochlea.