AAV-mediated gene cocktails enhance supporting cell reprogramming and hair cell regeneration. Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs leads to severe hearing impairment. Neighboring supporting cells (SCs) can regenerate damaged HCs, making functional HC regeneration crucial for restoring auditory function. This study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS (Gfi1, Pou4f3, and Six1), is effective in achieving functional HC regeneration in the mouse cochlea. Key transcription factors Atoh1, Gfi1, Pou4f3, and Six1 are essential for SCs destined for HC development and maturation. AAV-ie, a novel and efficient AAV variant, was used to overexpress these genes in SCs of the juvenile mouse cochlea, leading to the regeneration of OHC-like and IHC-like cells. Single-cell nuclear sequencing and lineage tracing showed that GPAS-induced HCs were more mature and closer to native HCs. Electrophysiological analysis confirmed that GPAS-induced HCs had similar electrophysiological characteristics to native HCs. Additionally, GPAS could induce HC regeneration in a mouse model with AAV-DTR-induced HC damage. The study highlights the potential of AAV-mediated gene regulation as a promising therapeutic strategy for sensorineural hearing loss. The results suggest that AAV-mediated co-regulation of multiple genes, such as GPAS, can promote the maturation and functional regeneration of HCs in the cochlea.AAV-mediated gene cocktails enhance supporting cell reprogramming and hair cell regeneration. Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs leads to severe hearing impairment. Neighboring supporting cells (SCs) can regenerate damaged HCs, making functional HC regeneration crucial for restoring auditory function. This study demonstrates that AAV-mediated co-regulation of multiple genes, such as GPAS (Gfi1, Pou4f3, and Six1), is effective in achieving functional HC regeneration in the mouse cochlea. Key transcription factors Atoh1, Gfi1, Pou4f3, and Six1 are essential for SCs destined for HC development and maturation. AAV-ie, a novel and efficient AAV variant, was used to overexpress these genes in SCs of the juvenile mouse cochlea, leading to the regeneration of OHC-like and IHC-like cells. Single-cell nuclear sequencing and lineage tracing showed that GPAS-induced HCs were more mature and closer to native HCs. Electrophysiological analysis confirmed that GPAS-induced HCs had similar electrophysiological characteristics to native HCs. Additionally, GPAS could induce HC regeneration in a mouse model with AAV-DTR-induced HC damage. The study highlights the potential of AAV-mediated gene regulation as a promising therapeutic strategy for sensorineural hearing loss. The results suggest that AAV-mediated co-regulation of multiple genes, such as GPAS, can promote the maturation and functional regeneration of HCs in the cochlea.