Precise genetic control of ATOH1 enhances maturation of regenerated hair cells in the mature mouse utricle

Abstract Vestibular hair cells are mechanoreceptors critical for detecting head position and motion. In mammals, hair cell loss causes vestibular dysfunction as spontaneous regeneration is nearly absent. Constitutive expression of exogenous ATOH1, a hair cell transcription factor, increases regeneration of hair cells, but these cells fail to mature. To mimic native hair cells which downregulate endogenous ATOH1 as they mature, we engineered viral vectors carrying the supporting cell promoters GFAP and RLBP1. In utricles damaged ex vivo , both CMV-ATOH1 and GFAP-ATOH1 increased regeneration more effectively than RLBP1-ATOH1, while GFAP-ATOH1 and RLBP1-ATOH1 induced hair cells exhibiting more mature transcriptomes. In utricles damaged in vivo , GFAP-ATOH1 induced regeneration of hair cells expressing genes representing maturing type II hair cells, and more hair cells with bundles and synapses than untreated organs. Together our results demonstrate the efficacy of spatiotemporal control of ATOH1 overexpression in inner ear regeneration.