In Vivo and Ex Vivo Utricles treated with ATOH1 under different promoters

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. With the aim of developing an optimized AAV-based ATOH1 gene therapy, we used single-cell RNA-Seq and explored the impact of defined promoters on ATOH1 transgene expression level, timing, effects in distinct cell types, and the maturity of regenerated vestibular hair cells ex vivo and in vivo. Overall design: Gentamicin-damaged utricle explants were transduced with AAV8-CMV-ATOH1, AAV8-GFAP-ATOH1, or AAV8-RLBP1-ATOH1 and then were maintained in culture for 7, 12, or 20 days before being processed for 10X singlecell RNA-Seq. In addition, in vivo utricles and cristae were collected from IDPN-damaged mice treated with AAV8-GFAP-ATOH1-H2B-EGFP (IDPN and GFAP-ATOH1), IDPN damaged mice (IDPN), or naive mice (Control) at 2 weeks and 4 weeks post-treatment (4 weeks and 6 weeks after IDPN administration).