Abstract: Sensorineural hearing loss affects 1.5 billion people worldwide, with the primary pathology being the irreversible loss of cochlear hair cells and supporting cells. Although hearing aids and cochlear implants can improve hearing, we currently lack the ability to reverse the underlying pathology of hearing loss-hair cell and supporting cell loss. Recent studies found that defined transcription factors can reprogram endogenous cochlear supporting cells to directly acquire a hair cell fate, however, the hair cells formed are limited both in number and degree of maturation. Moreover, this non-mitotic approach, coined direct transdifferentiation, leads to a loss of the overall supporting cell population. Thus, a better understanding of 1) mitotic regeneration of supporting cells and 2) how regenerated hair cells in the cochlea mature is critical. In this proposal, we will test whether singular or combinatorial application of transcription factors can replenish hair cells and supporting cells in the immature and mature mouse cochlea. In preliminary and recently published data using transgenic mouse models, we found that 1) greater epithelial ridge (GER) cells, instead of being a transient structure during development, migrate into the organ of Corti to regenerate lost supporting cells and mature to become the supporting cell subtype inner phalangeal cells, which are critical for the survival of inner hair cells, 2) damage induces GER cells to robustly proliferate and upregulate transcription factors associated with proliferation, 3) Atoh1 overexpression robustly induces new hair cell formation in the GER, which mature to become inner hair cell- and outer hair cell-like cells. The first aim will test the hypothesis that damage-responsive transcription factors promote mitotic regeneration in the neonatal and damaged mature cochlea. In the second aim, we will use regenerated hair cells in the GER as a model system to characterize the spatiotemporal features by which regenerated hair cells mature and then test whether the outer hair cell factor Ikzf2 enhances an outer hair cell fate. Moreover, we will examine the ability of combination of hair cell transcription factors to induce hair cell regeneration and maturation in the damaged mature cochlea. To gain an unbiased insight into the genetic signature of ectopic supporting cells and hair cells, the third aim will probe the transcriptomes of GER-derived hair cells and supporting cells. We will reveal their genetic landscape using bioinformatic approaches to define genes marking progenitors and regenerated supporting cells and hair cells and candidate genes driving regeneration. In summary, we will apply state-of-the-art technologies (live cell imaging, electrophysiology, snRNA-seq, inner ear surgery, viral transduction) to study the mechanisms of supporting cell and hair cell regeneration. We have assembled a team of experts who have worked together to collect promising preliminary data. At the...