Project Summary/Abstract The goal of this project is to investigate whether the loss of key supporting cell fate genes will enhance the reprogramming and regeneration of cochlear cells into hair cells as a strategy for hearing restoration. We have found that supporting cells of the mature cochlea can be reprogrammed into hair cell-like cells following combined damage AND reprogramming with Atoh1+Gfi1+Pou4f3. We have also found that non-sensory cells of the inner and outer sulcus can be reprogrammed with Atoh1+Gfi1+Pou4f3 and provide a second potential population of cells for hearing restoration. Here, we are targeting two Sox-family genes that regulate hair cell fate but are expressed in supporting cells and other non-sensory cells in the mature cochlea. Sox-family members Sox2 and Sox10 are both linked to hair cell fate decisions as they each produce ectopic hair cells when one allele is lost. Supporting cells nearest to the inner hair cells appear to be particularly susceptible to the combined loss of both Sox2 AND Sox10 in the production of ectopic inner hair cells. Moreover, preliminary data suggests that reprogrammed hair cell-like cells do not turn off Sox2 or Sox10 possibly limiting their complete fate conversion. Remarkably, the loss of one allele of Sox2 increases the number of regenerated hair cells suggesting that it is preventing hair cell fate in these cells. Here, we hypothesize that supporting cell network genes, Sox2 and Sox10, are restricting the cell’s ability to turn into a hair cell following reprograming. To test this hypothesis, we will use genetically modified mouse models to reprogram and regenerate cochlear cells into hair cell-like cells. In addition to reprogramming we will: i) completely delete Sox2 from cochlear supporting cells in the organ of Corti, ii) delete one copy of Sox10 from non-sensory cells of the inner and outer sulcus, and iii) delete one copy of Sox2 PLUS one copy of Sox10 from supporting cells in the organ of Corti. Following each of these genetic manipulations, we will investigate whether the number or the maturity of hair cell-like cells has improved. We anticipate that our studies will reveal a unique role for supporting cell network genes as a target for hearing restoration therapies.