The conceptual framework of this project wrestles with a persistent challenge facing the HRP consortium: We must verify that the candidate genes we advance as regenerative genes actually perform as advertised. Is our altering of the gene expression of a candidate gene truly the trigger that turns supporting cells into hair cells? Our solution is to devise a mammalian model system that meets several definitive criteria. First, we need deafened adult mammalian inner ears to detect the production of new hair cells; we achieve this genetically by specifically killing hair cells that are uniquely sensitive to a bacterial toxin. Second, we need a way to turn on or turn off the candidate gene after the hair cells are dead; we achieve this by chemically activating a gene that in turn unmasks the expression of the proposed candidate. Third, we need a way to detect newly produced hair cells in the cochlea; we achieve this by using a tissue clearing and staining procedure developed with HRP funding that allows us to detect hair cells produced from supporting cells.

This entire approach is called a model system for validating candidate genes for hair cell regeneration. But one size does not fit all, and we need to continually adapt the core model system to achieve full functionality. In this proposal, we aim to test our model system in healthy ears to see if tweaking our candidate genes can produce hair cells from supporting cells in the absence of widespread hair cell death. The idea here is to make sure that the bacterial toxin–mediated destruction of hair cells is not interfering with our candidate gene activity and new hair cell production. Our second goal is to test a new virus delivery system that will allow us to evaluate larger candidate genes. Presently, we can only express very small genes with the virus we are using, restricting candidate gene verification. Our final goal is to evaluate a modified viral vector that is encased in lipid membranes to learn if it can express candidate genes more efficiently in supporting cells. The benefit of this approach is that viral production is quick, inexpensive, and requires no special training or expertise. Successful completion of this proposal will establish a comprehensive, cost-effective approach to aggressively validate candidate genes for hair cell regeneration.