DESCRIPTION (provided by applicant): Congenital hearing loss is a common disorder, with approximately 1 out of every 600 children suffering from profound deafness. A central player in hearing is the protein otoferlin. More than 60 pathogenic mutations in otoferlin are known, and up to 8% of all forms of prelingual autosomal recessive hearing loss are due to otoferlin mutations. Currently the only known bodily function for otoferlin is in mediating neurotransmitter release from hair cells, making it a hearing specific protein. While otoferlin is essential for hearing, current approaches to study otoferlin in vivo have not elucidated the exact function of the protein or provided a molecular level explanation for mutations associated with deafness. Further, the large size of otoferlin has prevented rescue experiments and prohibited viral-based gene transfer, representing a major hurdle toward the use of gene therapy as a treatment. To screen for the effects of pathogenic mutations on otoferlin function, develop truncated forms of otoferlin that can be packaged into existing gene delivery vehicles, and determine the function of otoferlin, we will use zebrafish as a model system. A major advantage of using zebrafish as a model is the ability to easily transfect hair cells with mutant and truncated forms of otoferlin, something that cannot be achieved easily in a mouse model. This work builds off studies conducted under a K99/R00 award devoted to characterizing otoferlin using recombinant protein. Our development of zebrafish as a model for testing results of recombinant protein studies uniquely positions us to pursue a powerful two-pronged approach to probe and engineer otoferlin on both the molecular and organismal level. In specific aim 1 of this proposal we will engineer and test truncated forms of otoferlin to determine the minimal regions of the protein required for hearing. Determination of the minimal sequence of otoferlin capable of restoring hearing will be critical for the design of therapeutics, including the design of truncated forms of otoferlin small enough to be packaged for viral mediated gene therapy. Specific aim 2 will use zebrafish and recombinant proteins to study pathological missense mutations associated with deafness in human patients. The goal of this aim will be to establish the molecular basis for why certain otoferlin missense mutations result in hearing loss in humans. Specific aim 3 will characterize the biophysical properties of otoferlin, with the goal of determining the unique functional properties of otoferlin that are needed for hearing. The results of these studies will directly impact the development of therapeutics for treating deafness, explain the basis for several human pathological missense mutations, and establish the mechanisms otoferlin utilizes for the encoding of sound. The PI is well suited for the proposed work, having already established zebrafish as a model for otoferlin studies, and having carri...