PROJECT SUMMARY My long-term goal is to help develop and deliver regeneration therapeutics useful for the clinical treatment of hearing loss. Unfortunately, at this time there is no high throughput, non-destructive method of therapeutic delivery into the cochlea/inner ear. The objectives of this proposal, the next step toward the attainment of this long-term goal, are to use a relevant large animal model (the pig) to i) develop an ex-vivo model system, which mimics human round window membrane (RWM), to track the passage of therapy-related substances through the RWM, and ii) create a method to enhance the intratympanic delivery of therapeutics into the inner ear using nanovesicles including extracellular vesicles (exosomes). The central hypothesis, supported by our preliminary data, is that an ex-vivo porcine RWM model can be used to determine optimal conditions for the transport of exosomes (or other nanovesicles) to deliver cargo through the RWM into the inner ear, thereby facilitating the high-efficiency delivery of therapeutics. The rationale for this proposal is that its successful completion is likely to offer a framework whereby a new pool of therapeutics can be tested for non-surgical delivery into the inner ear, in a large animal model that has the preclinical advantage over rodents in terms of size, physiology, and genetic similarity (amino-acid sequences of common deafness genes) to humans. The following specific aims will be pursued during K99 (aim 1 and 2) and R00 phase: Aim 1) To further validate the ex-vivo porcine RWM model to demonstrate drug permeability equivalent to that reported for human tissue; Aim 2) To identify and evaluate exosomes and other nanovesicles enhancing cargo transport across porcine RWM in-vitro; Aim 3) To evaluate in-vivo transport across porcine RWM by nanovesicles. Under the first aim, we will measure the permeability of therapeutics with known delivery efficiencies and some promising therapy related materials using a preliminary viability-verified ex-vivo RWM model and compare those with values reported for human tissue. For the second aim, nanovesicles including: RWM exosomes (successfully isolated and characterized as a preliminary result), mesenchymal stem cell exosomes (gold standard), and liposomes (FDA approved), that are loaded with promising therapeutics will be evaluated for the efficiency of transport using the ex-vivo RWM model. Finally, for the last aim, once the parameters associated with optimal transport through the RWM are established with the ex-vivo model, we will inject therapeutic-loaded nanovesicles through the porcine tympanic membrane and measure their passage across RWM and uptake by cochlear cells in vivo. Upon completion of the K99, the expected outcomes are 1. Availability of a safe and translatable platform to test transport of therapeutics into the inner ear, and 2. Data on the efficiency of nanovesicles as novel nonsurgical transport of promising therapeutics to the ear. These...