Severe vocal fold (VF) mucosal injury and its associated voice impairment (dysphonia) is a debilitating and challenging clinical problem with few treatment options, none of which are consistently effective. In patients with such tissue impairment or loss, an engineered VF mucosa would provide a biomechanically appropriate tissue graft for voice restoration. During our previous funding cycle, we engineered such a VF mucosa via organotypic culture of primary vocal fold fibroblasts and epithelial cells from human donors. In a series of preclinical experiments, the engineered mucosa recapitulated the structure and physiologic function of native human tissue. In the current application, we propose a natural extension of our prior work by advancing characterization of the in vitro tissue engineering process at the biological system level (Aim 1), studying remodeling and the adaptive immune response to the engineered mucosa following in vivo heterotopic implantation (Aim 2), and studying long-term physiologic and immunologic outcomes following in vivo orthotopic implantation (Aim 3). These experiments will generate important preclinical safety and efficacy data. We will combine the planned experimental work with regulatory and clinical manufacturing milestones (Aim 4), with a final goal of holding a pre-Investigational New Drug meeting with the FDA by the end of the grant. The tissue engineering technology outlined in this application has the potential to transform treatment of the most recalcitrant VF tissue disorders encountered in the clinic.