PROJECT SUMMARY Bladder reconstruction (BR) is essential to restore urinary function in patients with neurogenic bladder, congenital disorders, and as sequalae to the surgical treatment of bladder or pelvic malignancies. The current standard of care is to use the patient's own intestinal tissue (ileal segment; IS) as graft material during BR to create a neobladder or urinary diversion. While IS grafts are non-immunogenic and readily available, post- surgical persistence of intestinal cells in the graft impedes regeneration into bladder wall, resulting in stone formation, metabolic acidosis and risk of secondary cancers. Our objective is to develop new technology for intrasurgical tissue engineering of the IS by knockdown of cellular components using irreversible electroporation (IRE) and identify factors fundamental for regeneration of functional bladder wall. IRE is used in patients for tumor ablation by inducing cell death with ultrashort electric pulses. Our proposed strategy builds upon our preliminary data showing (i) IRE can knockdown intestinal cells in an IS graft, aiding repopulation with urothelium in a rat model of BR, (ii) feasibility of new pulse application strategies for the focal knockdown of mucosa, or decellularization while preserving vasculature and ECM in the IS, and (iii) phenotypic changes in IRE treated IS following urothelialization, that were not observed in sham controls under physiologic conditions of bladder filling and voiding. In specific aim 1, we will Define the impact of graft perfusion on bladder wall regeneration by performing vasculature sparing IRE of the IS. In specific aim 2, Elucidate the role of IS mucosa in post-BR complications by knockdown with IRE. In specific aim 3, Investigate the role of mechanotransduction in bladder function development in IRE treated IS. Intrasurgical creation of a perfused, histocompatible graft (Aim 1) and focal decellularization of the mucosa while sparing the underlying layers in the IS (Aim 2) are the first examples of in vivo knockdown tissue engineering using IRE. The study and application of mechanotransduction principles to augment urinary barrier function development in IS grafts (Aim 3) is previously undescribed. Knowledge gained from proposed research will yield a simple intrasurgical technique (mt-IRE or vs-IRE) that combines technology (IRE) and grafting technique (with IS) that are already in the clinic, enabling rapid translation for the immediate benefit of patients undergoing BR. Eventually, we anticipate our work to advance the concept of in vivo production of functionalized grafts using the patient as the source of biomaterial and the bioreactor, with application to reconstructive surgery involving other tubular organs such as the esophagus, trachea or large blood vessels.