During the last funding cycle, we developed a novel gene therapy for diabetes in streptozotocin (STZ) mice by induced islet neogenesis in the liver. We used the powerful helper-dependent adenovirus (HDAd) to first deliver Pdx1, which induced both endocrine and exocrine pancreas development in the liver, leading to fulminant hepatitis. Delivery of NeuroD/Beta 2 (ND/beta2), in the presence of an islet growth factor, betacellulin (Btc), led to islet neogenesis and reversed the diabetes. The liver islets induced by ND/a2 produced all 4 major islet hormones, insulin, glucagon, somatostatin and pancreatic polypeptide. ND/a2 gene transfer activated both downstream and upstream transcription factors. In this application, we will pursue these observations, trace the origin of the newly formed islets, and develop parallel gene therapy regimens that will shed light on the mechanism of islet neogenesis and possibly lead to even better regimens for diabetes. The specific aims are: (1) (a) To characterize the long-term response of STZ mice to HDAd therapy using (i) ND/beta2, (ii) ND/beta2+Btc, (iii) Pdx1`+Btc, and (iv) Pdx1+ND/beta2+Btc. (b) To test the effect of reinstatement of transgene expression by repeated administration of HDAd in the two ND/beta2 protocols. There are 3 possible long-term outcomes, (i) neogenesis that lasts as long as the vector-derived transcription factor(s) is expressed, (ii) permanent islet neogenesis persisting after disappearance of transgene expression, and (iii) return of pancreatic islets after 6 or more months with removal of hyperglycemia. These outcomes address important issues in islet biology and gene therapy. (2) To (i) isolate the insulin-producing cells in the liver induced by ND/beta2+/-Btc by flow cytometry and study the purified cell populations in vitro, and (ii) study the cellular origin of the newly formed insulin-positive cells following NDbeta2+/-Btc treatment by bone marrow and stem cell transplantation. (3) To test the effect of HDAd-mediated transfer of the Neurogenin3 gene (ngn3) and (4) of Pax4 to STZ mice. The in-vivo islet neogenesis response of transcription factors that are upstream and downstream to ND/a2 may reveal alternate pathways and feedback loops in the islet transcription network. The development of powerful HDAds in our laboratory will help dissect the lifetime in vivo effect. Finally, the best regimens identified by the experiments proposed are strong candidates for clinical trials in the future.