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An ideal solution to the treatment or cure of type 1 diabetes mellitus would be the formation of new functioning ?-cells from the patient?s own tissues that are not attacked by the autoimmunity, thereby avoiding the need for any immunosuppression. Abundant recent data have suggested that ?-cells are a viable potential source for endogenous transdifferentiation into ?-cells. Here, we describe a pancreatic intraductal viral delivery system in the mouse, wherein a single infusion of an adeno-associated virus (AAV), carrying a pdx1/mafA expression vector, is given to a toxin-induced (alloxan) diabetic mouse. This AAV gene therapy induced robust and durable ?-cell transdifferentiation into ?-cell-like cells through neogenesis, with recovery of over 60% of the ?-cell mass within 4 weeks, and with persistent, durable euglycemia. Serendipitously, when this ?-cell-like cell neogenesis was similarly induced in the autoimmune NOD mouse model, the mice became euglycemic for 4 months or more, without any additional therapy or immunosuppression. To our knowledge, no clinically applicable ?-cell replacement therapy in NOD mice has been successful without immunosuppression. We suspect that the neogenic ?-cell-like cells may not be attacked by the autoimmunity because they are ?imperfect? ?-cells by RNA-seq analysis. Since pancreatic duct injection is routinely performed in humans as a relatively simple, non-surgical procedure, and since numerous viral gene therapy trials are currently ongoing for several diseases, we feel that our approach may be rapidly translatable to humans with type 1 diabetes mellitus. In this proposal, we will perform important proof-of-principle studies in non-human primates as last steps in preparation for human gene therapy clinical trials. The primate pancreas has a very different texture and consistency than the mouse pancreas (and is very similar to the human pancreas). Thus, the mechanics of the viral delivery will likely require substantial alterations. In addition, a glucagon promoter is preferable to the CMV promoter for expression of pdx1 and mafA, so we will strive to develop and optimize a glucagon promoter vector that is effective in primates. Further studies will investigate this pancreatic ductal infusion approach in the context of AAV neutralizing antibodies. We will also perform detailed analyses of the new ?-cell-like cells, including physiology, gene expression phenotype, and anatomy. In summary, we feel that the proposed studies, if successful, should position us well in preparation for clinical trials in humans with type 1 diabetes mellitus.
