Type 1 diabetes (T1D) is characterized by the autoimmune-mediated destruction of the insulin producing ? cells of the islets of Langerhans. Our work and that of others in murine models has shown that transferring genes for immunoregulatory molecules to ? cells in vivo in the endogenous pancreas or in vitro in islet grafts, effectively suppresses autoimmunity and promotes islet transplantation tolerance, respectively. We have been using adeno-associated virus (AAV) vector gene transfer to manipulate the immunogenicity of murine ? cells in vivo and in vitro. AAV vector technology has rapidly advanced and clinical trials have demonstrated successful application of this gene delivery strategy. With this in mind, the goal of our R21 proposal is to develop AAV vectors with increased tropism for human ? cells in vivo and in vitro. A two-pronged approach will be taken. First, we will employ a panel of wild-type AAV capsid proteins to identify those capsids that efficiently transduce human ? cells in vitro, and in an in vivo model. Secondly, an effort will be made to engineer novel AAV capsid variants that selectively transduce human ? cells in vivo and in vitro, and which evade human AAV neutralizing antibodies. Here "DNA shuffling" of AAV capsid genes combined with directed evolution will be used to develop novel ? cell-specific AAV capsids. Experiments will also include testing the efficacy of AAV vector treatment to block immune-mediated destruction of human ? cells in vivo. Together, this work will provide a better understanding of the transduction properties of AAV capsids for human ? cells, and will identify/generate capsids that can be directly tested in the clinic. In this way, initial steps wil be taken towards our long-term goal of using AAV vectors to establish ? cell-specific and/or islet graft tolerance for human T1D.