Development of Human beta cell-specific AAV Vectors for Type I Diabetes

描述（由申请人提供）：1型糖尿病（T1D）以自身免疫介导的朗格汉斯胰岛胰岛素产生细胞的破坏为特征。我们和其他人在小鼠模型中的研究表明，将免疫调节分子基因转移到体内内源性胰腺细胞或体外胰岛移植物中，分别有效抑制自身免疫和促进胰岛移植耐受性。我们利用腺相关病毒（AAV）载体基因转移技术在体内和体外操纵小鼠细胞的免疫原性。AAV载体技术迅速发展，临床试验已经证明这种基因传递策略的成功应用。考虑到这一点，我们的R21提案的目标是开发在体内和体外对人类细胞具有更高趋向性的AAV载体。将采取双管齐下的办法。首先，我们将使用一组野生型AAV衣壳蛋白来鉴定那些在体外和体内模型中有效转导人类细胞的衣壳。其次，将努力设计新的AAV衣壳变体，在体内和体外选择性转导人类细胞，并逃避人类AAV中和抗体。在这里，AAV衣壳基因的“DNA洗牌”结合定向进化将用于开发新的细胞特异性AAV衣壳。实验还将包括测试AAV载体治疗在体内阻断免疫介导的人类细胞破坏的功效。总之，这项工作将更好地了解人类细胞中AAV衣壳的转导特性，并将识别/生成可直接在临床中测试的衣壳。通过这种方式，我们将朝着使用AAV载体建立人类T1D细胞特异性和/或胰岛移植耐受的长期目标迈出初步步骤。

DESCRIPTION (provided by applicant): 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.