IMMUNE

治疗性蛋白质的基因转移是治疗遗传性疾病的一种有前途的方法。腺相关 病毒（AAV）介导的凝血因子IX（F.IX）基因体内转移至小鼠的肌肉或肝脏， 目前正在临床试验中评价重度血友病B受试者。治疗的主要问题 是诱导CDS* 细胞毒性T淋巴细胞应答（CTL），其可引起转导的 表达转基因产物的细胞（肌纤维或肝细胞）。有趣的是，AAV基因转移 已经显示不引起对许多转基因产物的CTL应答，这可能是由于免疫应答导致的。 宽容或无知。我们将确定对人F.IX转基因的耐受性诱导的要求 IX基因转移，随后用腺病毒载体（a. 用于诱导CTL的有效载体）表达相同的转基因。CTL应答将通过 靶细胞的抗原特异性裂解和转导组织的免疫化学分析。我们将定义 耐受性诱导的要求（靶器官、转基因表达水平、系统性蛋白质表达水平） 交付）。我们假设AAV介导的肝基因转移导致系统性F.IX蛋白表达， 递送可导致免疫耐受，从而防止对F. IX的CTL应答。而且我们 假设免疫显性F.IX T细胞表位内源性表达有助于耐受性 从而降低CTL应答的风险。潜在突变的影响， 将在一系列表达非功能性F.IX的转基因小鼠中测试表达的F.IX对CTL应答的影响。 从血友病B数据库获得的人F.IX的变体。最后，提出了一系列的实验， 鉴定可抑制对F. IX的CTL应答的发展的调节性T细胞亚群。这些 研究是基于肝AAV-F.IX基因转移诱导调节性CD 4 + T细胞 能够抑制CTL转化为F. IX。这些调节细胞的详细特征和 他们的诱导要求将导致改进的基因转移方案，并将是一个实验性的 增强调节性T细胞应答的方法的基础。更好地理解细胞 免疫应答的潜在机制或免疫应答的缺乏应该为免疫应答的设计提供基础。 防止接受病毒基因转移的细胞被破坏的策略。

Gene transfer of a therapeutic protein is a promising approach toward treatment of genetic disease. Adenoassociated viral (AAV) mediated in vivo transfer of a coagulation factor IX (F.IX) gene to muscle or liver of subjects with severe hemophilia B is currently being evaluated in clinical trials. A major concern of treatment is induction of CDS* cytotoxic T lymphocyte responses (CTL), which may cause elimination of transduced cells (muscle fibers or hepatocytes) expressing the transgene product. Interestingly, AAV gene transfer has been shown not to cause CTL responses to a number of transgene products, which may be due to immune tolerance or ignorance. We will determine requirements for tolerance induction to a human F.IX transgene product in mice, which will receive AAV-F.IX gene transfer followed by challenge with an adenoviral vector (a potent vector for induction of CTL) expressing the identical transgene. CTL responses will be measured by antigen-specific lysis of target cells and immunochemical analyses of transduced tissues. We will define requirements for tolerance induction (target organ, levels of transgene expression, systemic protein delivery). We hypothesize that AAV-mediated hepatic gene transfer resulting in systemic F.IX protein delivery can lead to immune tolerance thereby preventing CTL responses to F.IX. Furthermore, we hypothesize that endogenous expression of immunodominant F.IX T cell epitopes contributes to tolerance thereby reducing the risk of CTL responses. The effect of the underlying mutation in endogenously expressed F.IX on CTL responses will be tested in a series of transgenic mice expressing non-functional variants of human F.IX taken from the hemophilia B database. Finally, a series of experiments is; proposed to identify subsets of regulatory T cells that can suppress development of CTL responses to F.IX. These studies are based on the hypothesis that hepatic AAV-F.IX gene transfer induces regulatory CD4+ T cells capable of suppressing CTL to F.IX. A detailed characterization of these regulatory cells and the requirements for their induction will lead to improved gene transfer protocols and will be an experimental basis for approaches to augment regulatory T cell responses. Improved understanding of cellular mechanisms underlying immune responses or absence thereof should provide the basis for design of strategies that preclude destruction of cells receiving viral gene transfer.