Immunology of Factor IX Gene Transfer to Liver

描述：血友病B是一种X连锁出血性疾病， 功能性凝血因子IX（F.IX）。动物临床前研究 模型显示腺相关病毒介导的基因转移 (AAV)载体导致F.IX的持续表达和F. IX的部分校正。 凝血缺陷已经进行了I期临床试验， 在向患有严重血友病B的患者肌内施用载体时， 以及肝脏定向基因转移的I期试验（通过输注载体 进入肝循环）现已获得批准。目前，最严重的 通过基于蛋白质的疗法治疗血友病的并发症是 形成抗凝血因子的抑制性抗体。使用鼠 和犬模型，我们已经证明了持续的F.IX表达与 在F.IX错义突变的背景下，肌肉定向方法，而 在F.IX基因缺失/无效突变的情况下， 抑制剂形成然而，在同一品系的动物中， 已经使用以下方法实现了无抑制剂形成的表达： 肝脏定向基因治疗因此，基因转移的免疫学结果 取决于载体和靶组织的组合。我们发现 腺相关病毒介导的基因转移到肝脏可以诱导免疫反应， 对F.IX无反应，这可以通过耐受性或 抑制机制抗F.IX的形成依赖于CD 4 + T辅助细胞。 因此，我们提出了一种基于转基因小鼠的基因转移模型， 卵清蛋白CD 4 β-限制性T细胞受体，以确定事件 导致抗原特异性免疫或在AAV介导的免疫后无应答性， 分泌蛋白的基因转移。我们将研究潜在的机制 耐受诱导（克隆缺失，T细胞无反应性）或抑制/免疫 肝脏基因转移的偏离（例如，通过激活调节细胞）， 与中和抗体应答相关的T细胞引发相反， 注射肌肉的淋巴结。对于卵清蛋白和F.IX系统，我们 将进行过继淋巴细胞转移实验， 和肝脏定向基因转移的抑制机制。在最近 产生表达人F.IX的肝衍生变体的转基因小鼠， 肝脏定向基因治疗中抑制物形成的风险可以直接 与其他治疗方式相比， 血友病B小鼠在适当的遗传背景。最后一个风险是 抑制剂的形成可以通过肝定向的 基因转移和瞬时免疫调节。综合来看，这些研究 将提供转基因产物特异性T细胞应答的详细分析 在AAV介导的肝基因转移之后。

DESCRIPTION: Hemophilia B is the X-linked bleeding disorder caused by absence of functional coagulation factor IX (F.IX). Pre-clinical studies in animal models have shown that gene transfer mediated by an adeno-associated viral (AAV) vector results in sustained expression of F.IX and partial correction of the coagulation deficiency. A Phase I clinical trial has been carried out based on intramuscular administration of vector to patients with severe hemophilia B, and a Phase I trial for liver-directed gene transfer (by infusion of the vector into the hepatic circulation) is now approved. Currently, the most serious complication of treatment for hemophilia by protein-based therapy is the formation of inhibitory antibodies against the coagulation factor. Using murine and canine models, we have demonstrated sustained F.IX expression with the muscle-directed approach in the context of a F.IX missense mutation, while expression in the context of a F.IX gene deletion/null mutation was limited by inhibitor formation. However, in animals of the same strain, sustained expression without inhibitor formation has been accomplished using liver-directed gene therapy. Thus, the immunological outcome of gene transfer is dependent on the combination of vector and target tissue. We found that AAV-mediated gene transfer to the liver can induce immunological unresponsiveness to F.IX, which may be explained by either a tolerance or a suppression mechanism. Anti-F.IX formation is dependent on CD4+ T helper cells. Therefore, we are proposing a gene transfer model based on mice transgenic for an ovalbumin CD4 about-restricted T cell receptor in order to define the events leading to antigen-specific immunity or unresponsiveness after AAV-mediated gene transfer of a secreted protein. We will investigate potential mechanisms of tolerance induction (clonal deletion, T cell anergy) or suppression/immune deviation (e.g. by activation of regulatory cells) in hepatic gene transfer as opposed to T cell priming associated with a neutralizing antibody response in lymph nodes of injected muscle. For both the ovalbumin and the F.IX system, we will perform adoptive lymphocyte transfer experiments to distinguish tolerance and suppression mechanisms in liver-directed gene transfer. In recently generated transgenic mice expressing liver-derived variants of human F.IX, the risk of inhibitor formation in liver-directed gene therapy can be directly compared to other treatment modalities after mice have been crossed with hemophilia B mice on the appropriate genetic background. Finally, the risk of inhibitor formation may be further reduced by a combination of liver-directed gene transfer and transient immune modulation. Taken together, these studies will provide a detailed analysis of transgene product-specific T cell responses following AAV-mediated hepatic gene transfer.