Immunology of Factor IX Gene Transfer to Liver

描述：血友病B是由缺勤引起的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 模型表明，由腺相关病毒介导的基因转移 (AAV)载体导致F.IX的持续表达和部分纠正 凝血功能缺陷。已经进行了一期临床试验， 重型乙型血友病患者肌肉注射载体的研究 肝脏导向基因转移(通过输注载体)的I期试验 进入肝脏循环)现已获批。目前，最严重的 蛋白治疗血友病的并发症是 形成抑制凝血因子的抗体。用小鼠 和犬类模型，我们已经证明了F.IX的持续表达 在F.IX错义突变的背景下的肌肉定向方法，而 在F.IX基因缺失/零突变的背景下，表达受到以下限制 抑制剂的形成。然而，在相同品系的动物中，持续 在没有抑制物形成的情况下，表达已经使用 肝脏导向的基因治疗。因此，基因转移的免疫学结果 取决于载体和靶组织的结合。我们发现 AAV介导的肝内基因转移可诱导免疫性 对F.IX无反应，这可以用容忍或 抑制机制。抗F.IX抗体的形成依赖于CD4+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。 一种卵清蛋白CD4约限制性T细胞受体以定义事件 在AAV介导后导致抗原特异性免疫或无反应 一种分泌蛋白质的基因转移。我们将研究潜在的机制 耐受诱导(克隆性缺失、T细胞无能)或抑制/免疫 肝脏基因转移中的偏差(例如，通过激活调节细胞) 与与中和抗体反应相关的T细胞启动相反 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以区分耐受性 以及肝脏导向基因转移的抑制机制。在最近 产生了表达人FIX肝脏衍生变体的转基因小鼠， 肝脏导向基因治疗中抑制物形成的风险可以直接 与其他治疗方法相比，在小鼠与 血友病B小鼠在适当的遗传背景下。最后，风险是 抑制物的形成可通过肝脏导向的联合应用而进一步减少 基因转移和瞬时免疫调节。总而言之，这些研究 将提供转基因产物特异性T细胞反应的详细分析 在AAV介导的肝脏基因转移后。

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.