Optimization of AAV vector to deliver FVa for hemophilia with inhibitors

摘要 腺相关病毒（AAV）载体的基因治疗已成功应用于血友病的治疗 患者然而，具有抑制物（抗凝血因子抗体）的患者被排除在这些之外。 审判也已经探索了AAV载体用于在临床前递送旁路产物FVIIa转基因， 动物模型虽然实现了止血的长期改善，但完全表型 没有观察到校正。在凝血级联过程中，FV（FVa）作为FXa的辅因子起作用， 扩增凝血酶生成。我们开创了一项研究，其中FVa由肝脏特异性启动子驱动， 构建并包装成AAV病毒体。将AAV/FVa载体注射入血友病小鼠后， 在28周内实现了完全的表型矫正，没有明显的并发症。在这一提议中， 我们将探索一种有效的策略，使用AAV载体提供旁路产品，治疗 血友病与抑制剂。首先，我们将通过利用不同的肝细胞启动子来优化FVa构建体 以及FV重链和轻链之间的接头序列的修饰（Aim 1）。接下来我们就 探索AAV/FVa和AAV/FVIIa的组合是否对改善肿瘤细胞增殖具有协同作用。 血友病环境中的止血（目的2）。由于在小鼠实验中获得的结果往往不 翻译到大型动物模型，我们建议检查长期表型校正效果， 使用单独的AAV/FVa载体或与AAV/FVIIa组合的AAV/FVa载体在血友病狗中的应用（Aim 3）。总体而言，研究 本项目提出的方法为今后AAV载体介导的旁路产物基因转移奠定了基础 在血友病患者中进行抑制剂的临床试验。 !

ABSTRACT Gene therapy with adeno-associated virus (AAV) vectors have been successfully applied in hemophilia patients. However, the patients with inhibitors (antibodies against coagulation factors) are excluded from these trials. AAV vectors have also been explored for delivery of a bypass product, FVIIa transgene, in preclinical animal models. Although long-term improvement of hemostasis was achieved, the complete phenotypic correction was not observed. During the coagulation cascade, FV (FVa) functions as a co-factor of FXa to amplify thrombin generation. We pioneered a study in which FVa driven by a liver specific promoter was constructed and packaged into AAV virions. After injection of AAV/FVa vectors into hemophilic mice, completely phenotypic correction was achieved over 28 weeks without obvious complications. In this proposal, we will explore an effective strategy using AAV vectors to deliver bypass products in the treatment of hemophilia with inhibitors. First, we will optimize FVa constructs by utilization of different hepatocyte promoters and modification of linker sequences between the FV heavy chain and light chain (Aim 1). Next, we will explore whether the combination of AAV/FVa and AAV/FVIIa has a synergistic effect on the improvement of hemostasis in hemophilic settings (Aim 2). Since the results obtained in mice experiments often do not translate to large animal models, we propose to examine the long term phenotypic correction effect in hemophilic dogs using AAV/FVa vector alone or in combination with AAV/FVIIa (Aim 3). Overall, the studies proposed in the project will establish the basis for AAV vector-mediated bypass product gene transfer in future clinical trials in hemophilia patients with inhibitors. !