Optimization of AAV vector to deliver FVa for hemophilia with inhibitors

摘要 腺相关病毒载体的基因治疗已成功应用于血友病 病人。然而，使用抑制物(抗凝血因子抗体)的患者被排除在这些范围之外。 审判。AAV载体也被探索用于在临床前传递旁路产物FVIIa转基因 动物模型。虽然止血取得了长期的改善，但完全表型 未观察到矫正。在凝血级联过程中，Fv(FVA)作为FXA的辅助因子发挥作用 放大凝血酶的生成。我们开创了一项研究，在这项研究中，由肝脏特异性启动子驱动的FVA 构建并包装成AAV病毒粒子。将AAV/FVA载体注射到血友病小鼠体内后， 完全表型矫正超过28周，没有明显的并发症。在这份提案中， 我们将探索一种有效的策略，使用AAV载体来提供旁路产品治疗 使用抑制剂的血友病。首先，我们将利用不同的肝细胞启动子来优化FVA的构建 以及FV重链和轻链之间的接头序列的修饰(目的1)。接下来，我们将 探讨AAV/FVA和AAV/FVIIa联合应用是否具有协同效应 血友病环境中的止血(目标2)。因为在小鼠实验中得到的结果通常不是 转化为大型动物模型，我们建议检查长期的表型校正效应 单独使用AAV/FVA载体或与AAV/FVIIa联合使用的血友病犬(目标3)。总体而言，这些研究 本项目的提出将为今后AAV载体介导的旁路产物基因转移奠定基础 血友病患者使用抑制剂的临床试验。 好了！

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. !