Gene Therapy for Hemophilia Using Muscle-Expressed FVIIa

工程项目2简介 血友病是一种遗传性出血性疾病，目前正在通过蛋白质替代疗法进行治疗，这种疗法提供了 只是暂时的纠正，由于需要频繁输液，这是一个重大的财政负担。对于那些 随着注意力的集中，疾病管理中最严重的并发症是 在血友病A患者中，高达30%的患者会出现针对输注因子的中和抗体。 对于这类患者，通过输注大剂量重组激活因子V11(RFV11a)来控制出血会导致 有效的镇静作用。然而，由于蛋白质的半衰期很短，这种治疗方法的成本很高。 以及重复给药的必要性。为了解决这些缺陷，我们以前开发了一种新的基因 一种转移方法，通过这种方法，我们可以使用FVII转基因来止血，这种转基因被设计成在 活性，双链形式(FVIIa)。以肝脏为靶组织，我们证明了这种持续表达 腺相关病毒(AAV)基因转移后的FV11a可纠正血友病的表型缺陷 老鼠和狗。这种方法具有比因子IX(FIX)或因子V11(FV11)基因更好的免疫学优势 由于接受者对转基因产品具有耐受性，因此应采取相应的策略。此外，它还能起到止血作用，即使在 面对FVIII或FIX的中和抗体。此外，由于rFV11a用于止血管理 其他凝血缺陷(如血小板紊乱)，与基于FVIII/FIX基因的方法相反，FVIIa 基因转移可以将几种凝血缺陷的治疗统一到一种产品上。然而，a 相当大比例的血友病患者由于病毒性肝炎而患有晚期肝病，因此呈现 肝脏是不适合病毒载体介导的基因转移的组织.我们开发了一种换能器 通过表达生物活性的犬的AAV载体广泛的肌肉区域固定和展示了多个 血友病B犬一年的止血效果。因此，作为这个项目的主题，我们建议使用 以骨骼肌作为FV11a表达和分泌的靶组织，建立犬FV11a的动物模型(S 血友病与人类相似的血友病。首先，我们希望进行一项系统的研究 启动子和AAV血清型对FV11a肌肉表达的影响，并研究其止血效果和安全性 犬FVlla(CFVlla)在最佳条件下的表达，包括使用抑制剂的狗(目标1)。在……里面 在接下来的实验中，我们将确定一种活性增强的cFV11a变体的治疗指数， 作为一种手段来降低每细胞的有效载体剂量/病毒载量，从而解决任何潜在的限制 肌肉进行cFVlla生物活性所需的翻译后修饰，因为增加了 抗原表达(目标2)。最后，我们将开发一个肌肉cFV11a表达的调控系统，通过一个 可提高FVIIa基因转移安全性的小型口服生物利用药(多西环素)(目标3)。 总体而言，项目中提出的实验将全面建立临床前 肌源性FV11a基因转移的基础。

Description of Project 2 Hemophilia, an inherited bleeding disorder, is currently treated by protein replacement therapy that provides only transient correction and is a significant financial burden due to the need for frequent infusions. For those with access to concentrate, the most serious complication in the management of the disorder is the development of neutralizing antibodies to the infused factor that occur in up to 30% of those with hemophilia A. For such patients, control of bleeds by infusion of high-dose recombinant activated Factor Vll (rFVlla) results in effective liemostasis. However, this treatment carries a substantial cost due to the short half-life of the protein and the need for repeated dosing. To address these shortcomings we previously developed a novel gene transfer approach whereby we can effect hemostasis using a FVII transgene, engineered to be secreted in the active, two chain form (FVIIa). Using the liver as the target tissue, we demonstrated that continuous expression of FVlla following adeno-associated virus (AAV) gene transfer can correct the phenotypic defect in hemophilic mice and dogs. This approach has immunological advantages over Factor IX (FIX) or Factor Vlll (FVlll) gene strategies since the recipient is tolerant to the transgene product. Moreover it can effect hemostasis even in the face of neutralizing antibodies to FVIII or FIX. Additionally, since rFVlla is used for hemostasis management for other coagulation defects (such a platelet disorders), in contrast to FVIII/FIX gene-based approaches, FVIIa gene transfer may unify the treatment of several coagulation defects to a single product. However, a substantial proportion of hemophilia patients have advanced liver disease due to viral hepatitis, thus rendering the liver an unsuitable tissue for viral vector-mediated gene transfer. We developed a method for transducing extensive areas of muscle by an AAV vector expressing biologically active canine FIX and demonstrated multi- year hemostatic efficacy in hemophilia B dogs. Therefore, as a theme for this project, we propose to use skeletal muscle as the target tissue for expression and secretion of FVlla, using the canine model(s) of hemophilia that closely mimic the human condition. Initially, we wish to perform a systematic study of promoters and AAV serotypes for muscle expression of FVlla, and study the hemostatic efficacy and safety of canine FVlla (cFVlla) expression under optimal conditions, including dogs with inhibitors (Aim 1). In subsequent experiments, we will determine the therapeutic index of a variant of cFVlla with increased activity, as a means to reduce the effective vector dose/viral load per cell and thus address any potential limitations of muscle to perform post-translational modifications necessary for cFVlla biological activity, due to increased antigen expression (Aim 2). Lastly, we will develop a regulated system for cFVlla expression from muscle via a small, orally bioavailable drug (doxycycline) that will enhance the safety of FVIIa gene transfer (Aim 3). Overall, the experiments proposed in the project will establish in a comprehensive manner the pre-clinical basis for muscle-derived FVlla gene transfer.