Gene Therapy for Hemophilia Using Muscle-Expressed FVIIa

使用肌肉表达的 FVIIa 进行血友病基因治疗

• 批准号: 8185314
• 负责人: Katherine A High
• 金额: $ 37.15万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8185314
• 关键词: Address; Adult; Animal Disease Models; Animal Model; Antigens; Area; Bioavailable; Biological; Blood Coagulation Disorders; Blood Coagulation Factor; Blood Platelet Disorders; Bypass; Canis familiaris; Cells; Chronic Hepatitis; Cleaved cell; Clinic; Coagulation Process; Complication; Data; Defect; Dependovirus; Development; Disease; Disease Management; Dose; Doxycycline; Engineering; Enhancers; Extravasation; FDA approved; Factor IX; Funding; Gene Transfer; Genes; Half-Life; Hemophilia A; Hemophilia B; Hemorrhage; Hemostatic Agents; Hemostatic function; Human; Immune response; In Vitro; Infusion procedures; Inherited; Inherited Blood Coagulation Disorders; Isolated limb perfusion; Kinetics; Knowledge; Liver; Liver diseases; Longevity; Mediating; Methods; Modeling; Mus; Muscle; Patients; Periderm; Pharmaceutical Preparations; Population; Post-Translational Protein Processing; Property; Proteins; Recombinants; Replacement Therapy; Safety; Series; Serotyping; Skeletal Muscle; System; Techniques; Testing; Therapeutic; Therapeutic Index; Thrombin; Tissues; Transgenes; Ursidae Family; Variant; Viral Load result; Viral Vector; Viral hepatitis; Vitamin K; adeno-associated viral vector; base; clinical application; cost; disease phenotype; enzyme replacement therapy; gene replacement; gene therapy; inhibitor/antagonist; innovation; muscular system; neutralizing antibody; novel; novel strategies; pre-clinical; preclinical study; promoter; research study; success; therapeutic transgene; vector

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.

项目描述2 血友病是一种遗传性出血性疾病，目前通过蛋白质替代疗法治疗， 仅仅是短暂的矫正，并且由于需要频繁的输注，这是一个巨大的经济负担。对于那些 在获得浓缩物的情况下，管理这种疾病最严重的并发症是 在高达30%的血友病A患者中出现针对输注因子的中和抗体。 对于这样的患者，通过输注高剂量重组活化因子VII I（rFVIIa）控制出血导致 有效止血。然而，由于蛋白质的半衰期短，这种治疗带来了相当大的成本 以及需要重复给药。为了解决这些缺点，我们以前开发了一种新的基因， 转移方法，由此我们可以使用FVII转基因实现止血，所述FVII转基因被工程化以在血管内皮细胞中分泌。 活性双链形式（FVIIa）。使用肝脏作为靶组织，我们证明了连续表达 腺相关病毒（AAV）基因转移后的FVIIa的表达可以纠正血友病患者的表型缺陷， 老鼠和狗该方法具有优于因子IX（FIX）或因子VIII（FVIII）基因的免疫学优势 由于受体对转基因产物具有耐受性，因此可以使用这些策略。此外，它甚至可以在 面对FVIII或FIX的中和抗体。此外，由于rFVIIa用于治疗的止血管理， 其他凝血缺陷（如血小板疾病），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为单一产品。但 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， 肝脏不适合病毒载体介导的基因转移。我们发明了一种方法 广泛的肌肉区域的AAV载体表达生物活性犬FIX，并证明了多- 血友病B犬的年止血有效性。因此，作为本项目的主题，我们建议使用 骨骼肌作为FVIIa表达和分泌的靶组织，使用犬模型， 非常类似人类的血友病首先，我们希望进行系统的研究， 启动子和AAV血清型用于FVIIa的肌肉表达，并研究 犬FVIIa（cFVIIa）在最佳条件下的表达，包括具有抑制剂的犬（Aim 1）。在 在随后的实验中，我们将确定具有增加的活性的cFVIIa变体的治疗指数， 作为减少每个细胞的有效载体剂量/病毒载量的手段， 肌肉进行cFVIIa生物活性所必需的翻译后修饰，由于增加的 抗原表达（目的2）。最后，我们将开发一个通过肌肉表达cFVlla的调节系统。 小，口服生物可利用的药物（强力霉素），将提高FVIIa基因转移的安全性（目标3）。 总体而言，该项目中提出的实验将全面建立临床前 肌源性FVIIa基因转移的基础。