Safety & Efficacy of Intravas. Del. of AAV-F.IX to Skeletal Muscle

项目2的目标是研究区域血管内途径将编码因子IX(F.IX)的AAV载体输送到骨骼肌的安全性和有效性。要检查的两个主要安全问题包括对转基因产品因子IX的免疫反应，以及使用这种传递方法的媒介DMA生殖系传播的风险。在之前的资助期间，我们探索了AAV-F.IX直接肌肉注射在动物和严重血友病B患者中的安全性和有效性。尽管这种方法在人类测试的所有剂量(高达2×10[12]Vg/kg)下被证明是安全的，但达到治疗剂量(1 x 10[13]Vg/kg)所需的大量注射使继续研究变得不切实际。因此，我们在血友病B的大型动物模型中探索了将大剂量载体输送到骨骼肌的替代方法。我们已经显示了血友病B犬的长期表达水平为4%-14%，使用了一种通过股动脉动脉内输送载体的技术，也显示了将LacZ转基因用于第二种基于载体输送到远端静脉的技术的有效性。然而，第一种技术需要用环磷酰胺进行短暂的免疫抑制，以防止抑制物的形成。在这项应用中，我们将确定第二种血管内给药技术是否也需要瞬时免疫抑制，并将表征以下参数中的因子IX对免疫反应的影响：载体的给药途径 (隔离肢体灌注与顺行灌注)；载体传递时免疫抑制的存在或不存在；AAV血清型；载体剂量；以及决定对转基因产品的免疫耐受度的因子IX基因的潜在突变。这些研究必须在FIX基因缺陷的动物身上进行，但不能在老鼠身上进行，因为它们太小了，不适合分娩过程。犬类免疫学的最新进展现在使对该物种的详细免疫学研究成为可能。第二个目标将集中在检查生殖系传播的风险作为血清型、剂量和给药方法的函数。在第三个目标中，我们将利用狗肌肉中F.IX的高水平表达来确定在骨骼肌中可以合成的全功能因子IX蛋白的上限，并从生物化学的角度表征肌肉合成的F.IX。

The goal of Project 2 is to investigate the safety and efficacy of regional intravascular approaches for the delivery of an AAV vector encoding Factor IX (F.IX) to skeletal muscle. Two major safety issues to be examined include immune response to the transgene product, Factor IX, and the risk of germline transmission of vector DMA using this delivery approach. In the previous funding period, we explored the safety and efficacy of direct intramscular injection of AAV-F.IX in animals and patients with severe hemophilia B. Although this approach proved safe at all doses tested in humans (up to 2 x 10[12] vg/kg), the large number of injections required to reach a therapeutic dose (1 x 10[13] vg/kg) made continuation of the study impractical. We therefore explored in a large animal model of hemophilia B alternative methods for delivering large doses of vector to skeletal muscle. We have shown long-term expression at levels of 4-14% in hemophilia B dogs using a technique involving intra-arterial delivery of vector via the femoral artery, and have also shown efficacy using a lacZ transgene for a second technique based on vector delivery to a distal vein. However, transient immunosuppression with cyclophosphamide was required to prevent inhibitor formation with the first technique. In this application, we will determine whether transient immunosuppression is also required with the second intravascular delivery technique and will characterize the effect on the immune response to Factor IX of the following parameters: route of administration of vector (isolated limb perfusion vs. anterograde perfusion); presence or absence of immunosuppression at time of vector delivery; AAV serotype; vector dose; and underlying mutation in the Factor IX gene, which determines the degree of immunologic tolerance to the transgene product. These studies must be carried out in an animal genetically deficient in F.IX, but cannot be done in mice, as they are too small for the delivery procedure. Recent advances in canine immunology now enable detailed immunologic studies in this species. A second aim will be focused on examining the risk of germline transmission as a function of serotype, dose, and delivery method. In the third aim we will take advantage of the high levels of F.IX expression in dog muscle to determine the upper limit of fully functional Factor IX protein that can be synthesized in skeletal muscle and to characterize muscle-syntheszied F.IX biochemically.