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入路将编码因子IX （F.IX）的AAV载体传递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，以及使用这种递送方法的载体DMA的种系传播风险。在之前的资助期内，我们探索了直接肌内注射AAV-F的安全性和有效性。尽管该方法在人体试验的所有剂量（高达2 × 10[13] vg/kg）下都被证明是安全的，但达到治疗剂量（1 × 10[13] vg/kg）所需的大量注射使得继续研究变得不切实际。因此，我们在大型血友病B动物模型中探索了将大剂量载体递送到骨骼肌的替代方法。我们已经在血友病B犬中使用经股动脉动脉内传递载体的技术显示了4-14%的长期表达水平，并且在基于远端静脉传递载体的第二种技术中使用lacZ转基因也显示了有效性。然而，在第一种技术中，需要环磷酰胺的短暂免疫抑制来防止抑制剂的形成。在本应用中，我们将确定是否还需要第二次血管内给药技术进行短暂免疫抑制，并将描述以下参数对因子IX免疫反应的影响：载体给药途径

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.