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

安全

• 批准号: 6959245
• 负责人: Katherine A High
• 金额: $ 32.22万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6959245
• 关键词: coagulation factor IX; disease /disorder proneness /risk; dogs; dosage; gene deletion mutation; gene delivery system; gene expression; gene mutation; hemophilia B; immune response; immune tolerance /unresponsiveness; immunogenetics; immunosuppression; isolation perfusion; perfusion; protein biosynthesis; protein structure function; serotyping; striated muscles; transfection /expression vector

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.

项目2的目标是研究用于将编码因子IX（F.IX）的AAV载体递送至骨骼肌的区域血管内方法的安全性和有效性。两个主要的安全性问题进行检查，包括免疫反应的转基因产品，因子IX，和生殖系传播的载体DMA使用这种交付方法的风险。在之前的资助期，我们探索了在患有重度血友病B的动物和患者中直接肌内注射AAV-F.IX的安全性和有效性。虽然这种方法在人体中测试的所有剂量下都是安全的（高达2 x 10[12] vg/kg），但达到治疗剂量（1 x 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，我们在血友病B的大型动物模型中探索了将大剂量载体递送至骨骼肌的替代方法。我们已经使用涉及通过股动脉动脉内递送载体的技术在血友病B狗中显示了4-14%水平的长期表达，并且还显示了使用lacZ转基因用于基于载体递送至远端静脉的第二种技术的功效。然而，需要环磷酰胺的短暂免疫抑制来防止第一种技术的抑制剂形成。在本申请中，我们将确定第二种血管内递送技术是否也需要瞬时免疫抑制，并将表征以下参数对因子IX的免疫应答的影响： （隔离肢体灌注对比顺行灌注）;载体递送时免疫抑制的存在或不存在; AAV血清型;载体剂量;和因子IX基因中的潜在突变，其决定对转基因产物的免疫耐受程度。这些研究必须在F.IX基因缺陷的动物中进行，但不能在小鼠中进行，因为它们太小而无法进行传递过程。犬免疫学的最新进展现在可以在这个物种中进行详细的免疫学研究。第二个目标将集中在检查生殖系传播的风险作为血清型，剂量和交付方法的函数。在第三个目标中，我们将利用犬肌肉中高水平的F.IX表达来确定可在骨骼肌中合成的全功能因子IX蛋白的上限，并以生物化学方式表征肌肉合成的F.IX。