AAV mediated muscle directed gene therapy for Hemophilia B

血友病B是一种因凝血因子IX缺乏而导致的严重出血素质。目前这种疾病的治疗方法是预防性地或在出血时输注凝血因子浓缩物。这种治疗方法是次优的，原因有很多，部分原因是蛋白质在循环中的半衰期相对较短。通过基因疗法治疗血友病的能力将代表着这种疾病治疗的相当大的进步，因为它将确保患者持续保持足以防止大多数自发性出血的凝血因子水平。这一应用的基本假设是，AAV介导的、肌肉定向的凝血因子IX基因转移可以实现这一治疗目标。之前在小鼠和血友病狗身上的研究支持这一假设，尽管在狗身上实现的表达水平处于治疗范围的下限。因此，要以安全有效的方式将这些有希望的临床前结果成功地转化为大量临床人群，将需要对载体的生物学和肌肉中因子IX的生物合成有更详细的了解。本申请中概述的具体目标建立在上一个资助期已完成的工作的基础上，并包括1)确定限制肌肉中生物活性F.IX表达的翻译后修饰，并确定是否可以通过提供限制酶(S)的反式限制酶来增强F.IX在肌肉中的表达；2)确定导入肌肉中的载体DNA是以高分子量Episome形式存在还是整合到染色体DNA中；以及3)确定在肌肉内注射重组AAV载体后，引入肌肉特异性启动子/增强子元件是否可以导致F.IX的高水平表达。项目1将利用标准的分子生物学技术，血友病B的小动物和大动物模型，以及通过核心对凝血因子的测量。项目1将与项目4进行广泛的合作，在可行的情况下，增强F.IX表达的发现将被转化到床边。项目1还在血友病B的皮肤定向基因转移领域与项目2持续合作，并开发一种新的再转座子载体。

Hemophilia B is a severe bleeding diathesis caused by the absence of blood coagulation factor IX. The disease is currently treated by infusion of clotting factor concentrates either prophylactically or in response to bleeds. This approach to treatment is sub-optimally for a number of reasons, owing partly to the fact that the protein has a relatively short half-life in the circulation. The ability to treat hemophilia by a gene therapy approach will represent a considerable advance in the treatment of the disease, since it will insure that patients maintain continuously a level of clotting factor adequate to prevent most spontaneous bleeds. The underlying hypothesis of this application is that AAV-mediate, muscle- directed gene transfer of blood coagulation Factor IX can achieve this therapeutic goal. Previous work in mice and in hemophilic dogs supports this hypothesis although the levels of expression achieved in dogs were at the lower limit of the therapeutic range. Thus, successful translation of these promising pre-clinical results to a large clinical population in a safe and efficacious manner will require a more detailed understanding of the biology of the vector and of the biosynthesis of Factor IX in muscle. The specific aims outlined in this application build on the work accomplished in the previous funding period , and include 1) to identify those post- translational modifications that are limiting for expression of biologically active F.IX in muscle, and to determine whether F.IX expression in muscle can be enhanced by supplying the limiting enzyme(s) in trans; 2) to determine whether vector DNA introduced into muscle persists as a high molecular weight episome or is integrated into chromosomal DNA; and 3) to determine whether introduction of muscle-specific promoter/enhancer elements can result in higher levels of expression of F.IX following intramuscular injection of a recombinant AAV vector. Project #1 will make use of standard molecular biology techniques, of small and large animal models of hemophilia B, and of measurements of clotting factors through the Core. Project #1 will have extensive collaborations with Project #4 in that discoveries that enhance expression of F.IX will be translated to the bedside as feasible. Project #1 also has ongoing collaborations with Project #2 in the area of skin-directed gene transfer for hemophilia B, and development of a novel re transposon vector.