AAV mediated muscle directed gene therapy for Hemophilia B

血友病B是一种严重的出血素质所造成的缺乏凝血因子IX。目前，通过输注凝血因子浓缩物来治疗该疾病，无论是抗凝治疗还是对出血作出反应。由于许多原因，这种治疗方法是次优的，部分原因是蛋白质在循环中的半衰期相对较短。通过基因治疗方法治疗血友病的能力将代表该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。本申请的基本假设是血液凝固因子IX的AAV介导的肌肉定向基因转移可以实现该治疗目标。 先前在小鼠和血友病犬中的工作支持这一假设，尽管在犬中达到的表达水平处于治疗范围的下限。因此，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的临床群体将需要更详细地了解载体的生物学和肌肉中因子IX的生物合成。本申请中概述的具体目标建立在前一资助期完成的工作基础上，并且包括1）鉴定限制生物活性F.IX在肌肉中表达的那些翻译后修饰，并确定是否可以通过提供反式限制酶来增强肌肉中的F.IX表达; 2）确定导入肌肉的载体DNA是否作为高分子量附加体持续存在或整合到染色体DNA中;和3）确定肌肉特异性启动子/增强子元件可导致肌内注射重组AAV载体后F.IX的更高水平表达。项目#1将使用标准分子生物学技术、血友病B的小型和大型动物模型以及通过Core测量凝血因子。 项目#1将与项目#4进行广泛的合作，因为增强F.IX表达的发现将在可行的情况下转化到床边。项目#1还与项目#2在血友病B的皮肤定向基因转移领域以及新型转座子载体的开发方面进行合作。

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.