Development of highly efficient factor VIII mini-gene therapy

 描述(申请人提供)：在人类人口中，大约每5000名男性中就有一人患有凝血障碍，即血友病A。这种疾病主要是由位于X染色体上的第VIII因子基因缺失引起的，很难用传统药物治疗。目前通过静脉输注因子浓缩物治疗血友病A非常昂贵，并且有开发抑制剂的潜在副作用。另一方面，基因疗法可以潜在地防止目前治疗方法的这些局限性。虽然重组腺相关病毒(RAAV)载体在携带因子VIII基因方面有很好的应用前景，但由于其大小限制(限于~5kb)和因子VIII蛋白的低效分泌，使其在血友病A基因治疗中的应用滞后于其他遗传病。为了利用rAAV载体改善因子VIII的基因传递，我们将开发一种 具有增强表达和分泌功能的新型人类第VIII因子分子。这项建议的具体目的是：1)。开发分泌和表达能力增强的人因子分子；开发一种氨基酸变化最小、比活性增强的人第VIII因子分子；3)优化AAV-VIII因子包装和表达框，开展血友病动物模型的临床前研究。这一提议的成功可能会导致使用AAV载体进行血友病A的临床试验。

 DESCRIPTION (provided by applicant): Approximately one in 5000 males in human population suffers from coagulation disorder, hemophilia A. This disease is primarily caused by deficiency in the factor VIII gene located in the X-chromosome and is difficult to treat by conventional medicine. Current treatment of hemophilia A by intravenous infusion of factor VIII concentrates is very costly and has a potential side effect of developing inhibitors. Gene therapy, on the other hand, can potentially prevent these limitations of current treatments. Although recombinant adeno-associated virus (rAAV) vectors are promising for deliver factor VIII gene, applying AAV vector technology to Hemophilia A gene therapy lagged behind other genetic diseases because of this size constraint (limited to ~5kb) and inefficient secretion of factor VIII protein. To improve factor VIII gene delivery utilizing rAAV vectors, we will develop a novel human factor VIII molecules with enhanced expression and secretion. The specific aims for this proposal are: 1). To develop a human factor VIII molecule with improved secretion and expression; 2). To develop a human factor VIII molecule with enhanced specific activity with minimal amino acid alteration; 3). To optimize the AAV factor VIII packaging and expression cassette and carry out preclinical studies in Hemophilia Animal Model. The success of this proposal may lead to a clinical trial of hemophilia A using AAV vectors.