Molecular engineering of factor VIII gene for rAAV delivery

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。目前通过静脉输注凝血因子VIII的方法治疗血友病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载体在携带因子基因方面有很好的应用前景，但由于大小的限制(限于~5kb)，将重组腺相关病毒载体技术应用于血友病A落后于其他遗传病。为了利用rAAV载体改善因子VIII的基因传递，我们开发了新型的工程化因子VIII分子，可用于双载体和单载体策略。这项建议的具体目的是：1)。开发一种高效分泌因子VIII重链分子；开发并鉴定一种新的携带rAAV的微型因子VIII基因；分析针对基因工程第VIII因子转基因产物的免疫反应。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 lagged behind other genetic diseases because of this size constraint (limited to ~5kb). To improve factor VIII gene delivery utilizing rAAV vectors, we have developed novel engineered factor VIII molecules for use in both dual vectors and single vector strategy. The specific aims for this proposal are: 1). To develop a Factor VIII heavy chain molecule efficient for secretion; 2). To develop and characterize a novel mini factor VIII gene for rAAV delivery; 3). To analyze immune responses against the engineered factor VIII transgene products. The success of this proposal may lead to a clinical trial of hemophilia A using AAV vectors.