Study about factor VIII expression and immunological influence in the gene therapy of hemophilia A

1.因子VIII表达：构建由巨细胞病毒增强子/鸡-肌动蛋白启动子/兔-球蛋白3'UTR、犬因子VIII基因B结构域缺失和兔-球蛋白聚A组成的重组腺病毒载体。转染COS1、HepG2和Hela细胞，表达FVIII。在第4天和第7天，COS1细胞分别检测到约0.6单位/ml和1.0单位/ml的因子VIII。HepG2细胞在第4天和第7天分别检测到0.8单位/ml和1.0单位/ml的因子VIII。Hela细胞未检测到因子VIII。免疫影响：分析抗VIII因子同种异体抗体的免疫学和生物学特性。特别是(1)IgG亚类特异性，(2)同种抗体结合区，(3)对因子VIII辅因子功能的抑制机制。我们首次证明了对因子IX结合的抑制作用，因子VIII与血管性血友病因子和活化血小板的结合，以及凝血酶和活化因子Xa对因子VIII活化的抑制作用。此外，我们在血浆中检测到因子VIII/抗因子VIII自身抗体免疫复合物。血友病A患者因子VIII基因型与抗因子VIII同种抗体发展的相互作用：我们发现HLA-DR4.1、DQ4和DQA1与抑制剂发展密切相关。此外，我们还发现某些因子VIII基因型与抑制剂的发生有关。

1.Factor VIII expression : Recombinant adeno viral vector consisting of Cytomegalovirus enhancer/chiken _-actin promoter/rabbit _-globulin 3'UTR, B domain less canine factor VIII gene and rabbit _-globulin poly A was constructed. COS1, HepG2 and Hela cells were transfected and FVIII was expressed. Approximately 0.6 unit/ml and 1.0 unit/ml of factor VIII was detected by COS1 cells at day 4 and 7, respectively. Furthermore, 0.8 unit/ml of and 1.0unit/ml of factor VIII was detected by HepG2 cells at day 4 and day 7, respectively. Factor VIII was not detected by Hela cells.2.Immunological influence : Immunological and biological characterizations of anti-factor VIII alloantibodies were analyzed. Especially, (1)IgG subclass specificity, (2)binding region of the alloantibodies, (3)inhibitory mechanism on factor VIII cofactor function were determined. We demonstrated for the first time the inhibitory effects on factor IX binding, factor VIII binding to von Willebrand factor and activated platelets and inhibitory effects on factor VIII activation by thrombin and activated factor Xa. Furthermore, we detected factor VIII/anti-factor VIII autoantibodies immune complex in plasma.3.Interaction between factor VIII genotype of heamophilia A patient and development of anti-factor VIII alloantibodies : We found a close relationship between HLA-DR4.1, DQ4 and DQA1 and inhibitor development. Furthermore we also found that some factor VIII genotype was associated with the development of inhibitor.