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

1.因子VIII表达：构建了由巨细胞病毒增强子/鸡肌动蛋白启动子/兔球蛋白3 '非翻译区、无B结构域的犬凝血因子VIII基因和兔球蛋白polyA组成的重组腺病毒载体。转染COS 1、HepG 2和Hela细胞并表达FVIII。在第4天和第7天，COS 1细胞分别检测到约0.6单位/ml和1.0单位/ml的因子VIII。HepG 2细胞在第4天和第7天分别检测到0.8U/ml和1.0U/ml的因子VIII。2.免疫学影响：分析抗凝血因子VIII同种抗体的免疫学和生物学特性。特别地，（1）IgG亚类特异性，（2）同种抗体的结合区域，（3）对因子VIII辅因子功能的抑制机制被确定。我们首次证明了对因子IX结合、因子VIII与血管性血友病因子和活化血小板结合的抑制作用，以及对凝血酶和活化因子Xa对因子VIII活化的抑制作用。3.血友病A患者凝血因子VIII基因型与抗凝血因子VIII同种抗体产生的相互作用：HLA-DR 4.1、DQ 4和DQA 1与抑制因子产生密切相关。此外，我们还发现，一些因子VIII基因型与抑制剂的发展。

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.