外源人凝血因子Ⅷ高效转移和稳定持久表达体系的建立

Objective and methods: The study mainly aims at the problems of using the retroviral vector mediated gene expression, namely the inactivation of the viral promoter and gene silencing. In order to enhance and prolong the expression of viral vector mediated trans-gene ,the retroviral vector backbone of MFG was modified and some elements capable of changing the conformation of the chromatin were inserted into the vector. Results and conclusions:1)The viral vector with the enhancer and promoter of 3'U3 replaced with cmv promoter has a much lower virus production and gene expression. Further studies show that there may be some important signals in the U3 region of 3'LTR for the processing ,maturation and stabilization of the MoMLV retroviral transcribed RNA.2)The scaffold/matrix attachment region(S/MAR) from the upstream of human β IFN can significantly enhance the viral titer of MFG ,and enhance the expression of viral vector mediated reporter and human coagulation factor VIII.(about 58%,420±32ng/106 24h,p,0.01).3)The results also show that there is probably a negative regulation element within the 3'AU rich region of PI3KγmRNA 3'UTR,which has a high homology with the reversed S/MAR from the upstream of human β IFN gene.

本研究将去除FⅧcDNA5"端非翻译区上游行翻译区的B链部分抑制序列并增加SV40内含子结构及骨髓基质细胞组成型启动子序列，用新的能产生GALV病毒假型病毒外壳蛋白包装细胞株PG13和普通双嗜性包装细胞株PA317，构建含B链缺失型FⅧcDNA并在骨髓基质细胞中高效表达亩帜孀疾《驹靥澹芯科湓谔迥谕庾菩斜泶铮巡的基因治疗探索有效的途丁