病毒载体-2014-10研究.ppt

Viral Vectors病毒载体 Institute of Molecular Biology, CTGU 刘朝奇 2014-10 病毒学基本概念 病毒载体 定义 临床应用 基本过程 载体优化 常用载体 载体(vector) 是携带外源DNA 进入宿主细胞进行扩增和表达的DNA,它们一般是通过改造质粒、噬菌体或病毒等构建的。 载体应具备以下条件： １、 能在适当的宿主细胞中复制； ２、 具有多种限制酶的单一切点（即所谓多克隆 位点）以便外源DNA 插入； ３、 具有筛选标志以区别阳性与阴性重组分子； ４、 载体分子较小，以便体外基因操作，同时载体DNA 与宿主DNA 便于分离； ５、 对于表达型载体还应具有与宿主细胞相适应的启动子、增强子、加尾信号等基因表达元件。 Gene therapy Recent years for several types of vectors, particularly viral vectors, which have been used in 70% of gene therapy clinical trials as of January 2007 Obstacles to systemic targeting The first: Reactions with the complement system and pre-existing antibodies The next: the endothelial cell layer Additional: the stroma and the basal-membrane-like structure to reach the tumor Viruses as Vectors interest on four types retroviruses (lentiviruses) adenoviruses adeno-associated viruses herpes simplex virus type 1 Retrovirus Vectors a class of enveloped viruses ssRNA molecule as the genome,10kb integrates into the host genome three genes: gag (coding for core proteins), pol (coding for reverse transcriptase) env (coding for the viral envelope protein) long terminal repeats (LTRs) packaging sequences 包装细胞(packaging cell) 将缺失了包装信号及相关序列的缺陷型逆转录病毒导入哺乳动物细胞，产生大量病毒包装蛋白。NIH/3T3，ψ—2，PA317，ψCRIP 逆转录病毒载体导入包装细胞后，载体转录出病毒基因组的部分序列、标记基因、外源基因，被包装成病毒颗粒。 缺陷型的逆转录病毒颗粒 一过性感染 病毒滴度106CFU/ml以上 逆转录病毒载体的特点 缺陷型的逆转录病毒感染细胞 RNA进入靶细胞后，变成前病毒并整合到宿主细胞的染色体上，可稳定表达 只能感染处于增殖期的细胞 安全性问题 逆转录病毒感染的可能性 污染的可能性 逆转录病毒在靶细胞基因组上的整合 破坏细胞正常生长的必须基因/抑癌基因 LTR激活原癌基因 染色体重排激活原癌基因 Moloney murine leukaemia virus (Mo-MLV) amphotrophic virus The essential regions include the 5 3 LTRs the packaging sequence lying downstream of the 5 LTR. Transgene expression can either be driven by the promoter/enhancer region in the 5 LTR, or by alternative viral (e.g. cytomegalovirus, Rous sarcoma virus) or cellular (e.g. beta actin, tyrosine) promoters selectable markers, such as neomycin beta galactosidase, can be included The available carrying capacity for retro