Morozov V A, Noguiez-Hellin P, Laune S, Tamboise E, Salzmann J L, Klatzmann D
Laboratoire de Biologie et Thérapeutique des Pathologies Immunitaires, Université P. et M. Curie, CNRS ERS 107, Hôpital Pitié-Salpêtrière, CERVI, Paris, France.
Cancer Gene Ther. 1997 Sep-Oct;4(5):286-93.
We recently described a novel nonviral/viral vector for gene transfer, the plasmovirus (Noguiez-Hellin P, Robert-le Meur M, Laune S, et al. C R Acad Sci Paris, Sciences de la Vie. 1996;319:45-50; Noguiez-Hellin P, Robert-le Meur M, Salzmann J-L, et al. Proc Natl Acad Sci USA. 1996;93:4175-4180). Plasmoviruses are plasmids capable of expressing all the viral genes required for generating infectious particles and packaging a defective genome containing a transgene. Transfected as plasmids, plasmoviruses transform the transduced cells into packaging cells that release infectious replication-defective retrovirus vectors (RV) containing a transgene, which are capable of infecting nearby cells. We previously showed that such a vector can efficiently "propagate" the transgene after transfection. Here we examine in greater detail the different steps of plasmovirus replication in vitro in human (143 B TK-) and murine (NIH 3T3 TK-) cells. Molecular-biological analysis revealed plasmovirus-coded protein expression starting from 24 hours post-transfection, followed by the detection of infectious RV 48 hours post-transfection. The gag proteins were correctly processed in the released particles. Electron microscopic analysis revealed typical type C particles. Nonintegrated plasmovirus DNA was not toxic for the cells and could be detected for at least 14 days post-transfection. While the transfected gag gene and the transgene could also be detected throughout this period, we observed that env-coded proteins decreased after 72 hours post-transfection. Nevertheless, the production of RV resulted in the propagation of the transgene in the culture, with stable integration of plasmovirus proviral DNA into the host genome of infected cells. We show that this propagation results in a major improvement in therapeutic efficacy using an HSV1-TK transgene and ganciclovir treatment, when compared to that of plasmovirus constructs that cannot propagate. Altogether, these results demonstrate the functionality of this gene transfer method and suggest that improvements in the vector design enhance its efficacy.
我们最近描述了一种用于基因转移的新型非病毒/病毒载体——质粒病毒（诺吉耶斯 - 埃林P，罗伯特 - 勒米尔M，洛内S等。《法国科学院院报，生命科学》。1996年；319：45 - 50；诺吉耶斯 - 埃林P，罗伯特 - 勒米尔M，萨尔兹曼J - L等。《美国国家科学院院刊》。1996年；93：4175 - 4180）。质粒病毒是能够表达产生感染性颗粒所需的所有病毒基因并包装含有转基因的缺陷基因组的质粒。作为质粒进行转染时，质粒病毒将转导的细胞转化为包装细胞，这些包装细胞释放含有转基因的感染性复制缺陷型逆转录病毒载体（RV），该载体能够感染附近的细胞。我们之前表明，这样的载体在转染后能够有效地“传播”转基因。在此，我们更详细地研究了质粒病毒在人（143 B TK -）和小鼠（NIH 3T3 TK -）细胞中体外复制的不同步骤。分子生物学分析显示，转染后24小时开始表达质粒病毒编码的蛋白质，随后在转染后48小时检测到感染性RV。gag蛋白在释放的颗粒中被正确加工。电子显微镜分析显示典型的C型颗粒。未整合的质粒病毒DNA对细胞无毒，并且在转染后至少14天可以检测到。虽然在此期间也可以检测到转染的gag基因和转基因，但我们观察到转染后72小时后env编码的蛋白质减少。然而，RV的产生导致转基因在培养物中传播，质粒病毒前病毒DNA稳定整合到感染细胞的宿主基因组中。我们表明，与不能传播的质粒病毒构建体相比，这种传播在使用HSV1 - TK转基因和更昔洛韦治疗时导致治疗效果有显著改善。总之，这些结果证明了这种基因转移方法的功能性，并表明载体设计的改进提高了其功效。
