Duisit G, Salvetti A, Moullier P, Cosset F L
Laboratoire de Thérapie Génique, CHU Hotel Dieu, Nantes, France.
Hum Gene Ther. 1999 Jan 20;10(2):189-200. doi: 10.1089/10430349950018986.
We have generated three different E1-deleted replication-defective adenoviral vectors expressing either Moloney murine leukemia virus (Mo-MuLV) Gag-Pol core particle proteins, gibbon ape leukemia virus (GALV) envelope glycoproteins, or an MuLV-derived retroviral vector genome encoding mCD2 antigen, a murine cell surface marker easily detectable by flow cytometry. Each of the three vectors was first characterized individually by infection of cells providing the complementary retroviral function(s) and able to induce the production of retroviral vectors with an efficiency similar to or higher than that of FLY stable retroviral packaging cells [Cosset, F.-L., Takeuchi, Y., Battini, J.-L., Weiss, R.A., and Collins, M.K.L., (1995). J. Virol. 69, 7430-7436]. In small-scale pilot experiments, TE671 cells simultaneously coinfected with the three adenoviral vectors efficiently released helper-free retroviral vectors in their supernatant, with titers greater than 10(6) infectious particles per milliliter by end-point titrations. Our results also indicated that in contrast to retroviral vector-packageable RNAs, the adenovirus-mediated overexpression of both Gag-Pol and Env packaging functions had limited impact on retroviral titers. The primary mechanism suspected is the premature intracellular cleavage of the Pr65gag precursor that we found in gag-pol-expressing cells, which in turn may impair the normal incorporation of high loads of functional Env. Last, the characterization of the adenoviral/retroviral chimeric vectors allowed the screening of various primate cells for retroviral production and we found that three hepatocyte-derived cell lines were highly efficient in the assembly and release of infectious retroviral particles.
我们构建了三种不同的E1缺失型复制缺陷腺病毒载体，分别表达莫洛尼鼠白血病病毒（Mo-MuLV）的Gag-Pol核心颗粒蛋白、长臂猿白血病病毒（GALV）的包膜糖蛋白，或编码mCD2抗原的源自MuLV的逆转录病毒载体基因组，mCD2抗原是一种可通过流式细胞术轻松检测到的鼠细胞表面标志物。首先，通过感染提供互补逆转录病毒功能且诱导产生逆转录病毒载体的效率与FLY稳定逆转录病毒包装细胞相似或更高的细胞，分别对这三种载体进行了表征[科塞特，F.-L.，竹内，Y.，巴蒂尼，J.-L.，魏斯，R.A.，以及柯林斯，M.K.L.，（1995年）。《病毒学杂志》69卷，7430 - 7436页]。在小规模预实验中，同时用这三种腺病毒载体共感染的TE671细胞在其上清液中高效释放无辅助逆转录病毒载体，通过终点滴定法测得滴度大于每毫升10⁶个感染性颗粒。我们的结果还表明，与可包装逆转录病毒载体的RNA不同，腺病毒介导的Gag-Pol和Env包装功能的过表达对逆转录病毒滴度的影响有限。怀疑的主要机制是我们在表达gag-pol的细胞中发现的Pr65gag前体的细胞内过早切割，这反过来可能会损害高负载功能性Env的正常掺入。最后，腺病毒/逆转录病毒嵌合载体的表征使得能够筛选各种灵长类细胞用于逆转录病毒生产，并且我们发现三种源自肝细胞的细胞系在传染性逆转录病毒颗粒的组装和释放方面效率很高。
