Trobridge G D, Russell D W
Markey Molecular Medicine Center and Department of Medicine, University of Washington, Seattle 98195-7720, USA.
Hum Gene Ther. 1998 Nov 20;9(17):2517-25. doi: 10.1089/hum.1998.9.17-2517.
Retroviral vectors based on human foamy virus (HFV) have been developed and show promise as gene therapy vehicles. Here we describe a method for the production of HFV vector stocks free of detectable helper virus. The helper and vector plasmid constructs used both lack the HFV bel genes, so recombination between these constructs cannot create a wild-type virus. A fusion promoter that combines portions of the cytomegalovirus (CMV) immediate-early and HFV long terminal repeat (LTR) promoters was used to drive expression of both the helper and vector constructs. The CMV-LTR fusion promoter allows for HFV vector production in the absence of the Bel-1 trans-activator protein, which would otherwise be necessary for efficient transcription from the HFV LTR. Vector stocks containing either neomycin phosphotransferase or alkaline phosphatase reporter genes were produced by transient transfection at titers greater than 10(5) transducing units/ml. G418-resistant BHK-21 cells obtained by transduction with neo vectors contained randomly integrated HFV vector proviruses without detectable deletions or rearrangements. The vector stocks generated were free of replication-competent retrovirus (RCR), as determined by assays for LTR trans-activation and a marker rescue assay developed here for the detection of Bel-independent RCR.
基于人泡沫病毒（HFV）的逆转录病毒载体已被开发出来，并显示出作为基因治疗载体的潜力。在此，我们描述了一种生产无检测到辅助病毒的HFV载体储备液的方法。所使用的辅助质粒和载体质粒构建体均缺失HFV bel基因，因此这些构建体之间的重组不会产生野生型病毒。一种融合启动子，它结合了巨细胞病毒（CMV）立即早期启动子和HFV长末端重复序列（LTR）启动子的部分序列，用于驱动辅助质粒和载体质粒构建体的表达。CMV-LTR融合启动子允许在没有Bel-1反式激活蛋白的情况下生产HFV载体，否则该蛋白对于从HFV LTR进行有效转录是必需的。通过瞬时转染产生了含有新霉素磷酸转移酶或碱性磷酸酶报告基因的载体储备液，滴度大于10(5)转导单位/毫升。用新霉素载体转导获得的对G418耐药的BHK-21细胞含有随机整合的HFV载体原病毒，没有可检测到的缺失或重排。通过LTR反式激活测定和在此开发的用于检测不依赖Bel的复制型逆转录病毒（RCR）的标志物拯救测定确定，所产生的载体储备液不含具有复制能力的逆转录病毒（RCR）。
