Dubensky T W, Driver D A, Polo J M, Belli B A, Latham E M, Ibanez C E, Chada S, Brumm D, Banks T A, Mento S J, Jolly D J, Chang S M
Department of Viral and Genetic Therapeutics, Viagene, Inc., San Diego, California 92121, USA.
J Virol. 1996 Jan;70(1):508-19. doi: 10.1128/JVI.70.1.508-519.1996.
Several DNA-based Sindbis virus vectors were constructed to investigate the feasibility and potential applications for initiating the virus life cycle in cells transfected directly with plasmid DNA. These vectors, when transfected into mammalian cells, have been used to produce virus, to express heterologous genes, and to produce infectious vector particles. This approach involved the conversion of a self-replicating vector RNA (replicon) into a layered DNA-based expression system. The first layer includes a eukaryotic RNA polymerase II expression cassette that initiates nuclear transcription of an RNA which corresponds to the Sindbis virus vector replicon. Following transport of this RNA from the nucleus to the cytoplasm, the second layer, autocatalytic amplification of the vector, proceeds according to the Sindbis virus replication cycle and results in expression of the heterologous gene. The Sindbis virus DNA vectors expressed reporter genes in transfected cells at levels that were comparable to those of in vitro-transcribed RNA replicons and were approximately 10-fold higher than the levels produced by conventional RNA polymerase II-dependent plasmids in which the promoter and reporter gene were linked directly. Reporter gene expression was also observed in rodent muscle following injection with Sindbis virus DNA vectors. In a second application, packaged vector particles were produced in cells cotransfected with complementing replicon and defective helper DNAs. The Sindbis virus-derived DNA vectors described here increase the utility of alphavirus-based vector systems in general and also provide a vector with broad potential applications for genetic immunization.
构建了几种基于DNA的辛德毕斯病毒载体，以研究直接用质粒DNA转染细胞启动病毒生命周期的可行性和潜在应用。这些载体转染到哺乳动物细胞后，已用于产生病毒、表达异源基因以及产生感染性载体颗粒。这种方法涉及将自我复制的载体RNA（复制子）转化为基于分层DNA的表达系统。第一层包括一个真核RNA聚合酶II表达盒，它启动与辛德毕斯病毒载体复制子相对应的RNA的核转录。该RNA从细胞核转运到细胞质后，第二层，即载体的自催化扩增，按照辛德毕斯病毒复制周期进行，并导致异源基因的表达。辛德毕斯病毒DNA载体在转染细胞中表达报告基因的水平与体外转录的RNA复制子相当，比传统的依赖RNA聚合酶II的质粒（其中启动子和报告基因直接相连）产生的水平高约10倍。在用辛德毕斯病毒DNA载体注射后，在啮齿动物肌肉中也观察到了报告基因的表达。在第二个应用中，在与互补复制子和缺陷辅助DNA共转染的细胞中产生了包装载体颗粒。本文所述的源自辛德毕斯病毒的DNA载体总体上提高了基于甲病毒的载体系统的实用性，并且还提供了一种具有广泛遗传免疫应用潜力的载体。
