Wahlfors J J, Xanthopoulos K G, Morgan R A
Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Hum Gene Ther. 1997 Nov 20;8(17):2031-41. doi: 10.1089/hum.1997.8.17-2031.
Retroviral vectors are efficient tools for gene transfer studies. Their major advantage is that they can permanently integrate the transgene into the target cell's genome. However, because of the compulsory nuclear expression phase of their life cycle, it can be difficult for retroviruses to carry complex expression cassettes. In a attempt to mimic the structural features of most eukaryotic genes and obtain a potentially self-amplifying system for retrovirus production, we tested the feasibility of Semliki Forest virus (SFV) expression to mediate cytoplasmic synthesis of retrovirus vector RNA. An equivalent of a retrovirus virion RNA (retrovirus vector cassette, RVC) was cloned under the SFV 26S promoter, and full-length chimeric SFV-RVC RNA was produced in vitro. This RNA was introduced into retrovirus packaging cells, either via electroporation or transduction in SFV virions, and supernatants were analyzed for the presence of biologically active retroviruses. We demonstrate that this strategy can be used for cytoplasmic retrovirus production. The resulting viral particles are fully functional; they can transduce target cells, undergo reverse transcription, and integrate into genomic DNA. We also demonstrate that the SFV virion-based RVC delivery into packaging cells can yield high transient titers, in this case more than 10(5) G418R cfu/ml. This study shows that a simple, one-plasmid, heterologous viral RNA production system can be used to create functional retroviral RNA outside the cell nucleus.
逆转录病毒载体是基因转移研究的有效工具。它们的主要优点是能够将转基因永久整合到靶细胞基因组中。然而，由于其生命周期中必需的核表达阶段，逆转录病毒携带复杂的表达盒可能会很困难。为了模拟大多数真核基因的结构特征并获得一个潜在的用于逆转录病毒生产的自我扩增系统，我们测试了用塞姆利基森林病毒（SFV）表达来介导逆转录病毒载体RNA胞质合成的可行性。将相当于逆转录病毒病毒体RNA的片段（逆转录病毒载体盒，RVC）克隆到SFV 26S启动子下游，并在体外产生全长嵌合SFV-RVC RNA。通过电穿孔或SFV病毒体转导将该RNA导入逆转录病毒包装细胞，然后分析上清液中是否存在具有生物活性的逆转录病毒。我们证明该策略可用于胞质逆转录病毒的生产。产生的病毒颗粒功能完全正常；它们能够转导靶细胞、进行逆转录并整合到基因组DNA中。我们还证明基于SFV病毒体的RVC导入包装细胞能够产生高瞬时滴度，在这种情况下超过10(5) G418R cfu/ml。这项研究表明，一个简单的单质粒异源病毒RNA生产系统可用于在细胞核外产生功能性逆转录病毒RNA。
