Chromatin-Binding Regions of EBNA1 Protein Facilitate the Enhanced Transfection of Epstein–Barr Virus-Based Vectors

作者：Sara E. Howden, Hady Wardan, Lucille E. Voullaire, Samuel McLenachan, Robert Brady Williamson, Panos A. Ioannou, Jim Vadolas · 发表于：Human Gene Therapy · 年份：2006 · DOI：10.1089/hum.2006.17.833 · 被引用次数：21 · 研究领域：Plant Virus Research Studies、Viral-associated cancers and disorders、Cytomegalovirus and herpesvirus research

Epstein-Barr virus (EBV)-based vectors can stably maintain large genomic fragments in mammalian cells, offering great potential for the treatment/correction of many acquired and inherited disorders. Numerous studies report marked increases in the transfection efficiency of EBV-based vectors after delivery into cell lines constitutively expressing Epstein-Barr nuclear antigen-1 (EBNA1), compared with cells not expressing EBNA1. We employ a novel strategy, involving the transfection of mRNA encoding EBNA1, to transiently express EBNA1 protein in human cells. Subsequently we show that the transfection efficiency of a 21-kb EBVbased vector is improved significantly when codelivered with mRNA encoding EBNA1. Similar increases in transfection efficiency were observed after delivery of the plasmid into cells constitutively expressing EBNA1. We also investigate the mechanism by which EBNA1 facilitates the transfection of EBV-based vectors, using mRNA encoding modified versions of the protein. Previous studies suggest that the EBNA1 DNA-binding domain (DBD), together with the nuclear localization signal (NLS), may enhance transfection of EBV plasmids by facilitating their nuclear transport. We demonstrate that an EBNA1 derivative comprising only the NLS and DBD does not facilitate transfection of EBV-based vectors. However, cells expressing an EBNA1 derivative devoid of a functional NLS but retaining the chromatin-binding regions, domains A and B, enhances plasmid transfection efficie...