Studies on Epstein-Barr virus vector for human gene therapy

目前还没有像Winch这样的感染系统，可以在Epstein-Barr病毒(EBV)感染后产生病毒，因此，还不可能生产大量用于基因治疗的重组EBV。在这项研究中，我们的目标是建立一个EBV扩增系统，在病毒复制阶段包含复制起点的DNA序列和包装信号在病毒复制过程中被有效扩增并包装成病毒颗粒。我们产生了基因组大小超过200kbp的重组EBV感染细胞。如此大的EBV基因组超过了包装成病毒颗粒的大小限制。因此，该细胞应成为扩增病毒的理想包装细胞。我们证实，携带巨大EBV基因组的Akata细胞在抗LG抗体处理后能产生裂解抗原，但不能产生后代病毒。在此基础上，我们克隆了含有EBV和GFP基因复制起点和包装信号的质粒，并将其导入携带巨大EBV基因组的Akata细胞。1998年6月，高田博士和伊迈博士访问了Hammerschmidt博士的实验室，我们证实，经抗LG处理后，转基因质粒被包装并产生为传染性病毒。在访问期间，我们讨论了相互实验的结果，并相互获得了许多重要信息。

There has been no infection system such as that winch produces viruses following Epstein-Barr virus (EBV infection, Therefore, it has not been possible to produce a large amount of recombinant EBV for gene therapy. In this study, we aimed to generate an amplicon system of EBV.DNA sequences containing the replication origin in a virus replicative phase and packaging signals are efficiently amplified and packaged into virus particles during virus replication. We generated cells infected with recombinant EBV with more than 200kbp genome size. Such large EBV genomes exceed the size limitation for being packaged into virus particles. Therefore, such cells should become an ideal packaging cells for propagating amplicon virus. We confirmed that Akata cells carrying huge EBV genome produce lyric antigen but do not produce progeny viruses upon treatment with anti-lg antibodies. Base on these results, we genierated a plasmid containing the replication origin and packaging signal of EBV and GFP gene, and transfected it into the Akata cells carrying huge EBV genome. We confirmed that the transfected plasmid is packaged and produced as infectious viruses after anti-lg treatment.Dr.Takada and Dr.lmai visited Dr.Hammerschmidt's laboratory in June, 1998. During its visit, we discussed about mutual experimental results, and obtained many important informations each other.