Research of EBV virus vector for gene therapy

我们缺乏支持EB病毒（EBV）有效复制的宿主细胞。最近，我们从Akata细胞系（简称Akata-）中分离了EBV阴性细胞克隆，发现Akata-细胞是EBV繁殖的良好宿主，适合于重组EBV的克隆繁殖，这成为确定EBV遗传学的有力工具，并使得将EBV用作基因治疗的载体成为可能。生产重组EBV的程序如下。EBV阳性Akata细胞每个细胞具有约20个拷贝的EBV质粒。在通过同源重组将耐药基因插入EBV阳性秋田细胞的EBV质粒后，将产生的病毒制剂，野生型EBV和重组EBV的混合物用于感染EBV阴性Akata细胞。在选择性培养基中孵育3周后，很容易分离出许多耐药克隆，其中大多数仅用重组EBV感染。通过抗Ig抗体处理细胞产生大量的重组EBV，为了作为人类基因治疗的载体，我们需要开发一种缺失具有潜在致癌活性的EBV基因的EBV载体。为此，在本研究中，我们的目的是产生用于EBV增殖的包装细胞。众所周知，包装到病毒颗粒中的EB病毒基因组存在大小限制。EBV的基因组大小约为170 kbp，超过200 kbp的基因组不包装成病毒颗粒。因此，我们打算并成功地将30 kbp的外源DNA插入Akata细胞中的20个EBV质粒之一中。目前我们正试图分离出只含有重组质粒的细胞克隆，这将成为繁殖缺失所有转化基因的EBV重组体的理想宿主。

We lack a host cell supporting an efficient replication of Epstein-Barr virus (EBV). Recently, we isolated EBV-negative cell clones from the Akata cell line (referred as Akata-) , and found that Akata-cells are good hosts for EBV propagation and are suitable for propagation of recombinant EBV clonally, which becomes a powerful tool for determining EBV genetics and which makes it possible to use EBV as a vector for gene therapy. The procedure for producing recombinant EBV is as follows. EBV-positive Akata cells have about 20 copies of EBV plasmid per cell. After insertion of the drug-resistant gene into an EBV plasmid of EBV-positive Akita cells by homologous recombination, a virus preparation produced, a mixture of wild-type EBV and recombinant EBV is used to infect EBV-negative Akata cells. After 3 weeks of incubation in the selective media, many drug-resistant clones are isolated very easily, and most of them are infected with recombinant EBV only. By treatment of cells with anti-Ig antibodies a large amount of recombinant EBV is produced.For use as a vector for human gene therapy, we need to develop an EBV vector deleted of EBV genes that have potentially oncogenic activities. For that purpose, in the present study, we aimed to generate a packaging cell for EBV propagation. It is known that there is a size limitation for the EBV genome being packaged into virus particle. The genome size of EBV is about 170 kbp, and the genome over 200 kbp is not packaged into virus particles. Therefore, we intended and succeeded to insert a 30 kbp of foreign DNA into one of 20 EBV plasmid in Akata cells. Now we are trying to isolate cell clones that contained a recombinant plasmid only, which should become an ideal host for propagating EBV recombinants deleted of all the transforming genes.