权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Research of EBV virus vector for gene therapy

EB病毒基因治疗载体的研究

基本信息

批准号：
07557029
负责人：
TAKADA Kenzo
金额：
$ 5.76万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557029/
关键词：
EB virus Viral vector Gene therapy ウイルススペクター

项目摘要

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.

我们缺乏支持 Epstein-Barr 病毒 (EBV) 有效复制的宿主细胞。最近，我们从Akata细胞系（简称Akata-）中分离出EBV阴性细胞克隆（简称Akata-），发现Akata-细胞是EBV繁殖的良好宿主，适合重组EBV的克隆繁殖，成为确定EBV遗传学的有力工具，使EBV作为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 细胞每个细胞约有 20 个 EBV 质粒拷贝。将耐药基因通过同源重组插入EBV阳性秋田细胞的EBV质粒中，制成病毒制剂，用野生型EBV和重组EBV的混合物感染EBV阴性秋田细胞。在选择性培养基中培养3周后，很容易分离出许多耐药克隆，其中大多数仅感染重组EBV。通过用抗Ig抗体处理细胞，产生大量重组EBV。为了用作人类基因治疗的载体，我们需要开发删除具有潜在致癌活性的EBV基因的EBV载体。为此，在本研究中，我们旨在生成用于 EBV 传播的包装细胞。已知被包装成病毒颗粒的EBV基因组存在大小限制。 EBV的基因组大小约为170kbp，超过200kbp的基因组不被包装成病毒颗粒。因此，我们打算并成功地将30 kbp的外源DNA插入到Akata细胞中的20个EBV质粒之一中。现在我们正在尝试分离只含有重组质粒的细胞克隆，它应该成为繁殖删除了所有转化基因的EBV重组体的理想宿主。