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

Development of adeno-associated virus (AAV) vector

腺相关病毒（AAV）载体的开发

基本信息

批准号：
02454181
负责人：
HANDA Hiroshi
金额：
$ 3.71万
依托单位：
Tokyo Institute of Technology (1991)The University of Tokyo (1990)
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

The human parvovirus adeno-associated virus (AAV) grows in the presence of helper functions provided by helper adenovirus. AAV is not pathogenic to human beings. When AAV infects cells in the absence of a helper virus, it frequently integrates its genome into the host cell genome. AAV has several advantages as virus vectors. To exploit AAV as virus vectors, we have worked on three main projects, (1) reconstruction of AAV genome, (2) reconstruction of helper adenovirus genome, (3) regulation of expression of foreign genes inserted into AAV genome.(1) To reconstruct AAV genome, we prepared a recombinant plasmid pAAV which contained whole AAV genome. Several foreign gents, were inserted into the envelope protein region of PAAV. Human thymus epithelial cells were immortalized with the plasmid pAAV-SV40 DNA, which contained the SV40 early gene in the envelope region of PAAV. It indicated that the AAV vector was able to be developed from the recombinant plasmid.(2) We also made a recombinant helper adenovirus, which expressed AAV envelope proteins to complement the defectiveness of recombinant AAV vectors. We have tried to produce recombinant AAV vectors by co-introduction of pAAV-SV40 DNA and the recombinant helper adenovirus into 293 cells. The recombinant AAV virus was obtained but the virus titer was low, because of low efficiency of production of AAV envelope proteins.(3) To study the regulation of gene expression of foreign genes inserted into AAV genome, we have developed the affinity latex particles to purify DNA-binding transcription factors. By using the particles, we were able to purify multiple members of the ATF/E4TF3 family or two subunits of E4TF1 directly from crude cell extracts. The particles would be useful for studying various transcription factors.On the basis of this study, we will try further to exploit AAV as useful virus vectors.

人类细小病毒腺相关病毒 (AAV) 在辅助腺病毒提供的辅助功能的存在下生长。 AAV 对人类不具有致病性。当 AAV 在没有辅助病毒的情况下感染细胞时，它经常将其基因组整合到宿主细胞基因组中。 AAV 作为病毒载体有几个优点。为了利用AAV作为病毒载体，我们开展了三个主要项目：（1）AAV基因组的重建，（2）辅助腺病毒基因组的重建，（3）插入AAV基因组的外源基因的表达调控。（1）为了重建AAV基因组，我们制备了包含整个AAV基因组的重组质粒pAAV。几个外源基因被插入到 PAAV 的包膜蛋白区域。用质粒pAAV-SV40 DNA使人胸腺上皮细胞永生化，该质粒在PAAV的包膜区域中含有SV40早期基因。 (2)我们还制备了重组辅助腺病毒，它表达AAV包膜蛋白，以弥补重组AAV载体的缺陷。我们尝试通过将pAAV-SV40 DNA和重组辅助腺病毒共同导入293细胞来产生重组AAV载体。获得了重组AAV病毒，但由于AAV包膜蛋白的生产效率低，病毒滴度较低。(3)为了研究插入AAV基因组的外源基因的基因表达调控，我们开发了亲和乳胶颗粒来纯化DNA结合转录因子。通过使用这些颗粒，我们能够直接从粗细胞提取物中纯化 ATF/E4TF3 家族的多个成员或 E4TF1 的两个亚基。这些颗粒可用于研究各种转录因子。在此研究的基础上，我们将进一步尝试将AAV作为有用的病毒载体。