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Development of effectiveness evaluation and intracellular dynamics evaluation method of plasmid DNA for gene therapy

基因治疗用质粒DNA有效性评价及细胞内动力学评价方法的开发

基本信息

批准号：
11672258
负责人：
NAKAGAWA Shinsaku
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11672258/
关键词：
Cytoplasmic gene expression T7 promoter Fusogenic liposome Gene therapy T7 RNA polymerase スペルミジン細胞内物質導入

项目摘要

It is necessary to develop a more efficient gene expression system for gene therapy. A plasmid DNA, using eukaryoic or mammalian promoters, requires to localize into nuclear for gene expression. However, it is difficult to entry into nuclear, because nuclear pore size is not sufficient against the size of plasmid DNA.In this study, to develop a novel cytoplasmic gene expression system that dose not require nuclear localization of plasmid DNA to transcription, we examined the characterization of T7 cytoplasmic gene expression system. When co-transfected with pT7-IRES-L (luciferase expression plasmid containing T7 promoter) and T7 RNA polymerase into LLCMK_2 cells, the gene expression of pT7-IRES-L was observed rapidly within 6h after transfection and significant level of luciferase activity was detected. In contrast, pRSV-L, a common plasmid DNA consist of luciferase expression plasmid and Rous sarcoma virus promoter, required 24-48h for induction of gene expression. The gene expression level of the T7 system was enhanced with an increase in the amount of T7 RNA polymerase. To increase and prolong the gene expression, a plasmid DNA (pT7 AUTO-2) which contained the T7 RNA polymerase gene driven by the T7 promoter was co-transfected with pT7-IRES-L and T7 RNA polymerase. The plasmid DNA (pT7 AUTO-2) dose-dependently enhanced the luciferase gene expression by pT7-IRES-L and T7 RNA polymerase. In addition, we attempted to optimize the cytoplasmic gene expression system. The optimal ratio for co transfection of pT7-IRES-L and pT7 AUTO-2 was 1 to 3 (mole ratio). These results suggest that T7 gene expression system may be useful in many gene therapies where transient but rapid efficient gene expression is required.

有必要开发更有效的基因表达系统用于基因治疗。使用真核或哺乳动物启动子的质粒 DNA 需要定位到核中以进行基因表达。然而，它很难进入细胞核，因为核孔径不足以对抗质粒DNA的大小。在本研究中，为了开发一种不需要质粒DNA核定位转录的新型细胞质基因表达系统，我们检查了T7细胞质基因表达系统的表征。将pT7-IRES-L（含有T7启动子的荧光素酶表达质粒）和T7 RNA聚合酶共转染LLLLMK_2细胞时，转染后6h内迅速观察到pT7-IRES-L的基因表达，并检测到显着水平的荧光素酶活性。相比之下，pRSV-L是一种由荧光素酶表达质粒和劳斯肉瘤病毒启动子组成的常见质粒DNA，需要24-48小时来诱导基因表达。 T7系统的基因表达水平随着T7 RNA聚合酶量的增加而增强。为了增加和延长基因表达，将含有由T7启动子驱动的T7 RNA聚合酶基因的质粒DNA(pT7 AUTO-2)与pT7-IRES-L和T7 RNA聚合酶共转染。质粒 DNA (pT7 AUTO-2) 通过 pT7-IRES-L 和 T7 RNA 聚合酶剂量依赖性地增强荧光素酶基因表达。此外，我们尝试优化细胞质基因表达系统。 pT7-IRES-L和pT7 AUTO-2共转染的最佳比例为1比3（摩尔比）。这些结果表明，T7 基因表达系统可能在许多需要瞬时但快速有效的基因表达的基因治疗中有用。