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

因此，有必要开发一种更高效的基因表达系统用于基因治疗。使用真核生物或哺乳动物启动子的质粒DNA需要定位到细胞核中以进行基因表达。然而，由于核孔大小与质粒DNA的大小不相匹配，很难进入细胞核。在本研究中，为了开发一种新型的细胞质基因表达系统，该系统不需要质粒DNA的核定位即可转录。将含有T7启动子的荧光素酶表达载体pT7-IRES-L与T7RNA聚合酶共转染LLCMK_2细胞后，在6h内迅速检测到pt7-IRES-L的基因表达，并检测到显著的荧光素酶活性。而pRSV-L是由荧光素酶表达载体和Rous肉瘤病毒启动子组成的通用质粒DNA，需要24-48h才能诱导基因表达。T7系统的基因表达水平随着T7 RNA聚合酶数量的增加而增强。为了提高和延长基因的表达，将T7启动子驱动的T7RNA聚合酶基因与pT7-IRES-L和T7RNA聚合酶共转染成pT7AUTO-2。PT7AUTO-2通过pT7-IRES-L和T7RNA聚合酶剂量依赖性地增强荧光素酶基因的表达。此外，我们还对细胞质基因表达系统进行了优化。PT7-IRES-L与pt7 AUTO-2共转染的最佳比例为1：3(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。

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.