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

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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.