Quantitative comparison of intracellular disposition between artificial and viral vector: Mechanism underlying the difference in dominant rate-limiting process
For developing of non-viral gene vectors sufficient for clinical application, it is necessary to understand why and to what extent non-viral vectors are inferior to the viral vectors, which in general exhibit more efficient transfection activity. This study describes a systematic and quantitative comparison of intracellular trafficking (iPK) of exogenous DNA transfected by viral and non-viral vectors in living cells, which is a hybrid of cellular uptake and subsequent intracellular distribution (e.g. endosome/lysosome, cytosol and nucleus). As a model, adenovirus and LipofectAMINE PLUS (LFN) was used for comparison since they are highly potent and widely used viral and non-viral vectors, respectively. Comparison of intracellular trafficking between adenovirus and LipofectAMINE PLUS (LFN) revealed that the three orders of magnitude lower transfection efficiency of LFN was dominantly rate-limited by the post-nuclear delivery process (Pharmacodynamics: PD). The contribution of transcription and translation processes to the overall differences in the trans-gene expression efficiency of PD was further evaluated by quantifying mRNA. As a result, transcription efficiency (Etranscript) of LFN, denoted as transgene expression divided by the amount of nuclear pDNA was about 16 times less than that for adenovirus. Furthermore, translation efficiency (Etranslate), denoted as transfection activity divided by mRNA expression was approximately 460 times less in LFN. Imaging of the decondensed form of DNA by in situ hybridization revealed that poor decondensation efficiency of LFN is involved in the inferior Etranscript. Collectively, an improvement in nuclear decondensation and the diminution of the interaction between vector and mRNA is essential for the development of new generations of non-viral vectors.
adenovirus non-viral vector lipoplex quantification intracellular trafficking gene vector
Hidetaka Akita
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Hokkaido 060-0812,Japan
国际会议
The 5th International Forum on Post-genome Technologies(5IFPT)(第五届国际后基因组生命科学技术学术论坛)
苏州
英文
2007-09-10（万方平台首次上网日期，不代表论文的发表时间）