Centre National de la Recherche Scientifique, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.
Gene Ther. 2013 Jan;20(1):62-8. doi: 10.1038/gt.2011.215. Epub 2012 Jan 19.
A major issue for successful human gene therapy or genetic vaccination is a safe high-transgene expression level. Plasmid-based (non-viral) physical methods of gene transfer offered attracting approaches but their low efficiencies have limited their use in human pre-clinical trials. One of the limits appears to be the size of the plasmid that must be transferred across the cell membrane to the nucleus for its processing. In the present work to enhance gene transfer and expression, we evaluated a new generation of DNA vector; the minicircle, combined with the electropulsation technique. Minicircle is a doubled-stranded circular DNA with reduced size as it is devoid of bacterial sequences. We showed that electrotransferred minicircle encoding green fluorescent protein had higher in vitro transfection level compared with full-length plasmid. We demonstrated that minicircle great efficiency was not because of cellular toxicity decrease but was correlated to more efficient vector uptake by cells. Vector electrotransfection was operated in vivo and, using fluorescence imaging, minicircle electrotransfer was shown to enhance the efficiency and duration of tissue-targeted gene delivery and expression. By combining powerful expression and delivery systems, we have provided a valuable method for new approaches in gene therapy and genetic vaccination.
对于成功的人类基因治疗或基因疫苗接种来说，一个主要的问题是安全的高转基因表达水平。基于质粒的（非病毒）基因转移物理方法提供了有吸引力的方法，但由于效率低，限制了它们在人类临床前试验中的应用。其中一个限制似乎是必须穿过细胞膜转移到细胞核进行加工的质粒的大小。在本研究中，为了增强基因转移和表达，我们评估了新一代 DNA 载体；微环，结合电脉冲技术。微环是一种双链环状 DNA，由于缺乏细菌序列，因此尺寸减小。我们表明，电转移编码绿色荧光蛋白的微环具有比全长质粒更高的体外转染水平。我们证明，微环的高效率不是因为细胞毒性降低，而是与细胞更有效地摄取载体有关。载体电转染在体内进行，并且通过荧光成像，显示微环电转移增强了组织靶向基因传递和表达的效率和持续时间。通过结合强大的表达和传递系统，我们为基因治疗和基因疫苗接种的新方法提供了一种有价值的方法。
