Wang Hongsheng, Li Xiaoqing, He Yuwen, Xie Bailu, Tang Wenying, Du Jun
Center of Microbiology, Biochemistry and Pharmacology, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510080, China.
Sheng Wu Gong Cheng Xue Bao. 2008 Aug;24(8):1333-9.
The most difficult field in gene therapy is that vector system should offer both a means of successful transfection and a maximum of safety for the patient. Viral vectors and plasmid vectors are traditional vectors; they may cause unwanted immunological side effects resulting from the expression of nontherapeutic genes. Our aim is to develop a new general gene therapy vector which is suggested to be called as Micro-Linear Vector. The gene expression cassette is capped by our designed cap, including promoter, enhancer, objective gene, and RNA-stabilizing sequence, so it can defend the exnuclease in the eukaryotic cell, at the same time, DNA not encoding the objective gene is reduced to a minimum. The GFP gene is separated from the pEGFP-N3 plasmid, and acts as a reporter gene to construct the Micro-Linear Vector, then both the new vector and the plasmid are transfected to cells, the results are tested by fluorescence microscope and flow cytometry. The results show that the Micro-Linear Vector has a high effective of transfection and safety in 293, 3T3, CNE2 and B95-8 cell lines, at the same time it is less toxicity than the plasmid. We can get the rudiments of conclusion that Micro-Linear Vector has high effection of the transfection and more safety than tradition plasmid in eukaryotic cell.
基因治疗中最困难的领域在于载体系统既要提供成功转染的手段,又要为患者提供最大程度的安全性。病毒载体和质粒载体是传统载体;它们可能会因非治疗性基因的表达而引发不必要的免疫副作用。我们的目标是开发一种新的通用基因治疗载体,建议将其称为微线性载体。基因表达盒由我们设计的帽结构封闭,包括启动子、增强子、目的基因和RNA稳定序列,因此它可以抵御真核细胞中的核酸外切酶,同时将不编码目的基因的DNA减少到最低限度。从pEGFP-N3质粒中分离出绿色荧光蛋白(GFP)基因,并将其作为报告基因构建微线性载体,然后将新载体和质粒都转染到细胞中,通过荧光显微镜和流式细胞术检测结果。结果表明,微线性载体在293、3T3、CNE2和B95-8细胞系中具有高转染效率和安全性,同时其毒性比质粒小。我们可以初步得出结论,微线性载体在真核细胞中具有高转染效率且比传统质粒更安全。