1] School of Pharmacy, University of Waterloo, Waterloo, Ontario, Canada [2] Waterloo Institute of Nanotechnology, University of Waterloo, Waterloo, Ontario, Canada.
School of Pharmacy, University of Waterloo, Waterloo, Ontario, Canada.
Mol Ther Nucleic Acids. 2014 May 27;3(6):e165. doi: 10.1038/mtna.2014.16.
Conventional plasmid DNA vectors play a significant role in gene therapy, but they also have considerable limitations: they can elicit adverse immune responses because of bacterial sequences they contain for maintenance and amplification in prokaryotes, their bioavailability is compromised because of their large molecular size, and they may be genotoxic. We constructed an in vivo platform to produce ministring DNA-mini linear covalently closed DNA vectors-that are devoid of unwanted bacterial sequences and encode only the gene(s) of interest and necessary eukaryotic expression elements. Transfection of rapidly and slowly dividing human cells with ministring DNA coding for enhanced green fluorescent protein resulted in significantly improved transfection, bioavailability, and cytoplasmic kinetics compared with parental plasmid precursors and isogenic circular covalently closed DNA counterparts. Ministring DNA that integrated into the genome of human cells caused chromosomal disruption and apoptotic death of possibly oncogenic vector integrants; thus, they may be safer than plasmid and circular DNA vectors.
常规的质粒 DNA 载体在基因治疗中发挥着重要作用，但它们也有相当大的局限性：由于它们包含用于原核生物维持和扩增的细菌序列，因此会引起不良的免疫反应；由于其较大的分子尺寸，其生物利用度受到损害；并且它们可能具有遗传毒性。我们构建了一个体内平台来生产 ministring DNA-迷你线性共价闭合 DNA 载体，这些载体不含不需要的细菌序列，仅编码感兴趣的基因（s）和必要的真核表达元件。与亲本质粒前体和同基因的环状共价闭合 DNA 对应物相比，用编码增强型绿色荧光蛋白的 ministring DNA 转染快速和缓慢分裂的人细胞，导致转染、生物利用度和细胞质动力学显著改善。整合到人细胞基因组中的 ministring DNA 导致染色体断裂和可能致癌的载体整合子的凋亡死亡；因此，它们可能比质粒和环状 DNA 载体更安全。
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