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• W1489141230 abstract "Nonviral gene therapy represents an attractive alternative to viral gene delivery due to a lack of specific immune response, low toxicity, and cheap large-scale vector production. However, the use of nonviral vectors bears the major disadvantage of low transfection efficiency. In this context, the influence of structural features of the plasmid DNA, such as topology, size, or concatemeric arrangement, on transfection efficiency, cellular trafficking, and transcription activity is of high interest. In vitro transfection of the three naturally occurring plasmid DNA topologies resulted in significantly higher transfection efficiency of compact supercoiled plasmid DNA compared to the open circular or the linear form. By comparing different application methods, this was attributed to the intracellular delivery process into the nucleus. Improved transfection efficiency could be achieved by stabilizing linear plasmid DNA against degradation from exonucleases indicating plasmid DNA stability as another important aspect in this context. Several in vitro studies using different transfection protocols revealed that a smaller plasmid size is beneficial to overcome intracellular barriers, particularly the nuclear envelope as a major barrier for intracellular plasmid DNA translocation. However, it has been shown that plasmid size is less critical for certain in vivo applications, such as intrapulmonary delivery of plasmid DNA polyplexes. Formation of plasmid DNA concatemers consisting of multiple repeats of monomeric plasmid copies can occur during plasmid DNA preparation not only in E. coli but also in mammalian cells after transfection of linear monomeric plasmid DNA. The latter leads to up to 100-fold higher transgene expression compared to transfection of circular monomeric plasmid DNA. Higher transgene expression was also found after transfection of undefined preformed plasmid concatemers compared to nonconcatemeric plasmid DNA. A systematic comparison between an EGFP encoding plasmid DNA monomer and its dimer revealed two major findings: transfection of smaller monomeric plasmid DNA resulted in higher numbers of transfected cells compared to the dimer indicating an inverse effect of plasmid size on transfection efficiency. However, the concatemeric arrangement of the dimer was superior regarding transgene expression per transgene copy on a single-cell level. These findings indicate a transcriptionally more active conformation of plasmid DNA concatemers compared to their monomeric forms. In conclusion, plasmid DNA topology, size, and concatemerization are critical for transfection efficiency and subsequently for transgene expression. High numbers of transfected cells can be targeted by preferably small constructs, whereas a concatemeric arrangement may increase the transgene expression once the plasmid reached the nucleus." @default.
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• W1489141230 date "2013-04-04" @default.
• W1489141230 modified "2023-09-25" @default.
• W1489141230 title "Plasmid DNA Concatemers: Influence of Plasmid Structure on Transfection Efficiency" @default.
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• W1489141230 doi "https://doi.org/10.1002/9783527670420.ch5" @default.
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