BACKGROUND: Intramuscular gene delivery through injection of plasmid DNA has long been considered a promising approach for safe and simple in vivo gene expression for vaccination and gene therapy purposes. Recently, intramuscular gene delivery has been improved by applying low-voltage electric pulses after plasmid injection, a procedure that has been variably called gene electro-transfer, in vivo electroporation or electrical stimulation. Different types of electrical treatments have been used with excellent results both in terms of transgene expression levels and immunization outcome. This approach, therefore, holds promise for safe gene delivery to animals and humans designed for non-viral gene therapy and DNA-based vaccination. The molecular mechanisms underlying this increment in transduction efficiency are, however, still unclear. METHODS: Plasmid DNA status and kinetics following gene electro-transfer was analyzed by different methods (Southern analysis, Q-PCR and transformation into competent bacteria). RESULTS: A large amount of plasmid DNA is degraded in the first 4 h post-injection, with or without electroporation; later, the amount of intramuscular plasmid DNA is higher in electroporated samples. On electroporation, plasmid is partially protected from degradation, presumably by its early compartmentalization into the nuclei of muscle cells. CONCLUSIONS: By investigating the intracellular outcome and persistence of plasmid DNA following simple injection or gene electro-transfer we provide useful information on the mechanisms of plasmid entry and expression and underline some of the steps that could be taken to further improve this methodology. Copyright 2002 John Wiley & Sons, Ltd.
BACKGROUND: Intramuscular gene delivery through injection of plasmid DNA has long been considered a promising approach for safe and simple in vivo gene expression for vaccination and gene therapy purposes. Recently, intramuscular gene delivery has been improved by applying low-voltage electric pulses after plasmid injection, a procedure that has been variably called gene electro-transfer, in vivo electroporation or electrical stimulation. Different types of electrical treatments have been used with excellent results both in terms of transgene expression levels and immunization outcome. This approach, therefore, holds promise for safe gene delivery to animals and humans designed for non-viral gene therapy and DNA-based vaccination. The molecular mechanisms underlying this increment in transduction efficiency are, however, still unclear. METHODS: Plasmid DNA status and kinetics following gene electro-transfer was analyzed by different methods (Southern analysis, Q-PCR and transformation into competent bacteria). RESULTS: A large amount of plasmid DNA is degraded in the first 4 h post-injection, with or without electroporation; later, the amount of intramuscular plasmid DNA is higher in electroporated samples. On electroporation, plasmid is partially protected from degradation, presumably by its early compartmentalization into the nuclei of muscle cells. CONCLUSIONS: By investigating the intracellular outcome and persistence of plasmid DNA following simple injection or gene electro-transfer we provide useful information on the mechanisms of plasmid entry and expression and underline some of the steps that could be taken to further improve this methodology. Copyright 2002 John Wiley & Sons, Ltd.
Authors: Daniel O Villarreal; Kendra T Talbott; Daniel K Choo; Devon J Shedlock; David B Weiner Journal: Expert Rev Vaccines Date: 2013-05 Impact factor: 5.217
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