New Insights in the Gene Electrotransfer Process: Evidence for the Involvement of the Plasmid DNA Topology

Escoffre, Jean‐Michel; Nikolova, Biliana; Mallet, Laetitia; Henri, Julien; Favard, Cyril; Golzio, Muriel; Teissié, Justin; Tsoneva, Iana; Rols, Marie‐Pierre

doi:10.2174/156652312802762554

Cited by 17 publications

(13 citation statements)

References 37 publications

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“…Assuming a Poisson distribution for the formation of LMDS within the population of plasmids (data not shown), at 100 Gy 18% of the irradiated plasmids have one LMDS per plasmid, while only 2% of the plasmids are not functional. There are contradictory conclusions in the literature concerning the effects of plasmid topology on transformation efficiency (98)(99)(100)(101), however our results in Fig. 4 clearly show that the transformation efficiency initially increases with dose, reaching a maximum at about 200 Gy.…”

Section: Discussioncontrasting

confidence: 69%

The Relative Contributions of DNA Strand Breaks, Base Damage and Clustered Lesions to the Loss of DNA Functionality Induced by Ionizing Radiation

et al. 2014

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The majority of studies on lethal radiobiological damage have focused on double-strand breaks (DSBs), a type of clustered DNA damage and the evaluation of their toxicity, while other types of clustered DNA damage have received much less attention. The main purpose of this study is to evaluate the contribution of different lesions induced by ionizing radiation to the loss of plasmid DNA functionality. We employed a simple model system comprising E. coli transformed with an irradiated plasmid [pGEM-3Zf (-)] to determine the effect of DSBs and other lesions including base damage and clustered lesions on the functionality ("viability") of the plasmid. The yields of γ-radiation-induced single-strand breaks (SSBs) and DSBs were measured by gel electrophoresis. We found that the transformation efficiency decreases with radiation dose, but this decrease cannot be explained by the formation of DSBs. For example, at doses of 500 and 700 Gy, the relative transformation efficiency falls from 100% to 53% and 26%, respectively, while only 5.7% and 9.1% of the plasmids contain a DSB. In addition, it is also unlikely that randomly distributed base lesions could explain the loss of functionality of the plasmid, since cells can repair them efficiently. However, clustered lesions other than DSBs, which are difficult to repair and result in the loss of information on both DNA strands, have the potential to induce the loss of plasmid functionality. We therefore measured the yields of γ-radiation-induced base lesions and cluster damage, which are respectively converted into SSBs and DSBs by the base excision repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg). Our data demonstrate that the yield of cluster damage (i.e., lesions that yield DSBs following digestion) is 31 times higher than that of frank DSBs. This finding suggests that frank DSBs make a relatively minor contribution to the loss of DNA functionality induced by ionizing radiation, while other toxic lesions formed at a much higher frequencies than DSBs must be responsible for the loss of plasmid functionality. These lesions may be clustered lesions/locally multiply damaged sites (LMDS), including base damage, SSBs and/or intrastrand and interstrand crosslinks, leading to the loss of vital information in the DNA. Using a mathematical model, we estimate that at least three toxic lesions are required for the inactivation of plasmid functionality, in part because even these complex lesions can be repaired.

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Section: Discussioncontrasting

confidence: 69%

The Relative Contributions of DNA Strand Breaks, Base Damage and Clustered Lesions to the Loss of DNA Functionality Induced by Ionizing Radiation

et al. 2014

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“…In mouse lyoma cells, electroporation in the presence of linear DNA reached higher level of transfection in comparison to cells electroporated with circular DNA. Following investigations in bacterial and mammalian cells, using two or three DNA isomers (linearized, circular supercoiled or circular relaxed DNA), showed that circular DNA, rather than linear DNA, is optimal for successful gene electrotransfer [169, 331, 332]. These studies revealed that DNA interaction with the membrane is not dependent on its topological isometry but DNA expression is significantly lower for linearized DNA.…”

Section: Gene Electrotransfermentioning

confidence: 99%

“…In bacterial cells, Xie et al observed a lower stability of the linear DNA, which is interpreted as a rapid degradation by intracellular enzymes [332]. Based on DNA geometry and stiffness, one can suggest other hypotheses [331]. Since circular DNA has a more congealed form and a bigger diameter than linear DNA (20-30 nm vs. 2-3 nm), its electrophoretic migration could be more efficient and/or increase the size/lifetime of membrane defects (pores) or cause larger membrane invaginations near the absorbed DNA.…”

Section: Gene Electrotransfermentioning

confidence: 99%

Gene Electrotransfer: A Mechanistic Perspective

Rosazza¹,

Meglič²,

Zumbusch³

et al. 2016

CGT

Self Cite

176

142

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Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

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“…These data indicate that even longterm storage of naked plasmid DNA at controlled conditions of -20°C preserves the plasmid topology as long as for 7 years and prevents the plasmid from degradation. This is of primary importance when plasmid DNA is used for gene therapy studies because it has been shown that ccc forms of plasmid DNA are required for effective gene transfer compared with oc or linearized forms (Remaut et al, 2006;Tolmachov, 2009;Escoffre et al, 2012).…”

Section: Analyses Of Plasmid Dna Topologies By Cge and Agementioning

confidence: 99%

A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability, Topology, andIn Vitro/In VivoFunctional Analysis

Walther

Schmeer²,

Kobelt³

et al. 2013

Human Gene Therapy Clinical Development

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The great interest for naked plasmid DNA in gene therapy studies is reflected by the fact that it is currently used in 18% of all gene therapy trials. Therefore, validation of topology and functionality of DNA resulting from its long-term stability is an essential requirement for safe and effective gene transfer. To this aim, we analyzed the stability of good manufacturing practice-grade pCMVb reporter plasmid DNA by capillary gel electrophoresis, agarose gel electrophoresis, and atomic force microscopy. The plasmid DNA was produced for a clinical gene transfer study started in 2005 and was stored for meanwhile 7 years under continuously monitored conditions at -20°C. The stability of plasmid DNA was monitored by LacZ transgene expression functional assays performed in vitro and in vivo on the 7-year-old plasmid DNA samples compared with plasmid batches newly produced in similar experimental conditions and quality standards. The analyses revealed that during the overall storage time and conditions, the proportion of open circular and supercoiled or covalently closed circular forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jetinjection of DNA showed unaltered functionality of the long-stored plasmid. In summary, the 7-year-old and the newly produced plasmid samples showed similar topology and expression performance. Therefore, our stable storage conditions are effective to preserve the integrity of the DNA to be used in clinical studies. This is an important prerequisite for the long-term performance of gene transfer materials used in trials of long duration as well as of the reference material used in standardization procedures and assays.

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New Insights in the Gene Electrotransfer Process: Evidence for the Involvement of the Plasmid DNA Topology

Cited by 17 publications

References 37 publications

The Relative Contributions of DNA Strand Breaks, Base Damage and Clustered Lesions to the Loss of DNA Functionality Induced by Ionizing Radiation

The Relative Contributions of DNA Strand Breaks, Base Damage and Clustered Lesions to the Loss of DNA Functionality Induced by Ionizing Radiation

Gene Electrotransfer: A Mechanistic Perspective

A Seven-Year Storage Report of Good Manufacturing Practice–Grade Naked Plasmid DNA: Stability, Topology, andIn Vitro/In VivoFunctional Analysis

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