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A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis

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Item Type:Article
Title:A seven-year storage report of good manufacturing practice-grade naked plasmid DNA: stability, topology, and in vitro/in vivo functional analysis
Creators Name:Walther, W. and Schmeer, M. and Kobelt, D. and Baier, R. and Harder, A. and Walhorn, V. and Anselmetti, D. and Aumann, J. and Fichtner, I. and Schleef, M.
Abstract:The great interest of 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 GMP-grade pCMV-{beta} 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 to 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 (oc) and supercoiled or covalently closed circular (ccc) forms is conserved without linearization or degradation of the plasmid. The in vitro transfection and the in vivo jet-injection 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 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.
Keywords:Circular DNA, Genetic Therapy, Plasmids, Quality Control, Reporter Genes, Specimen Handling, Time Factors, Transfection, Tumor Cell Line, Animals, Mice
Source:Human Gene Therapy Clinical Development
ISSN:2324-8637
Publisher:Mary Ann Liebert (U.S.A.)
Volume:24
Number:4
Page Range:147-153
Date:December 2013
Official Publication:https://doi.org/10.1089/humc.2013.067
PubMed:View item in PubMed

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