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| Item Type: | Article | ||||
|---|---|---|---|---|---|
| Title: | Capillary gel electrophoresis (CGE) for quality control of plasmid DNA in gene therapy: quality control of 20 years stored GMP-grade plasmid DNA | ||||
| Creators Name: | Schmeer, M. and Schleef, M. and Shankar, R. and Kobelt, D. and Walther, W. | ||||
| Abstract: | Plasmid DNA in any form (plasmid DNA, minicircle, miniplasmid) does experience renewed and increasing attention for use in gene therapy and DNA vaccination. For such applications, stability analyses and quality control are essential prerequisites for clinical use. In this context we analyzed the stability of good manufacturing practice (GMP)-grade pCMVβ reporter plasmid DNA by capillary gel electrophoresis. The plasmid DNA was produced for a clinical gene transfer study for treatment of malignant melanoma. The pCMVβ plasmid DNA was stored at -20 °C for 20 years under continuous, controlled monitoring. Another plasmid., pCMV-Luc, stored for 15 years, served as reference. The stability of plasmid DNA was analyzed by capillary gel electrophoresis (CGE) and functionally tested in vitro by LacZ functional assay. In this chapter we provide the detailed description of CGE and functional analysis of the GMP-grade pCMVβ and also pCMV-Luc plasmid DNA. By this the proportion of open circular and supercoiled or covalently closed circular forms of plasmid DNA is analyzed. Functionality of the plasmid was tested by in vitro transfection and LacZ functional assay. In result of this, the 20-year-old plasmid DNA showed topology and expression performance, which revealed significant alterations in topology while maintaining functionality regarding transgene expression. Therefore, stable storage conditions are effective to mainly preserve the integrity of the plasmid DNA as important parameter for long-term storage of, for example, reference samples. | ||||
| Keywords: | CGE Analysis, Clinical Trial, Covalently Closed Circular, GMP, Plasmid DNA, Supercoil | ||||
| Source: | Methods in Molecular Biology | ||||
| Series Name: | Methods in Molecular Biology | ||||
| Title of Book: | Gene therapy of cancer : methods and protocols | ||||
| ISSN: | 1064-3745 | ||||
| ISBN: | 9781071624401 | ||||
| Publisher: | Springer / Humana Press | ||||
| Volume: | 2521 | ||||
| Number: | 3 | ||||
| Page Range: | 317-328 | ||||
| Date: | 23 June 2022 | ||||
| Official Publication: | https://doi.org/10.1007/978-1-0716-2441-8_17 | ||||
| PubMed: | View item in PubMed | ||||
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