PlasmidFactory GmbH & Co. KG, Bielefeld, Germany.
Max-Delbrück-Center for Molecular Medicine, Berlin, Germany.
Methods Mol Biol. 2022;2521:317-328. doi: 10.1007/978-1-0716-2441-8_17.
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.
任何形式的质粒 DNA（质粒 DNA、小型环、微型质粒）都在基因治疗和 DNA 疫苗接种方面引起了人们的关注。对于此类应用，稳定性分析和质量控制是临床应用的必要前提。在这种情况下，我们通过毛细管凝胶电泳分析了符合良好生产规范 (GMP) 级别的 pCMVβ 报告质粒 DNA 的稳定性。该质粒 DNA 是为治疗恶性黑色素瘤的临床基因转移研究生产的。pCMVβ 质粒 DNA 在 -20°C 下连续、受控监测下储存了 20 年。另一个储存了 15 年的质粒 pCMV-Luc 用作参考。通过毛细管凝胶电泳 (CGE) 分析质粒 DNA 的稳定性，并通过体外 LacZ 功能测定进行功能测试。在本章中，我们提供了 GMP 级别的 pCMVβ 和 pCMV-Luc 质粒 DNA 的 CGE 和功能分析的详细描述。通过这种方法，可以分析质粒 DNA 的开环和超螺旋或共价闭合环状形式的比例。通过体外转染和 LacZ 功能测定测试质粒的功能。结果表明，20 年的质粒 DNA 显示出拓扑结构和表达性能，其拓扑结构发生了显著变化，而转基因表达的功能保持不变。因此，稳定的储存条件可以有效地主要保持质粒 DNA 的完整性，这是长期储存例如参考样本的重要参数。
