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Performance of High Quality Minicircle DNA for In Vitro and In Vivo Gene Transfer

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Abstract

Plasmid DNA is frequently used particularly for nonviral gene therapy. Conventional plasmid DNA contains bacterial backbone and resistance gene sequences, as well as immunogenic CpG motifs. These components are not required for transgene expression. They represent a potential risk for safe clinical application and reduce gene transfer rates as well as transgene expression. To overcome these drawbacks, the minicircle technology is removing such sequences, to improve performance and also to reduce DNA size. Here, we show the effective production of luciferase, GFP, or lacZ-carrying minicircle DNA with high yield and reproducible high quality. They are used for lipofection or electroporation gene transfer into human melanoma and colon carcinoma cell lines. Comparison of respective parental plasmid and minicircle-mediated luciferase gene transfer shows improved luciferase expression by minicircle in all cell lines. This is not associated with increase in intracellular minicircle copy numbers after lipofection or electroporation. The minicircles rather mediate enhanced transgene mRNA transcription compared to their parental plasmids. In addition, FACS analysis revealed increase in counts of GFP positive cells after minicircle gene transfer, indicating higher gene transfer rates. Furthermore, minicircle showed also improved performance in vivo after jet-injection gene transfer. Therefore, availability of minicircles with reproducible high quality and sufficient amount makes them an applicable and effective alternative to conventional plasmid gene vectors.

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Acknowledgements

We thank U. Stein for helpful discussion and J. Conde Lopez for assistance with the figures. This work was supported by the H.W. & J. Hector foundation, grant M48.2 (to WW) as well as by the CliniGene Network of Excellence funded by the European Commission FP6 Research Program under contract LSHB-CT-2006-018933 and the German Federal Ministry of Education and Research (BMBF) by the grant BioChancePLUS (0313749) to PlasmidFactory.

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Authors and Affiliations

  1. Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Dennis Kobelt & Wolfgang Walther

  2. PlasmidFactory GmbH & CO KG, Meisenstr. 96, 33607, Bielefeld, Germany

    Martin Schleef & Marco Schmeer

  3. Experimental and Clinical Research Center, Charité University Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany

    Jutta Aumann & Wolfgang Walther

  4. Charite Comprehensive Cancer Center, Charite, Invalidenstr. 80, 10117, Berlin, Germany

    Peter M. Schlag

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  1. Dennis Kobelt
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  2. Martin Schleef
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  6. Wolfgang Walther
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Correspondence to Wolfgang Walther.

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Kobelt, D., Schleef, M., Schmeer, M. et al. Performance of High Quality Minicircle DNA for In Vitro and In Vivo Gene Transfer. Mol Biotechnol 53, 80–89 (2013). https://doi.org/10.1007/s12033-012-9535-6

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