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Maintenance of Plasmid Expression in vivo Depends Primarily on the CpG Contents of the Vector and Transgene

  • MOLECULAR CELL BIOLOGY
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Abstract

Plasmid-mediated gene therapy, being a safe and relatively inexpensive therapeutic strategy, is plagued by a fast silencing of transgene expression. The silencing severely reduces the long-term efficiency of plasmid vectors. We have earlier constructed a low-CpG pMBR2 plasmid vector supporting prolonged expression of transgenes in mesenchymal stem cells in vitro. Long-term expression from the pMBR2 vector was studied for the wild-type mouse secreted alkaline phosphatase gene (mSEAPTwt) and its version devoid of CpGs (mSEAP0) after vector electroporation into mouse hindlimb muscles and hydrodynamic delivery to the liver. The mSEAP levels in the blood were measured over one year. With the pMBR2-mSEAP0 construct, the mSEAP levels in leg muscles increased more than 2.5-fold in the first two months and remained higher than the initial level until the end of the experiment. Far lower expression levels were observed with the control pCDNA3.1-mSEAP0 construct. Expression from pMBR2-mSEAPwt decreased to about 40% after 6 months and remained at similar levels thereafter. In the mouse liver, expression from pMBR2-mSEAP0 was approximately halved within the first 18 weeks and then decrease slowly to the final 17% level. Expression from pMBR2-mSEAPwt initially dropped to 18% and remained at approximately 10% thereafter. In contrast, expression from pCDNA3.1-mSEAP0 sharply dropped to 5% after 2 weeks and remained at nearly zero levels throughout the rest of the experiment. Thus, both vector and transgene should have significantly reduced CpG contents to ensure prolonged plasmid-mediated expression in the liver, while minimizing the vector CpG content is sufficient for expression in skeletal muscles. The results suggested additionally that the localization of S/MAR elements within the transcription unit, in contrast to their outside location, results in significant reduction of the level of secreted, but not cytoplasmic, proteins.

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Funding

This work was supported by the Russian Science Foundation (project no. 18-14-00300).

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Authors and Affiliations

  1. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991, Moscow, Russia

    A. V. Bruter, M. V. Kalashnikova, A. P. Prytyko & A. V. Belyavsky

  2. Blokhin National Cancer Research Center, Ministry of Health of the Russian Federation, 115478, Moscow, Russia

    A. V. Bruter & M. V. Kalashnikova

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Statement on the welfare of animals. Experiments with animals were performed in accordance with the ARRIVE guidance and the European Union directive 2010/63/EU on the protection of animals used for scientific purposes. The study protocol was approved by the Ethics Committee for animal research at the Blokhin National Cancer Research Center.

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Translated by T. Tkacheva

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Bruter, A.V., Kalashnikova, M.V., Prytyko, A.P. et al. Maintenance of Plasmid Expression in vivo Depends Primarily on the CpG Contents of the Vector and Transgene. Mol Biol 54, 427–435 (2020). https://doi.org/10.1134/S0026893320030048

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