Journal of Molecular Medicine

, Volume 89, Issue 5, pp 515–529 | Cite as

Development of S/MAR minicircles for enhanced and persistent transgene expression in the mouse liver

  • Orestis Argyros
  • Suet Ping Wong
  • Constantinos Fedonidis
  • Oleg Tolmachov
  • Simon N. Waddington
  • Steven J. Howe
  • Marcello Niceta
  • Charles Coutelle
  • Richard P. Harbottle
Original Article


We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.


Scaffold/matrix attachment region (S/MAR) Minicircle Plasmid Non-viral Gene therapy Liver Hydrodynamic delivery 



The work was supported by the Myrovlytis Trust.

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

109_2010_713_MOESM1_ESM.pdf (2.2 mb)
Supplementary Figures (PDF 2231 kb)


  1. 1.
    Gill DR, Smyth SE, Goddard CA, Pringle IA, Higgins CF, Colledge WH, Hyde SC (2001) Increased persistence of lung gene expression using plasmids containing the ubiquitin C or elongation factor 1alpha promoter. Gene Ther 8:1539–1546. doi: 10.1038/ PubMedCrossRefGoogle Scholar
  2. 2.
    Wolff JA, Ludtke JJ, Acsadi G, Williams P, Jani A (1992) Long-term persistence of plasmid DNA and foreign gene expression in mouse muscle. Hum Mol Genet 1:363–369PubMedCrossRefGoogle Scholar
  3. 3.
    Hodges BL, Taylor KM, Joseph MF, Bourgeois SA, Scheule RK (2004) Long-term transgene expression from plasmid DNA gene therapy vectors is negatively affected by CpG dinucleotides. Mol Ther 10:269–278. doi: 10.1016/j.ymthe.2004.04.018 PubMedCrossRefGoogle Scholar
  4. 4.
    Riu E, Chen ZY, Xu H, He CY, Kay MA (2007) Histone modifications are associated with the persistence or silencing of vector-mediated transgene expression in vivo. Mol Ther 15:1348–1355PubMedCrossRefGoogle Scholar
  5. 5.
    Zhou HS, Liu DP, Liang CC (2004) Challenges and strategies: the immune responses in gene therapy. Med Res Rev 24:748–761. doi: 10.1002/med.20009 PubMedCrossRefGoogle Scholar
  6. 6.
    Zhao H, Hemmi H, Akira S, Cheng SH, Scheule RK, Yew NS (2004) Contribution of Toll-like receptor 9 signaling to the acute inflammatory response to nonviral vectors. Mol Ther 9:241–248. doi: 10.1016/j.ymthe.2003.11.012 PubMedCrossRefGoogle Scholar
  7. 7.
    Chen ZY, Riu E, He CY, Xu H, Kay MA (2008) Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation. Mol Ther 16:548–556PubMedCrossRefGoogle Scholar
  8. 8.
    Chen ZY, He CY, Meuse L, Kay MA (2004) Silencing of episomal transgene expression by plasmid bacterial DNA elements in vivo. Gene Ther 11:856–864. doi: 10.1038/ PubMedCrossRefGoogle Scholar
  9. 9.
    Argyros O, Wong SP, Niceta M, Waddington SN, Howe SJ, Coutelle C, Miller AD, Harbottle RP (2008) Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector. Gene Ther 15:1593–1605PubMedCrossRefGoogle Scholar
  10. 10.
    Turker MS (2002) Gene silencing in mammalian cells and the spread of DNA methylation. Oncogene 21:5388–5393. doi: 10.1038/sj.onc.1205599 PubMedCrossRefGoogle Scholar
  11. 11.
    Curradi M, Izzo A, Badaracco G, Landsberge N (2002) Molecular mechanisms of gene silencing mediated by DNA methylation. Mol Cell Biol 22:3157–3173PubMedCrossRefGoogle Scholar
  12. 12.
    Yew NS, Zhao H, Przybylska M, Wu IH, Tousignant JD, Scheule RK, Cheng SH (2002) CpG-depleted plasmid DNA vectors with enhanced safety and long-term gene expression in vivo. Mol Ther 5:731–738. doi: 10.1006/mthe.2002.0598 PubMedCrossRefGoogle Scholar
  13. 13.
    Bigger BW, Tolmachov O, Collombet JM, Fragkos M, Palaszewski I, Coutelle C (2001) An araC-controlled bacterial cre expression system to produce DNA minicircle vectors for nuclear and mitochondrial gene therapy. J Biol Chem 276:23018–23027. doi: 10.1074/jbc.M010873200 PubMedCrossRefGoogle Scholar
  14. 14.
    Chen ZY, He CY, Ehrhardt A, Kay MA (2003) Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 8:495–500PubMedCrossRefGoogle Scholar
  15. 15.
    Darquet AM, Rangara R, Kreiss P, Schwartz B, Naimi S, Delaere P, Crouzet J, Scherman D (1999) Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer. Gene Ther 6:209–218. doi: 10.1038/ PubMedCrossRefGoogle Scholar
  16. 16.
    Nehlsen K, Broll S, Bode J (2006) Replicating minicircles: generation of nonviral episomes for the efficient modification of dividing cells. Gene Ther Mol Biol 10:233–244Google Scholar
  17. 17.
    Broll S, Oumard A, Hahn K, Schambach A, Bode J (2010) Minicircle performance depending on S/MAR-nuclear matrix interactions. J Mol Biol 395:950–965PubMedCrossRefGoogle Scholar
  18. 18.
    Vaysse L, Gregory LG, Harbottle RP, Perouzel E, Tolmachov O, Coutelle C (2006) Nuclear-targeted minicircle to enhance gene transfer with non-viral vectors in vitro and in vivo. J Gene Med 8:754–763. doi: 10.1002/jgm.883 PubMedCrossRefGoogle Scholar
  19. 19.
    Zhang X, Epperly MW, Kay MA, Chen ZY, Dixon T, Franicola D, Greenberger BA, Komanduri P, Greenberger JS (2008) Radioprotection in vitro and in vivo by minicircle plasmid carrying the human manganese superoxide dismutase transgene. Hum Gene Ther 19:820–826. doi: 10.1089/hum.2007.141 PubMedCrossRefGoogle Scholar
  20. 20.
    Harraghy N, Gaussin A, Mermod N (2008) Sustained transgene expression using MAR elements. Curr Gene Ther 8:353–366PubMedCrossRefGoogle Scholar
  21. 21.
    Bode J, Benham C, Knopp A, Mielke C (2000) Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements). Crit Rev Eukaryot Gene Expr 10:73–90PubMedGoogle Scholar
  22. 22.
    Bode J, Winkelmann S, Gotze S, Spiker S, Tsutsui K, Bi C, KP A, Benham C (2006) Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy. J Mol Biol 358:597–613PubMedCrossRefGoogle Scholar
  23. 23.
    Benham C, Kohwi-Shigematsu T, Bode J (1997) Stress-induced duplex DNA destabilization in scaffold/matrix attachment regions. J Mol Biol 274:181–196PubMedCrossRefGoogle Scholar
  24. 24.
    Jackson DA, Juranek S, Lipps HJ (2006) Designing nonviral vectors for efficient gene transfer and long-term gene expression. Mol Ther 14:613–626PubMedCrossRefGoogle Scholar
  25. 25.
    Jenke BH, Fetzer CP, Stehle IM, Jonsson F, Fackelmayer FO, Conradt H, Bode J, Lipps HJ (2002) An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. EMBO Rep 3:349–354. doi: 10.1093/embo-reports/kvf070 PubMedCrossRefGoogle Scholar
  26. 26.
    Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res 27:426–428PubMedCrossRefGoogle Scholar
  27. 27.
    Jacobs F, Snoeys J, Feng Y, Van Craeyveld E, Lievens J, Armentano D, Cheng SH, De Geest B (2008) Direct comparison of hepatocyte-specific expression cassettes following adenoviral and nonviral hydrodynamic gene transfer. Gene Ther 15:594–603PubMedCrossRefGoogle Scholar
  28. 28.
    Chen L, Woo SL (2008) Site-specific transgene integration in the human genome catalyzed by phiBT1 phage integrase. Hum Gene Ther 19:143–151. doi: 10.1089/hum.2007.110 PubMedCrossRefGoogle Scholar
  29. 29.
    Kass SU, Goddard JP, Adams RL (1993) Inactive chromatin spreads from a focus of methylation. Mol Cell Biol 13:7372–7379PubMedGoogle Scholar
  30. 30.
    Goetze S, Baer A, Winkelmann S, Nehlsen K, Seibler J, Maass K, Bode J (2005) Performance of genomic bordering elements at predefined genomic loci. Mol Cell Biol 25:2260–2272PubMedCrossRefGoogle Scholar
  31. 31.
    Luis Galbete J, Buceta M, Mermod N (2009) MAR elements regulate the probability of epigenetic switching between active and inactive gene expression. Mol Biosyst 5:143–150. doi: 10.1039/b813657b CrossRefGoogle Scholar
  32. 32.
    Limberis MP, Bell CL, Wilson JM (2009) Identification of the murine firefly luciferase-specific CD8 T-cell epitopes. Gene Ther 16:441–447PubMedCrossRefGoogle Scholar
  33. 33.
    Bates MK, Zhang G, Sebestyen MG, Neal ZC, Wolff JA, Herweijer H (2006) Genetic immunization for antibody generation in research animals by intravenous delivery of plasmid DNA. Biotechniques 40:199–208PubMedCrossRefGoogle Scholar
  34. 34.
    Wong SP, Argyros O, Coutelle C, Harbottle RP (2009) Strategies for the episomal modification of cells. Curr Opin Mol Ther 11:433–441PubMedGoogle Scholar
  35. 35.
    Chen ZY, Yant SR, He CY, Meuse L, Shen S, Kay MA (2001) Linear DNAs concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol Ther 3:403–410. doi: 10.1006/mthe.2001.0278 PubMedCrossRefGoogle Scholar
  36. 36.
    Wong SP, Argyros O, Coutelle C, Harbottle RP (2011) Non-viral S/MAR vectors replicate episomally in vivo when provided with a selective advantage. Gene Ther 18:82–87. doi: 10.1038/gt.2010.116 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Orestis Argyros
    • 1
  • Suet Ping Wong
    • 1
  • Constantinos Fedonidis
    • 1
  • Oleg Tolmachov
    • 2
  • Simon N. Waddington
    • 3
  • Steven J. Howe
    • 4
  • Marcello Niceta
    • 1
  • Charles Coutelle
    • 1
  • Richard P. Harbottle
    • 1
  1. 1.Gene Therapy Research Group, Section of Molecular Medicine, National Heart and Lung InstituteImperial College LondonLondonUK
  2. 2.Cardiovascular Science, Faculty of Medicine, National Heart and Lung InstituteImperial College LondonLondonUK
  3. 3.University College London, Institute for Women’s HealthLondonUK
  4. 4.Molecular Immunology Unit, Institute of Child HealthUniversity College LondonLondonUK

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