Efficient episomal gene transfer to human hepatic cells using the pFAR4–S/MAR vector

  • Aristeidis Giannakopoulos
  • Michael Quiviger
  • Eleana Stavrou
  • Meletios Verras
  • Corinne Marie
  • Daniel Scherman
  • Aglaia AthanassiadouEmail author
Original Article


Liver-directed gene therapy, using mainly viral vectors for the genetic cell modification, is a promising therapeutic approach for many genetic and metabolic liver diseases. The recent successful preclinical trials with AAV vectors expose the benefits as well as the limitations of the system. We focused on the development of an alternative non-viral episomal gene transfer system, by inserting the DNA element Scaffold/Matrix Attachment Region (S/MAR) into the free of antibiotic resistance gene miniplasmid vector (pFAR4). We produced pFAR4 derivative experimental vectors, carrying the eGFP gene driven by the composite HCRHPi liver-specific promoter and either lacking (pFAR4–noS/MAR) or containing the S/MAR element in an upstream (pFAR-S/MAR-IN) or downstream (pFAR4–S/MAR-OUT) configuration in relation to the poly-A signal of the eGFP expression cassette. Upon transfer into Huh7 cells by lipofection, vector pFAR4–S/MAR IN showed significantly higher transfection efficiency and eGFP expression than the control vector or the pFAR4–S/MAR-OUT (p < 0.005), estimated by fluorescent microscopy and flow cytometry. Stable transfections were produced only with cultures containing vector pFAR4–S/MAR IN, through the expansion of single colonies, which displayed sustained GFP expression and plasmid copy number per cell of 2.3 ± 0.4, at 3 months of culture. No vector integration events were detected in these cultures by FISH analysis, while the presence of free, circular plasmids was documented by plasmid rescue assay. The presence of S/MAR renders pFAR4 miniplasmid substantially more efficient regarding episomal gene transfer and is suitable for liver-directed studies towards gene therapy applications.


Liver gene therapy Non-viral vectors Episomes pFAR S/MAR 



This work was supported mainly by the Greek Secretariat of Research and Technology grant No. 12ERARE-11-72, through the TRANSPOSMART CONSORTIUM, of the E-RARE ERANET program. The help of M. Keramida with the FISH analysis is gratefully acknowledged and cell lines were a kind gift from professor Zoi Lygerou.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11033_2019_4777_MOESM1_ESM.docx (128 kb)
Supplementary material 1 (DOCX 128 kb)


  1. 1.
    Wolff JA, Malone RW, Williams P et al (1990) Direct gene transfer into mouse muscle in vivo. Science 247:1465–1468CrossRefGoogle Scholar
  2. 2.
    Yokoo T, Kamimura K, Abe H et al (2016) Liver-targeted hydrodynamic gene therapy: recent advances in the technique. World J Gastroenterol 22:8862–8868CrossRefGoogle Scholar
  3. 3.
    Sendra L, Miguel A, Pérez-Enguix D et al (2016) Studying closed hydrodynamic models of “in vivo” DNA perfusion in pig liver for gene therapy translation to humans. PLoS ONE 11:e0163898CrossRefGoogle Scholar
  4. 4.
    Ott MG, Schmidt M, Schwarzwaelder K et al (2006) Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12:401–409. CrossRefGoogle Scholar
  5. 5.
    Hacein-Bey-Abina S, Garrigue A, Wang GP et al (2008) Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J Clin Investig 118:3132–3142. CrossRefGoogle Scholar
  6. 6.
    Kaeppel C, Beattie SG, Fronza R et al (2013) A largely random AAV integration profile after LPLD gene therapy. Nat Med 19:889–891CrossRefGoogle Scholar
  7. 7.
    Nault J-C, Datta S, Imbeaud S et al (2015) Recurrent AAV2-related insertional mutagenesis in human hepatocellular carcinomas. Nat Genet 47:1187–1193CrossRefGoogle Scholar
  8. 8.
    Krieg AM (2002) CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20:709–760CrossRefGoogle Scholar
  9. 9.
    Chen Z-Y, Riu E, He C-Y et al (2008) Silencing of episomal transgene expression in liver by plasmid bacterial backbone DNA is independent of CpG methylation. Mol Ther 16:548–556CrossRefGoogle Scholar
  10. 10.
    Vandermeulen G, Préat V, Scherman D, Marie C (2011) New generation of plasmid backbones devoid of antibiotic resistance marker for gene therapy trials. Mol Ther 19:1942–1949CrossRefGoogle Scholar
  11. 11.
    Chen Z-Y, He C-Y, Ehrhardt A, Kay MA (2003) Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol Ther 8:495–500CrossRefGoogle Scholar
  12. 12.
    Marie C, Vandermeulen G, Quiviger M et al (2010) pFARs, plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells. J Gene Med 12:323–332CrossRefGoogle Scholar
  13. 13.
    Quiviger M, Arfi A, Mansard D et al (2014) High and prolonged sulfamidase secretion by the liver of MPS-IIIA mice following hydrodynamic tail vein delivery of antibiotic-free pFAR4 plasmid vector. Gene Ther 21:1001–1007CrossRefGoogle Scholar
  14. 14.
    Bode J, Winkelmann S, Götze S et al (2006) Correlations between scaffold/matrix attachment region (S/MAR) binding activity and DNA duplex destabilization energy. J Mol Biol 358:597–613CrossRefGoogle Scholar
  15. 15.
    Piechaczek C, Fetzer C, Baiker A et al (1999) A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res 27:426–428CrossRefGoogle Scholar
  16. 16.
    Jenke BHC, Fetzer CP, Stehle IM et al (2002) An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. EMBO Rep 3:349–354. CrossRefGoogle Scholar
  17. 17.
    Stehle IM, Scinteie MF, Baiker A et al (2003) Exploiting a minimal system to study the epigenetic control of DNA replication: the interplay between transcription and replication. Chromosome Res 11:413–421CrossRefGoogle Scholar
  18. 18.
    Schübeler D, Mielke C, Maass K, Bode J (1996) Scaffold/matrix-attached regions act upon transcription in a context-dependent manner. Biochemistry 35:11160–11169CrossRefGoogle Scholar
  19. 19.
    Giannakopoulos A, Stavrou EF, Zarkadis I et al (2009) The functional role of S/MARs in episomal vectors as defined by the stress-induced destabilization profile of the vector sequences. J Mol Biol 387:1239–1249CrossRefGoogle Scholar
  20. 20.
    Chen Z-Y, He C-Y, Kay MA (2005) Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. Hum Gene Ther 16:126–131CrossRefGoogle Scholar
  21. 21.
    Stenler S, Wiklander OP, Badal-Tejedor M et al (2014) Micro-minicircle gene therapy: implications of size on fermentation, complexation, shearing resistance, and expression. Mol Ther Nucleic Acids 3:e140. CrossRefGoogle Scholar
  22. 22.
    Stavrou EF, Lazaris VM, Giannakopoulos A et al (2017) The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells. Sci Rep 7:40673CrossRefGoogle Scholar
  23. 23.
    De Meyer SF, Vandeputte N, Pareyn I et al (2008) Restoration of plasma von Willebrand factor deficiency is sufficient to correct thrombus formation after gene therapy for severe von Willebrand disease. Arterioscler Thromb Vasc Biol 28:1621–1626CrossRefGoogle Scholar
  24. 24.
    Papapetrou EP, Ziros PG, Micheva ID et al (2006) Gene transfer into human hematopoietic progenitor cells with an episomal vector carrying an S/MAR element. Gene Ther 13:40–51CrossRefGoogle Scholar
  25. 25.
    Miao CH, Ye X, Thompson AR (2003) High-level factor VIII gene expression in vivo achieved by nonviral liver-specific gene therapy vectors. Hum Gene Ther 14:1297–1305CrossRefGoogle Scholar
  26. 26.
    Hashemi A, Roohvand F, Ghahremani MH et al (2012) Optimization of transfection methods for Huh-7 and Vero cells: comparative study. Tsitol Genet 46:19–27Google Scholar
  27. 27.
    Hagedorn C, Gogol-Döring A, Schreiber S et al (2017) Genome-wide profiling of S/MAR-based replicon contact sites. Nucleic Acids Res 45:7841–7854. CrossRefGoogle Scholar
  28. 28.
    Schaarschmidt D, Baltin J, Stehle IM et al (2004) An episomal mammalian replicon: sequence-independent binding of the origin recognition complex. EMBO J 23:191–201CrossRefGoogle Scholar
  29. 29.
    Stehle IM, Postberg J, Rupprecht S et al (2007) Establishment and mitotic stability of an extra-chromosomal mammalian replicon. BMC Cell Biol 8:33CrossRefGoogle Scholar
  30. 30.
    Friehs K (2004) Plasmid copy number and plasmid stability. Adv Biochem Eng Biotechnol 86:47–82Google Scholar
  31. 31.
    Griffiths PD (2002) Facilitating viral recombination. Rev Med Virol 12:335–336CrossRefGoogle Scholar
  32. 32.
    Kostyrko K, Neuenschwander S, Junier T et al (2017) MAR-Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering. Biotechnol Bioeng 114:384–396CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of General Biology, Medical SchoolUniversity of PatrasRion, PatrasGreece
  2. 2.CNRS, Unité de Technologies Chimiques et Biologiques pour la Santé (UTCBS), UMR 8258ParisFrance
  3. 3.Chimie Paris Tech, PSL, UTCBSParisFrance
  4. 4.Université Paris Descartes, Sorbonne-Paris-Cité, UTCBSParisFrance
  5. 5.INSERM, UTCBS U 1022ParisFrance
  6. 6.Department of PediatricsUniversity Hospital of PatrasRionGreece

Personalised recommendations