Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Therapeutic levels of erythropoietin (EPO) achieved after gene electrotransfer to skin in mice

Gene Therapy volume 17, pages 1077–1084 (2010)Cite this article

Subjects

Abstract

Gene electrotransfer refers to gene transfection by electroporation and is an effective non-viral method for delivering naked DNA into cells and tissues. This study presents data from gene electrotransfer with erythropoietin (EPO) to mouse skin. Nine-week-old female NMRI mice received one, two or three intradermal injections of 50 μg EPO plasmid and were subsequently electroporated. With plate electrodes and 100 μg of EPO, a significant increase in hemoglobin (P<0.01) was observed compared with controls. The level of hemoglobin peaked after 5 weeks but stayed significantly elevated for more than 3 months. Serum EPO was significantly increased (P<0.001) 24 h after the transfection and remained significantly different compared with controls until the maximum level of serum EPO was reached after 2 weeks. Eight weeks after the transfection serum EPO returned to baseline. In this study, we have established that gene electrotransfer to skin of even small amounts of DNA can lead to systemically therapeutic levels of protein. This means that in addition to DNA vaccinations, there is a potential utility for electroporation in alleviating systemic diseases such as cancer and protein deficiency disorders.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Kohn DB, Candotti F . Gene therapy fulfilling its promise. N Engl J Med 2009; 360: 518–521.

    Article  CAS  PubMed  Google Scholar 

  2. Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A et al. Direct gene transfer into mouse muscle in vivo. Science 1990; 247 (4949 Part 1): 1465–1468.

    CAS  PubMed  Google Scholar 

  3. Hengge UR, Chan EF, Foster RA, Walker PS, Vogel JC . Cytokine gene expression in epidermis with biological effects following injection of naked DNA. Nat Genet 1995; 10: 161–166.

    Article  CAS  PubMed  Google Scholar 

  4. Nicolau C, Le PA, Soriano P, Fargette F, Juhel MF . In vivo expression of rat insulin after intravenous administration of the liposome-entrapped gene for rat insulin I. Proc Natl Acad Sci USA 1983; 80: 1068–1072.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Alexander MY, Akhurst RJ . Liposome-medicated gene transfer and expression via the skin. Hum Mol Genet 1995; 4: 2279–2285.

    Article  CAS  PubMed  Google Scholar 

  6. Yang NS, Burkholder J, Roberts B, Martinell B, McCabe D . In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment. Proc Natl Acad Sci USA 1990; 87: 9568–9572.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Mavilio F, Pellegrini G, Ferrari S, Di NF, Di IE, Recchia A et al. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. Nat Med 2006; 12: 1397–1402.

    Article  CAS  PubMed  Google Scholar 

  8. Lucas ML, Heller L, Coppola D, Heller R . IL-12 plasmid delivery by in vivo electroporation for the successful treatment of established subcutaneous B16.F10 melanoma. Mol Ther 2002; 5: 668–675.

    Article  CAS  PubMed  Google Scholar 

  9. McCray AN, Ugen KE, Heller R . Enhancement of anti-melanoma activity of a plasmid expressing HIV-1 Vpr delivered through in vivo electroporation. Cancer Biol Ther 2007; 6: 1269–1275.

    Article  CAS  PubMed  Google Scholar 

  10. Hojman P, Gissel H, Gehl J . Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation. Gene Therapy 2007; 14: 950–959.

    Article  CAS  PubMed  Google Scholar 

  11. Trochon-Joseph V, Martel-Renoir D, Mir LM, Thomaidis A, Opolon P, Connault E et al. Evidence of antiangiogenic and antimetastatic activities of the recombinant disintegrin domain of metargidin. Cancer Res 2004; 64: 2062–2069.

    Article  CAS  PubMed  Google Scholar 

  12. Mir LM, Moller PH, Andre F, Gehl J . Electric pulse-mediated gene delivery to various animal tissues. In: Huang L, Hung MC, Wagner E, (eds). Nonviral Vectors for Gene Therapy, Part II. 2nd edn. Elsevier Academic Press: San Diego, CA, USA, 2005. pp. 83–114.

    Chapter  Google Scholar 

  13. Cemazar M, Sersa G . Electrotransfer of therapeutic molecules into tissues. Curr Opin Mol Ther 2007; 9: 554–562.

    CAS  PubMed  Google Scholar 

  14. Bodles-Brakhop AM, Heller R, Draghia-Akli R . Electroporation for the delivery of DNA-based vaccines and immunotherapeutics: current clinical developments. Mol Ther 2009; 17: 585–592.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Muramatsu T, Arakawa S, Fukazawa K, Fujiwara Y, Yoshida T, Sasaki R et al. In vivo gene electroporation in skeletal muscle with special reference to the duration of gene expression. Int J Mol Med 2001; 7: 37–42.

    CAS  PubMed  Google Scholar 

  16. Drabick JJ, Glasspool-Malone J, King A, Malone RW . Cutaneous transfection and immune responses to intradermal nucleic acid vaccination are significantly enhanced by in vivo electropermeabilization. Mol Ther 2001; 3: 249–255.

    Article  CAS  PubMed  Google Scholar 

  17. Roos AK, Moreno S, Leder C, Pavlenko M, King A, Pisa P . Enhancement of cellular immune response to a prostate cancer DNA vaccine by intradermal electroporation. Mol Ther 2006; 13: 320–327.

    Article  CAS  PubMed  Google Scholar 

  18. Dobano C, Widera G, Rabussay D, Doolan DL . Enhancement of antibody and cellular immune responses to malaria DNA vaccines by in vivo electroporation. Vaccine 2007; 25: 6635–6645.

    Article  CAS  PubMed  Google Scholar 

  19. Zhang L, Li L, Hoffmann GA, Hoffman RM . Depth-targeted efficient gene delivery and expression in the skin by pulsed electric fields: an approach to gene therapy of skin aging and other diseases. Biochem Biophys Res Commun 1996; 220: 633–636.

    Article  CAS  PubMed  Google Scholar 

  20. Glasspool-Malone J, Somiari S, Drabick JJ, Malone RW . Efficient nonviral cutaneous transfection. Mol Ther 2000; 2: 140–146.

    Article  CAS  PubMed  Google Scholar 

  21. Pavselj N, Preat V . DNA electrotransfer into the skin using a combination of one high- and one low-voltage pulse. J Control Release 2005; 106: 407–415.

    Article  CAS  PubMed  Google Scholar 

  22. Heller LC, Jaroszeski MJ, Coppola D, McCray AN, Hickey J, Heller R . Optimization of cutaneous electrically mediated plasmid DNA delivery using novel electrode. Gene Therapy 2007; 14: 275–280.

    Article  CAS  PubMed  Google Scholar 

  23. Lee PY, Chesnoy S, Huang L . Electroporatic delivery of TGF-beta1 gene works synergistically with electric therapy to enhance diabetic wound healing in db/db mice. J Invest Dermatol 2004; 123: 791–798.

    Article  CAS  PubMed  Google Scholar 

  24. Lin MP, Marti GP, Dieb R, Wang J, Ferguson M, Qaiser R et al. Delivery of plasmid DNA expression vector for keratinocyte growth factor-1 using electroporation to improve cutaneous wound healing in a septic rat model. Wound Repair Regen 2006; 14: 618–624.

    Article  PubMed  Google Scholar 

  25. Maruyama H, Ataka K, Higuchi N, Sakamoto F, Gejyo F, Miyazaki J . Skin-targeted gene transfer using in vivo electroporation. Gene Therapy 2001; 8: 1808–1812.

    Article  CAS  PubMed  Google Scholar 

  26. Gothelf A, Hojman P, Gehl J . Change in hemoglobin levels due to anesthesia in mice; an important confounder in studies on hematopoietic drugs. Biol Proced Online 2009; 11: 325–330.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Pagel H, Engel A, Jelkmann W . Erythropoietin induction by hypoxia. A comparison of in vitro and in vivo experiments. Adv Exp Med Biol 1992; 317: 515–519.

    Article  CAS  PubMed  Google Scholar 

  28. Whelan G, Flecknell PA . The use of etorphine/methotrimeprazine and midazolam as an anaesthetic technique in laboratory rats and mice. Lab Anim 1994; 28: 70–77.

    Article  CAS  PubMed  Google Scholar 

  29. Andre FM, Gehl J, Sersa G, Preat V, Hojman P, Eriksen J et al. Efficiency of high- and low-voltage pulse combinations for gene electrotransfer in muscle, liver, tumor, and skin. Hum Gene Ther 2008; 19: 1261–1272.

    Article  CAS  PubMed  Google Scholar 

  30. Lezon CE, Martinez MP, Conti MI, Bozzini CE . Plasma disappearance of exogenous erythropoietin in mice under different experimental conditions. Endocrine 1998; 8: 331–333.

    Article  CAS  PubMed  Google Scholar 

  31. Bugelski PJ, Nesspor T, Volk A, O'Brien J, Makropoulos D, Shamberger K et al. Pharmacodynamics of recombinant human erythropoietin in murine bone marrow. Pharm Res 2008; 25: 369–378.

    Article  CAS  PubMed  Google Scholar 

  32. Galson DL, Tan CC, Ratcliffe PJ, Bunn HF . Comparison of the human and mouse erythropoietin genes shows extensive homology in the flanking regions. Blood 1993; 82: 3321–3326.

    CAS  PubMed  Google Scholar 

  33. Wen D, Boissel JP, Tracy TE, Gruninger RH, Mulcahy LS, Czelusniak J et al. Erythropoietin structure-function relationships: high degree of sequence homology among mammals. Blood 1993; 82: 1507–1516.

    CAS  PubMed  Google Scholar 

  34. McMahon FG, Vargas R, Ryan M, Jain AK, Abels RI, Perry B et al. Pharmacokinetics and effects of recombinant human erythropoietin after intravenous and subcutaneous injections in healthy volunteers. Blood 1990; 76: 1718–1722.

    CAS  PubMed  Google Scholar 

  35. Woo S, Krzyzanski W, Jusko WJ . Pharmacokinetic and pharmacodynamic modeling of recombinant human erythropoietin after intravenous and subcutaneous administration in rats. J Pharmacol Exp Ther 2006; 319: 1297–1306.

    Article  CAS  PubMed  Google Scholar 

  36. Magnani M, Rossi L, Stocchi V, Cucchiarini L, Piacentini G, Fornaini G . Effect of age on some properties of mice erythrocytes. Mech Ageing Dev 1988; 42: 37–47.

    Article  CAS  PubMed  Google Scholar 

  37. Hoffmann-Fezer G, Mysliwietz J, Mortlbauer W, Zeitler HJ, Eberle E, Honle U et al. Biotin labeling as an alternative nonradioactive approach to determination of red cell survival. Ann Hematol 1993; 67: 81–87.

    Article  CAS  PubMed  Google Scholar 

  38. Gothelf A, Eriksen J, Hojman P, Gehl J . Duration and level of transgene expression after gene electrotransfer to skin in mice. Gene Therapy 2009; Accepted for publication.

  39. Medi BM, Hoselton S, Marepalli RB, Singh J . Skin targeted DNA vaccine delivery using electroporation in rabbits. I: efficacy. Int J Pharm 2005; 294: 53–63.

    Article  CAS  PubMed  Google Scholar 

  40. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK et al. Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 2008; 26: 5896–5903.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Ferraro B, Cruz YL, Coppola D, Heller R . Intradermal delivery of plasmid VEGF(165) by electroporation promotes wound healing. Mol Ther 2009; 17: 651–657.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lubbert H et al. Doxycycline-mediated quantitative and tissue-specific control of gene expression in transgenic mice. Proc Natl Acad Sci USA 1996; 93: 10933–10938.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Rizzuto G, Cappelletti M, Maione D, Savino R, Lazzaro D, Costa P et al. Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation. Proc Natl Acad Sci USA 1999; 96: 6417–6422.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Hojman P, Eriksen J, Gehl J . Tet-On Induction with Doxycycline after Gene Transfer in Mice: Sweetening of Drinking Water is not a Good Idea. Anim Biotechnol 2007; 18: 183–188.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The study was funded by the ANGIOSKIN project, EU 6th Frame Program and Copenhagen County Research Council. We thank Anne Boye, Lone Christensen, Marianne Fregil and Birgit Hertz for excellent technical assistance.

Author information

Authors and Affiliations

  1. Department of Oncology 54B1, Center for Experimental Drug and Gene Electrotransfer, Copenhagen University Hospital Herlev, Herlev, Denmark

    A Gothelf, P Hojman & J Gehl

  2. Centre of Inflammation and Metabolism, Copenhagen University Hospital, Copenhagen, Denmark

    P Hojman

Authors
  1. A Gothelf
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P Hojman
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J Gehl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J Gehl.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on Gene Therapy website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gothelf, A., Hojman, P. & Gehl, J. Therapeutic levels of erythropoietin (EPO) achieved after gene electrotransfer to skin in mice. Gene Ther 17, 1077–1084 (2010). https://doi.org/10.1038/gt.2010.46

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/gt.2010.46

Keywords

This article is cited by

Search

Advanced search

Quick links