Abstract
Electroporation-based gene transfer (electro gene transfer (EGT)) is gaining increasing momentum, in particular for muscle tissue, where long-term high-level expression is obtainable. Induction of expression using the Tet-On system was previously established; however, attempts to reach a predefined target dose – a prescription, have not been reported. We set three target haemoglobin levels (10, 12 and 14 mmol/l, base level was 8.2 mmol/l) and aimed at them by transferring the erythropoietin (EPO) gene to mouse tibialis cranialis (TC) muscle, and varying (1) DNA amount, (2) muscle mass transfected and (3) induction with the Tet-On system. Results showed that (a) using GFP, luciferase and EPO low DNA amounts were needed. In fact, 0.5 μg of DNA to one TC muscle led to significant Hgb elevation – this amount extrapolates to 1.4 mg of DNA in humans, (b) three prescribers hit the targets with average Hgb of 10.5, 12.0 and 13.7 mmol/l, (c) different approaches could be used, (d) undershooting could be corrected by retransferring, and (e) overshooting could be alleviated by reducing dose of inducer (doxycycline (dox)). In conclusion, this study shows that using EGT to muscle, a preset level of protein expression can be reached. This is of great interest for future clinical use.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Gehl J . Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 2003; 177: 437–447.
Bureau MF, Gehl J, Deleuze V, Mir LM, Scherman D . Importance of association between permeabilisation and electrophoretic forces for intramuscular DNA electrotransfer. Biochim Biophys Acta 2000; 1474: 353–359.
Satkauskas S, Bureau MF, Puc M, Mahfoudi A, Scherman D, Miklavcic D et al. Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilisation and DNA electrophoresis. Mol Ther 2002; 5: 133–140.
Satkauskas S, Andre F, Bureau M, Scherman D, Miklavcic D, Mir LM . Electrophoretic component of electric pulses determines the efficacy of in vivo DNA electrotransfer. Hum Gene Ther 2005; 16: 1194–1201.
Marty M, Sersa G, Garbay JR, Gehl J, Collins CG, Snoj M et al. Electrochemotherapy – an easy, highly effective and safe treatment of cutaneous and subcutaneous metastases: results of ESOPE (European Standard Operating Procedures of Electrochemotherapy) study. Eur J Cancer Suppl 2006; 4: 3–13.
Gehl J, Geertsen PF . Palliation of haemorrhaging and ulcerated cutaneous tumours using electrochemotherapy. Eur J Cancer Suppl 2006; 4: 35–37.
Mir LM . Bases and rationale of the electrochemotherapy. Eur J Cancer Suppl 2006; 4: 38–44.
Heller LC, Heller R . In vivo electroporation for gene therapy. Hum Gene Ther 2006; 17: 890–897.
Lu QL, Bou-Gharios G, Partridge TA . Non-viral gene delivery in skeletal muscle: a protein factory. Gene Therapy 2003; 10: 131–142.
Kreiss P, Bettan M, Crouzet J, Scherman D . Erythropoietin secretion and physiological effect in mouse after intramuscular plasmid DNA electrotransfer. J Gene Med 1999; 1: 245–250.
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.
Payen E, Bettan M, Rouyer-Fessard P, Beuzard Y, Scherman D . Improvement of mouse [beta]-thalassemia by electrotransfer of erythropoietin cDNA. Exp Hematol 2001; 29: 295–300.
Maruyama H, Sugawa M, Moriguchi Y, Imazeki I, Ishikawa Y, Ataka K et al. Continuous erythropoietin delivery by muscle-targeted gene transfer using in vivo electroporation. Hum Gene Ther 2000; 11: 429–437.
Terada Y, Tanaka H, Okado T, Inoshita S, Kuwahara M, Akiba T et al. Efficient and ligand-dependent regulated erythropoietin production by naked dna injection and in vivo electroporation. Am J Kidney Dis 2001; 38: S50–S53.
Muramatsu T, Arakawa S, Fukazawa K, Fujiwara Y, Yishida 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.
Maruyama H, Ataka K, Gejyo F, Higuchi N, Ito Y, Hirahara H et al. Long-term production of erythropoietin after electroporation-mediated transfer of plasmid DNA into the muscles of normal and uremic rats. Gene Therapy 2001; 8: 461–468.
Ataka K, Maruyama H, Neichi T, Miyazaki J, Gejko F . Effects of erythropoietin-gene electrotransfer in rats with adenine-induced renal failure. Am J Nephrol 2003; 23: 315–323.
Rizzuto G, Cappelletti M, Mennuni C, Wiznerowicz M, DeMartis A, Maione D et al. Gene electrotransfer results in a high-level transduction of rat skeletal muscle and corrects anemia of renal failure. Hum Gene Ther 2000; 11: 1891–1900.
Fattori E, Cappelletti M, Zampaglione I, Mennuni C, Calvaruso F, Arcuri M et al. Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates. J Gene Med 2005; 7: 228–236.
Magnani M, Rossi L, Stocchi V, Cucchiarini L, Piacentini G, Fornaini G . Effect of age on some properties of mice erythrocytes. Mech Ageing Develop 1988; 42: 37–47.
Toniatti C, Bujard H, Cortese R, Ciliberto G . Gene therapy progress and prospects: transcription regulatory systems. Gene Therapy 2004; 11: 649–657.
Gossen M, Freundlieb S, Bender G, Muller G, Hillen W, Bujard H . Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268: 1766–1769.
Perez N, Plence P, Millet V, Greuet D, Minot C, Noel D et al. Tetracycline transcriptional silencer tightly controls transgene expression after in vivo intramuscular electrotransfer: application to interleukin 10 therapy in experimental arthritis. Hum Gene Ther 2002; 13: 2161–2172.
Lamartina S, Roscilli G, Rinaudo CD, Sporeno E, Silvi L, Hillen W et al. Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators. Hum Gene Ther 2002; 13: 199–210.
Chenuaud P, Larcher T, Rabinowitz JE, Provost N, Joussemet B, Bujard H et al. Optimal design of a single recombinant adeno-associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle. Mol Ther 2004; 9: 410–418.
Miklavcic D, Semrov D, Mekid H, Mir LM . A validated model of in vivo electric field distribution in tissues for electrochemotherapy and for DNA electrotransfer for gene therapy. Biochim Biophys Acta 2000; 1523: 73–83.
Mir LM, Bureau MF, Gehl J, Rangara R, Rouy D, Caillaud JM et al. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 1999; 96: 4262–4267.
Bohl D, Naffakh N, Heard JM . Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nat Med 1997; 3: 299–305.
Moller PH, 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. In press.
Gothelf A, Mir LM, Gehl J . Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation. Cancer Treat Rev 2003; 29: 371–387.
Mir LM, Gehl J, Sersa G, Collins CG, Garbay J, Billard V et al. Standard operating procedures of the electrochemotherapy: instructions for the use of bleomycin or cisplatin administered either systemically or locally and electric pulses delivered by the CliniporatorTM by means of invasive or non-invasive electrodes. Eur J Cancer Suppl 2006; 4: 14–25.
Aihara H, Miyazaki J . Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 1998; 16: 867–870.
Gehl J, Sorensen TH, Nielsen K, Raskmark P, Nielsen SL, Skovsgaard T et al. In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochim Biophys Acta 1999; 1428: 233–240.
Samakoglu S, Fattori E, Lamartina S, Toniatti C, Stockholm D, Heard JM et al. betaMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in beta thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles. Blood 2001; 97: 2213–2220.
Durieux AC, Bonnefoy R, Freyssenet D . Kinetic of transgene expression after electrotransfer into skeletal muscle: Importance of promoter origin/strength. Biochim Biophys Acta 2005; 1725: 403–409.
Fabre E, Bigey P, Orsini C, Scherman D . Comparison of promoter region constructs for in vivo intramuscular expression. J Gene Med 2006; 8: 636–645.
Fisher JW . Erythropoietin: physiology and pharmacology update. Exp Biol Med 2003; 228: 1–14.
Zhou HS, Liu DP, Liang CC . Challenges and strategies: the immune responses in gene therapy. Med Res Rev 2004; 24: 748–761.
Mathiesen I . Electropermeabilisation of skeletal muscle enhances gene transfer in vivo. Gene Therapy 1999; 6: 508–514.
Donà M, Sandri M, Rossini K, Dell'Aica I, Podhorska-Okolow V, Carraro U . Functional in vivo gene transfer into the myofibers of adult skeletal muscle. Biochem Biophys Res Com 2003; 312: 1132–1138.
Wang XD, Tang JG, Xie XL, Yang JC, Li S, Ji JG et al. A comprehensive study of optimal conditions for naked plasmid DNA transfer into skeletal muscle by electroporation. J Gene Med 2005; 7: 1235–1245.
Molnar MJ, Gilbert R, Lu Y, Liu AB, Guo A, Larochelle N et al. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol Ther 2004; 10: 447–455.
Hartikka J, Sukhu L, Buchner C, Hazard D, Bozoukova V, Margalith M et al. Electroporation-facilitated delivery of plasmid DNA in skeletal muscle: plasmid dependence of muscle damage and effect of poloxamer 188. Mol Ther 2001; 4: 407–415.
Durieux A, Bonnefoy R, Busso T, Freyssenet D . In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage. J Gene Med 2004; 6: 809–816.
Wang M, Orsini C, Casanova D, Millan JL, Mahfoudi A, Thuillier V . MUSEAP, a novel reporter gene for the study of long-term gene expression in immunocompetent mice. Gene 2001; 279: 99–108.
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.
Perez N, Bigey P, Scherman D, Danos O, Piechaczyk M, Pelegrin M . Regulatable systemic production of monoclonal antibodies by in vivo muscle electroporation. Genet Vaccines Ther 2004; 2: 2.
Bettan M, Emmanuel F, Darteil R, Caillaud JM, Soubrier F, Delaère P et al. High-level protein secretion into blood circulation after electric pulse-mediated gene transfer into skeletal muscle. Mol Ther 2000; 2: 204–210.
Gossen M, Bujard H . Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc Natl Acad Sci USA 1992; 89: 5547–5551.
Kistner A, Gossen M, Zimmermann F, Jerecic J, Ullmer C, Lübbert 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.
Acknowledgements
We thank Vibeke Uhre and Marianne Fregil for excellent technical assistance. The study was supported by the Danish Research Agency (22-03-0367) and (22-02-0523).
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Hojman, P., Gissel, H. & Gehl, J. Sensitive and precise regulation of haemoglobin after gene transfer of erythropoietin to muscle tissue using electroporation. Gene Ther 14, 950–959 (2007). https://doi.org/10.1038/sj.gt.3302951
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/sj.gt.3302951
Keywords
This article is cited by
-
Induction of a local muscular dystrophy using electroporation in vivo: an easy tool for screening therapeutics
Scientific Reports (2020)
-
A combination of intradermal jet-injection and electroporation overcomes in vivodose restriction of DNA vaccines
Genetic Vaccines and Therapy (2012)
-
Human Erythropoietin Gene Delivery Using an Arginine-grafted Bioreducible Polymer System
Molecular Therapy (2012)