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In vivo NMR imaging evaluation of efficiency and toxicity of gene electrotransfer in rat muscle

Gene Therapy volume 12, pages 1434–1443 (2005)Cite this article

Abstract

In vivo gene electrotransfer (ET) is a simple method of gene delivery in various tissues relying on the injection of plasmid DNA followed by application of electric pulses. Noninvasive tools are needed to evaluate the ET efficiency and the resulting tissue damages. In this study, we performed ET of rat tibialis muscle after injection of either a plasmid coding for luciferase or a contrast agent (CA) detected by using magnetic resonance imaging (MRI). Plasmid expression and CA intracellular trapped quantity were compared throughout the electric field intensity range 0–300 V/cm. Although the CA trapped quantity reflects only the electropermeabilization step, both measurements were correlated. MRI measurements gave easy access to tridimensional visualization of the labelled zones where the CA has been injected and the applied electric field had a value allowing permeabilization. We also performed MRI measurements of the water transverse relaxation time T2 as an indicator of tissue modification, and tested whether another CA specific for necrosis could be used to detect muscle necrosis at high electric field intensity. In conclusion, MRI measurements may bring multiparametric information upon the efficiency and tissue toxicity of an ET protocol by using a simple and safe CA.

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References

  1. Bloquel C, Fabre E, Bureau MF, Scherman D . Plasmid DNA electrotransfer for intracellular and secreted proteins expression: new methodological developments and applications. J Gene Med 2004; 6 (Suppl 1): S11–S23.

    Article  CAS  Google Scholar 

  2. Rizzuto G 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.

    Article  CAS  Google Scholar 

  3. Bloquel C et al. Gene therapy of collagen-induced arthritis by electrotransfer of human tumor necrosis factor-alpha soluble receptor I variants. Hum Gene Ther 2004; 15: 189–201.

    Article  CAS  Google Scholar 

  4. Silvestre JS et al. Antiangiogenic effect of interleukin-10 in ischemia-induced angiogenesis in mice hindlimb. Circ Res 2000; 87: 448–452.

    Article  CAS  Google Scholar 

  5. Watanabe K et al. Protection against autoimmune myocarditis by gene transfer of interleukin-10 by electroporation. Circulation 2001; 104: 1098–1100.

    Article  CAS  Google Scholar 

  6. Rols MP et al. In vivo electrically mediated protein and gene transfer in murine melanoma. Nat Biotechnol 1998; 16: 168–171.

    Article  CAS  Google Scholar 

  7. Gehl J . Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol Scand 2003; 177: 437–447.

    Article  CAS  Google Scholar 

  8. Vilquin JT et al. Electrotransfer of naked DNA in the skeletal muscles of animal models of muscular dystrophies. Gene Therapy 2001; 8: 1097–1107.

    Article  CAS  Google Scholar 

  9. Widera G et al. Increased DNA vaccine delivery and immunogenicity by electroporation in vivo. J Immunol 2000; 164: 4635–4640.

    Article  CAS  Google Scholar 

  10. Mir LM . Therapeutic perspectives of in vivo cell electropermeabilization. Bioelectrochemistry 2001; 53: 1–10.

    Article  CAS  Google Scholar 

  11. Mir LM et al. High-efficiency gene transfer into skeletal muscle mediated by electric pulses. Proc Natl Acad Sci USA 1999; 96: 4262–4267.

    Article  CAS  Google Scholar 

  12. Aihara H, Miyazaki J . Gene transfer into muscle by electroporation in vivo. Nat Biotechnol 1998; 16: 867–870.

    Article  CAS  Google Scholar 

  13. Mathiesen I . Electropermeabilization of skeletal muscle enhances gene transfer in vivo. Gene Therapy 1999; 6: 508–514.

    Article  CAS  Google Scholar 

  14. Vicat JM et al. Muscle transfection by electroporation with high-voltage and short-pulse currents provides high-level and long-lasting gene expression. Hum Gene Ther 2000; 11: 909–916.

    Article  CAS  Google Scholar 

  15. Durieux AC, Bonnefoy R, Manissolle C, Freyssenet D . High-efficiency gene electrotransfer into skeletal muscle: description and physiological applicability of a new pulse generator. Biochem Biophys Res Commun 2002; 296: 443–450.

    Article  CAS  Google Scholar 

  16. Umeda Y et al. Skeletal muscle targeting in vivo electroporation-mediated HGF gene therapy of bleomycin-induced pulmonary fibrosis in mice. Lab Invest 2004; 84: 836–844.

    Article  CAS  Google Scholar 

  17. Payen E et al. Improvement of mouse beta-thalassemia by electrotransfer of erythropoietin cDNA. Exp Hematol 2001; 29: 295–300.

    Article  CAS  Google Scholar 

  18. Gehl J et al. In vivo electroporation of skeletal muscle: threshold, efficacy and relation to electric field distribution. Biochim Biophys Acta 1999; 1428: 233–240.

    Article  CAS  Google Scholar 

  19. Mir LM, Banoun H, Paoletti C . Introduction of definite amounts of nonpermeant molecules into living cells after electropermeabilization: direct access to the cytosol. Exp Cell Res 1988; 175: 15–25.

    Article  CAS  Google Scholar 

  20. Hartikka J 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.

    Article  CAS  Google Scholar 

  21. Paturneau-Jouas M et al. Electrotransfer at MR imaging: tool for optimization of gene transfer protocols – feasibility study in mice. Radiology 2003; 228: 768–775.

    Article  Google Scholar 

  22. Rinck P . Magnetic Resonance in Medicine: The Basic Textbook of the European Magnetic Resonance Forum, 4th edn. Blackwell Science: Berlin, Germany, 2001.

    Google Scholar 

  23. Wishnia A, Alameddine H, Tardif de Gery S, Leroy-Willig A . Use of magnetic resonance imaging for noninvasive characterization and follow-up of an experimental injury to normal mouse muscles. Neuromuscul Disord 2001; 11: 50–55.

    Article  CAS  Google Scholar 

  24. Ni Y et al. Magnetic resonance imaging-histomorphologic correlation studies on paramagnetic metalloporphyrins in rat models of necrosis. Invest Radiol 1997; 32: 770–779.

    Article  CAS  Google Scholar 

  25. Mathur-de-Vré R, Lemort M . Biophysical properties and clinical applications of magnetic resonance imaging contrast agents. Br J Radiol 1995; 68: 225–247.

    Article  Google Scholar 

  26. Rozijn TH et al. Determination of in vivo rat muscle Gd-DTPA relaxivity at 6.3 T. Magma 1999; 9: 65–71.

    CAS  PubMed  Google Scholar 

  27. Crich S et al. Relaxation efficiency of cell internalized Gd-HPDO3A. ESMRMB 21th Annual Meeting, Copenhagen, September 8–12, 2004, p S222.

  28. Gehl J, Mir LM . Determination of optimal parameters for in vivo gene transfer by electroporation, using a rapid in vivo test for cell permeabilization. Biochem Biophys Res Commun 1999; 261: 377–380.

    Article  CAS  Google Scholar 

  29. Paturneau-Jouas M et al. Electroporation-mediated delivery of a magnetic resonance imaging contrast agent into muscle to visualize electrotransfer. Acta Myologica 2001; 20: 174–178.

    Google Scholar 

  30. Van Donkelaar CC et al. Diffusion tensor imaging in biomechanical studies of skeletal muscle function. J Anat 1999; 194: 79–88.

    Article  Google Scholar 

  31. Wishnia A, Alameddine H, Tardif de Géry S, Leroy-Willig A . Use of magnetic resonance imaging for noninvasive characterisation and follow-up of experimental injury to normal mouse muscle. Neuromusc Disord 2001; 11: 50–55.

    Article  CAS  Google Scholar 

  32. Ferret E, Evrard C, Foucal A, Gervais P . Volume changes of isolated human K562 leukemia cells induced by electric field pulses. Biotechnol Bioeng 2000; 67: 520–528.

    Article  CAS  Google Scholar 

  33. Bureau MF et al. Importance of association between permeabilization and electrophoretic forces for intramuscular DNA electrotransfer. Biochim Biophys Acta 2000; 1474: 353–359.

    Article  CAS  Google Scholar 

  34. Allamand V et al. Early adenovirus-mediated gene transfer effectively prevents muscular dystrophy in alpha-sarcoglycan-deficient mice. Gene Therapy 2000; 7: 1385–1391.

    Article  CAS  Google Scholar 

  35. Palasz A, Czekaj P . Toxicological and cytophysiological aspects of lanthanides action. Acta Biochim Pol 2000; 47: 1107–1114.

    CAS  PubMed  Google Scholar 

  36. Crich SG et al. Improved route for the visualization of stem cells labeled with a Gd-/Eu-chelate as dual (MRI and fluorescence) agent. Magn Reson Med 2004; 51: 938–944.

    Article  CAS  Google Scholar 

  37. Bell JD, Taylor-Robinson SD . Assessing gene expression in vivo: magnetic resonance imaging and spectroscopy. Gene Therapy 2000; 7: 1259–1264.

    Article  CAS  Google Scholar 

  38. Soubrier F et al. pCOR: a new design of plasmid vectors for nonviral gene therapy. Gene Therapy 1999; 6: 1482–1488.

    Article  CAS  Google Scholar 

  39. Brasch RC . Rationale and applications for macromolecular Gd-based contrast agents. Magn Reson Med 1991; 22: 282–287.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by a grant of the Association Française contre les Myopathies. We thank Dr JM Caillaud (Biodoxis, Paris, France) for performing the histological analysis.

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Authors and Affiliations

  1. NMR Unit (AFM-CEA), Institute of Myology, IFR14, Pitié-Salpêtrière University Hospital, Paris, France

    A Leroy-Willig & P G Carlier

  2. Pharmacologie Chimique et Génétique, U640 INSERM and UMR CNRS 8151, Faculté de Pharmacie, Paris, France

    M F Bureau & D Scherman

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Appendix I: Quantification of the CA trapped quantity

Appendix I: Quantification of the CA trapped quantity

In normal untreated muscle, the mean muscle signal intensity in the absence of Gd-DOTA trapped quantity depends on the muscle water relaxation times T1o and T2o and on time intervals in the measurement sequence, TR and TE,22 according to:

where T1o, T2o are respectively the longitudinal and transverse relaxation times of muscle water, and So is the asymptotic value of signal at very long value of time TR and very short value of time TE, reflecting tissue water content. Sm (mean value of signal in untreated muscle) is measured in the reference untreated ROI of the slice of interest.

The increased signal intensity in the labelled muscle ROI with CA concentration C is Si. Signal increase is related to the shortening of longitudinal relaxation time, with actual value T1, by

T1 shortening is related to the tissue CA concentration C by 1/T1=1/T1o+KC, where K is the relaxivity of the CA.

T2 shortening induced by the CA, calculated from a similar equation, is negligible, with variation under 1 ms in the range of Gd-DOTA concentration for these experiments.

The relation between signal and local concentration is based upon an estimation of Gd-DOTA relaxivity in muscle cells. We assume the relaxivity of Gd-DOTA K to be 2.7/s/mM, deduced from Gd-DOTA relaxivity in water, and from experimental data obtained by Rozijn et al26 in rat muscle and in water solution, for the similar agent Gd-DTPA. The signal intensity is determined experimentally in water from a phantom with calibrated concentrations of Gd-DOTA, in acquisition conditions identical to those used in vivo. From the measurements obtained in the water phantom and from muscle relaxation times, the equation linking the signal increase R=(Si−Sm)/Sm to the concentration of Gd-DOTA C is

We here assume that So is not modified, and that the influence of T2 variation can be neglected since TE value is short. This is an acceptable approximation at value of electric field under 200 V/cm where T2 and T1 variations induced by oedema are weak. At higher electric field, the reference region is taken in a neighbouring muscle, also submitted to ET but not to injection, so that T2 variations linked to oedema do not interfere with determination of C. In case of increased T2, overestimation of C and then of Q would follow from this approximation.

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Leroy-Willig, A., Bureau, M., Scherman, D. et al. In vivo NMR imaging evaluation of efficiency and toxicity of gene electrotransfer in rat muscle. Gene Ther 12, 1434–1443 (2005). https://doi.org/10.1038/sj.gt.3302541

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