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. 1999 Apr 13;96(8):4262-7.
doi: 10.1073/pnas.96.8.4262.

High-efficiency gene transfer into skeletal muscle mediated by electric pulses

L M Mir  1 , M F Bureau, J Gehl, R Rangara, D Rouy, J M Caillaud, P Delaere, D Branellec, B Schwartz, D Scherman
Affiliations

High-efficiency gene transfer into skeletal muscle mediated by electric pulses

L M Mir et al. Proc Natl Acad Sci U S A. .

Abstract

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.

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Figures

Figure 1
Figure 1
Characteristics of DNA electrotransfer in muscle fibers. Bars represent the mean ± SEM of individual values (triangles), observed in the absence (white bars) or the presence (gray bars) of electric-pulse delivery. The ordinate scale is logarithmic. (A) Influence of the applied voltage-to-distance ratio (eight pulses; 20 ms per pulse; 1 Hz). (B) Influence of pulse length (eight pulses; 200 V/cm; 1 Hz). (C) Influence of pulse number (200 V/cm; 20 ms per pulse; 1 Hz). (D) Influence of the frequency of pulse delivery (eight pulses; 200 V/cm; 20 ms per pulse). (E) Dependence of luciferase gene expression on the amount of injected DNA in a constant volume of 30 μl (eight pulses; 200 V/cm; 20 ms per pulse; 1 Hz); (F) Kinetics of luciferase gene expression in the absence (open circles) or presence (filled squares) of electrotransfer (eight pulses; 200 V/cm; 20 ms per pulse; 2 Hz). Abscissa: time after plasmid injection. Mice were killed 7 days (A–E) or at the indicated times (F) after DNA transfer.
Figure 2
Figure 2
β-Galactosidase expression in mouse tibial cranial muscle after DNA injection with or without the application of electric pulses. (A and B) Legs from mice injected with DNA without (A) or with (B) exposure to the electric pulses (eight pulses; 200 V/cm; 20 ms per pulse; 1 Hz). (C and D) Macroscopic photographs of the whole tibial cranial muscle of mice injected with DNA without (C) or with (D) exposure to electric pulses.
Figure 3
Figure 3
Comparative effect of the time of injection vs. time of electric-pulse delivery on the in vivo 51Cr-EDTA uptake and on luciferase gene expression by muscle fibers. Bars represent the mean of the individuals values; error bars represent SEM. Gray bars represent the uptake of 51Cr-EDTA (A) or the uptake of plasmid DNA (B) as assessed by luciferase gene expression 7 days after the electric pulses (eight pulses; 200 V/cm; 20 ms per pulse; 1 Hz). White bars represent control values observed in the absence of electric pulses.
Figure 4
Figure 4
FGF1 expression in mouse tibial cranial muscle. A run of eight pulses of 200 V/cm and 20 ms per pulse at 2 Hz was used, and muscle was removed 7 days after electrotransfer of the pXL3096 plasmid.
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