Physiological effects of high- and low-voltage pulse combinations for gene electrotransfer in muscle.

Gene transfer by electroporation is gaining momentum now that high-level, long-term expression of transgenes is being obtained. Several different pulse regimens are efficient, yet little information is available about the physiological muscular response to gene electrotransfer. This paper provides a comprehensive evaluation of the physiological and molecular effects on host tissue after DNA electrotransfer. We have tested several pulse regimens with special emphasis on the pulse combination of a short (100 microsec) high-voltage (HV) pulse followed by a long low-voltage (LV) pulse used for DNA electrotransfer, comparing it with 8 HV pulses designed to ensure extensive permeabilization of the muscle membrane. Using both mouse and rat skeletal muscle tissue, we investigated cell permeabilization by the 51Cr-labeled EDTA assay, lactate dehydrogenase release, Na+ and Ca2+ influx, K+ efflux, ATP release, and water content, as well as muscle function both in vivo and ex vivo, Hsp70 induction, and histology. In all these assays, the HVLV pulse combination gave rise to minimal disturbance of cell function, in all cases significantly different from results when using 8 HV pulses. The evaluated parameters were normalized after 1 week. The addition of DNA caused significantly more transmembrane exchange, and this may be due to entrance of the DNA through the membrane. In conclusion, this study comprehensively documents the immediate effects of DNA electrotransfer and shows that only slight cell disturbances occur with the HVLV pulses used for gene transfer. This is highly important, as minimal perturbation of cell physiology is essential for efficient transgene expression.