Mechanisms of in Vivo DNA Electrotransfer: Respective Contributions of Cell Electropermeabilization and DNA Electrophoresis

Abstract

Efficient cell electrotransfection can be achieved using combinations of high-voltage (HV; 800 V/cm, 100 micros) and low-voltage (LV; 80 V/cm, 100 ms) pulses. We have developed equipment allowing the generation of various HV and LV combinations with precise control of the lag between the HV and LV pulses. We injected luciferase-encoding DNA in skeletal muscle, before or after pulse delivery, and measured luciferase expression after various pulse combinations. In parallel, we determined permeabilization levels using uptake of (51)Cr-labeled EDTA. High voltage alone resulted in a high level of muscle permeabilization for 300 seconds, but very low DNA transfer. Combinations of one HV pulse followed by one or four LV pulses did not prolong the high permeabilization level, but resulted in a large increase in DNA transfer for lags up to 100 seconds in the case of one HV + one LV and up to 3000 seconds in the case of one HV + four LV. DNA expression also reached similar levels when we injected the DNA between the HV and LV pulses. We conclude that the role of the HV pulse is limited to muscle cell permeabilization and that the LV pulses have a direct effect on DNA. In vivo DNA electrotransfer is thus a multistep process that includes DNA distribution, muscle permeabilization, and DNA electrophoresis.

DNA electrotransfer: its principles and an updated review of its therapeutic applications

“…42 It was shown that the duration of the high permeabilized state of the muscle fibres was the same for the three combinations tested, all of them including the same HV pulse. On the contrary, the authors found that efficacy was only achieved if at least an LV was delivered after the HV.…”

“…35,41,42 Moreover, for an efficient transfer, DNA must be injected before the electric pulses delivery. Thus, permeabilization of the cells is not sufficient even though it is necessary since efficient electrotransfer requires sufficiently intense electric fields (above cell permeabilization threshold).…”

Control by pulse parameters of DNA electrotransfer into solid tumors in mice

“…[10][11][12][13][14][15][16] A new protocol with high transfection efficacy in muscle and skin has recently been described, consisting of a combination of one high-field (HV, B1000 V cm À1 ) and one (or several) low-field (LV, B100 V cm À1 ) pulse. [17][18][19][20][21][22][23] For simplicity, we called all along the paper 'field' what indeed is the voltage to electrode width ratio, that is, an experimentally controlled parameter. In these studies, the authors suggested two functions of these electric pulses.…”

“…The 'HV' pulse permeabilizes the cells, whereas the 'LV' pulse drives plasmid DNA toward the cells and against the permeabilized membrane by the associated electrophoretic force. 19 In this new methodology, both types of pulses are necessary for successful gene expression in target cells, as described in the literature. The number of 'LV' pulses can vary, provided the overall duration of the pulses is kept constant.…”

“…The number of 'LV' pulses can vary, provided the overall duration of the pulses is kept constant. 19 The amplitude of efficient 'HV' and 'LV' pulses differs between muscle and skin, which might be attributed to the morphological and histological differences between these two tissues. Conditions used for skin transfection are known to be damaging for muscles.…”