Nanosecond electric pulses-induced enhancement of reporter gene expression after electrotransfer in skeletal muscle in vivo

We explored whether electric pulses of a duration of a few tens of nanoseconds (nsPEF) and of very high electric field amplitude (20 to 150 kV/cm), called nanopulses, could affect the expression of electrotransferred genes, improving the overall efficacy of non-viral, electric pulses-mediated, gene transfer. Contrary to the micro- and milliseconds pulses, the nanopulses act on the cell inside and may have targets other than the cell plasma membrane. DNA coding for the luciferase was electrotransferred into skeletal muscle of mice using conventional procedures of electrotransfer. Then tissues were exposed or not to nanopulses (10 ns duration) with a new exposure technique. nsPEF were applied by using insulated electrodes in order to overcome electrochemical reactions that could interfere with biological material and to prevent direct contact with high electric voltage. Muscle were processed 48 or 72 hours after. Measurements were performed using a luminometer and results were expressed as pg luciferase/mg muscle. An increase of two to three times of the luciferase activity in the extracts can be achieved with the application of a train of 30,000 nanosecond pulses of 30 kV (corresponding to a 40kV/cm static field), 0 to 60 minutes after plasmid electrotransfer. The time between DNA electrotransfer and nanopulse(s) delivery (between 0 and 60 minutes) seems to affect biological results. Other parameters influencing the luciferase gene expression as well as the mechanisms underlying such increase are under analysis. Cells and tissue manipulation by means of electric nanopulses delivery might be an efficient way to increase the overall efficacy of gene electrotransfer.