Abstract
Gene transfer into cells or tissue by application of electric pulses (i.e. gene electrotransfer (GET)) is a non-viral gene delivery method that is becoming increasingly attractive for clinical applications. In order to make GET progress to wide clinical usage its efficacy needs to be improved and the safety of the method has to be confirmed. Therefore, the aim of our study was to increase GET efficacy in skin, by optimizing electric pulse parameters and the design of electrodes. We evaluated the safety of our novel approach by assaying the thermal stress effect of GET conditions and the biodistribution of a cytokine expressing plasmid. Transfection efficacy of different pulse parameters was determined using two reporter genes encoding for the green fluorescent protein (GFP) and the tdTomato fluorescent protein, respectively. GET was performed using non-invasive contact electrodes immediately after intradermal injection of plasmid DNA into mouse skin. Fluorescence imaging of transfected skin showed that a sophistication in the pulse parameters could be selected to get greater transfection efficacy in comparison to the standard ones. Delivery of electric pulses only mildly induced expression of the heat shock protein Hsp70 in a luminescent reporting transgenic mouse model, demonstrating that there were no drastic stress effects. The plasmid was not detected in other organs and was found only at the site of treatment for a limited period of time. In conclusion, we set up a novel approach for GET combining new electric field parameters with high voltage short pulses and medium voltage long pulses using contact electrodes, to obtain a high expression of both fluorescent reporter and therapeutic genes while showing full safety in living animals.
Highlights
The skin is a physical barrier that shields the body from external agents
Low Voltage (LV) and High Voltage (HV) parameters used alone induced an almost undetectable expression of the protein whereas the application of Medium Voltage (MV) parameters induced a consistent expression of the tdTomato protein
The association of HV and LV parameters induced an improved tdTomato expression that was further increased using the combination of HV and MV parameters
Summary
The skin is a physical barrier that shields the body from external agents. This organ is an attractive target for gene therapy and vaccination, due to its accessibility, large surface area and the presence of numerous immune cells such as Langerhans cells and other dendritic cells that can elicit appropriate immune responses to defend the body[1,2]. Gene electrotransfer (GET) is obtained by a local and controlled injection of a small volume of a plasmid solution followed by electrical voltage pulses delivery between electrodes in contact with the target tissue[19]. Higher voltage to electrode distance ratio were needed to obtain the electrical conductivity across the SC and as result a way to affect the dermis and the epidermis. This technology was observed to be effective for pDNA expression in the skin. Our group introduced a few years ago another concept of non-invasive electrodes allowing the electrotreatment of a large skin area[43] These contact electrodes were observed to be highly safe and efficient for electroimmunization[44]
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