Safe and efficient novel approach for non-invasive gene electrotransfer to skin

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.

Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma

“…We recently showed that IL-12 expression was significantly increased following GET protocol [34]. Here, we verified that IL-12 expression can induce IFN-γ production (Fig.…”

“…Values for thermal stress positive control were determined between 39 and 45 degrees Celsius as already described [34] and luciferase activity was measured in the anesthetized animal using a cooled CCD camera (iKon M, Andor, Belfast, UK) as a measure of reporter gene expression. Reporter gene expression was assessed at 6 h post-heating or pIRE treatment, 5 min after intraperitoneal injection of luciferin (3 mg/mouse) in 100 μL of PBS.…”

“…Reporter gene expression was assessed at 6 h post-heating or pIRE treatment, 5 min after intraperitoneal injection of luciferin (3 mg/mouse) in 100 μL of PBS. Mean bioluminescence intensity in the treated area was determined using FIJI [34].…”

“…GET treatment was performed as described in Pasquet et al [34]. Briefly, tumor-surrounding skin was shaved and depilated using a depilatory cream the day prior treatment.…”

“…No thermal side effects were observed as demonstrated by the low activation of the thermo-inducible heat-shock protein (Hsp70) promoter 1B (Hspa1b), in double transgenic Hspa1b-LucF (+/+) Hspa1b-mPlum (+/+) mice. Our pIRE treatment was then combined with peri-tumoral IL-12 GET pre-treatment that was shown to induce a local and rapid production of IL-12 [10, 34] and resulted in a rapid production of IFNγ. This treatment induced (i) an increased survival rate of treated animals, (ii) a systemic activation of the adaptive immune system leading to a growth delay of distant tumors and (iii) the development of immune memory that protected against the emergence of a second tumor.…”