Abstract
Gene electrotransfer is an attractive method of non-viral gene delivery. However, the mechanism of DNA penetration across the plasma membrane is widely discussed. To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/low-amplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture. This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. Indeed, after centrifugation of the pulsed viral suspension and later on addition to cells, the activity was found mainly associated with the aluminum aggregates concentrated in the lower fraction and was proportional to generated quantities. Overall, this work focused on the use of electrotransfer to facilitate the adenovirus entry into cell, demonstrating that modifications of the penetrating agent can be more important than modifications of the target cell for transfer efficacy.
Highlights
Gene electrotransfer is an attractive method of non-viral gene delivery
The effects of several types of electric pulses on after the rAd-mediated gene transfer (AMGT) were tested for two cell lines: L929, weakly transduced by adenovirus type 5 (Ad5), and CHO-hCAR, which exhibits, on the contrary, an excellent rate of adenovirus infection
A minor influence of high voltage (HV) pulses delivered prior to low voltage (LV) ones was mainly observed for the fluorescence intensity, since 98% of cells were already Green Fluorescent Protein (GFP)+ with LV pulses alone (Table 1a)
Summary
The mechanism of DNA penetration across the plasma membrane is widely discussed To explore this process for even larger structures, like viruses, we applied various combinations of short/long and high/lowamplitude electric pulses to L929 cells, mixed with a human adenovirus vector expressing GFP. We observed a transgene expression increase, both in the number of GFP-converted cells and GFP levels, when we added a low-voltage/millisecond-pulse treatment to the adenovirus/cell mixture This increase, reflecting enhanced virus penetration, was proportional to the applied electric field amplitude and pulse number, but was not associated with membrane permeabilization, nor to direct cell modifications. We demonstrated that this effect is mainly due to adenovirus particle interactions with aggregated aluminum particles released from energized electrodes. In a given cell type, we can directly estimate the virus penetration into cells by measuring the expression of the reporter gene, inserted into the viral genome
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