545. Comparison between Different Tissues for Efficient and Safe DNA Electrotransfer In Vivo
Authors: Franck M. Andre, Lluis M. Mir
Journal: Molecular Therapy (2006)

The major limitation of gene therapy is the vector used for gene transfer. Viruses were used first and are still the most common approach. Due to adverse events in clinical trials, there was a need for non viral methods. Among them, DNA electrotransfer is very promising. In vivo DNA electrotransfer consist in the injection of a DNA plasmid, either locally or intravenously, followed by electric pulses directly applied to the targeted tissue. To obtain a good gene transfection in skeletal muscles, 8 short square Electric Pulses (EP) of 200 V/cm and 20ms duration delivered at a repetition frequency of 1Hz are often used. However, in our laboratory, a further increase in the efficacy of the gene transfer in skeletal muscles was obtained using combinations of short, High Voltage EP (HV; 800 V/cm, 100 μs) and long, Low Voltage EP (LV; 80 V/cm, 100 ms) [1]. The efficacy of the HV+LV combinations is due to the two different effects of the EP: the electropermeabilization of the target cells (mainly due to the HV) and the electrophoresis of the DNA (caused by the LV) towards or across the permeabilized plasma membranes. For optimal DNA transfer in the skeletal muscle and other tissues, the combinations of LV and HV have to be optimized for each tissue and a better understanding of the underlying mechanisms is required. To reach these goals, we compared DNA electrotransfer efficiency and safety in various tissues using combinations of HV and LV. We performed experiments in mice, on skeletal muscle, tumor (B16F1 melanoma), liver, kidney, and adipose tissue. We used HV and LV of different voltage to distance ratios and in some cases pulse number and duration was also varied. In all tissues low or even no transfection was obtained by means of plasmid injection only and the use of HV+LV pulses lead to an increase in transfer efficacy of up to 4 orders of magnitude compared to the injection alone. Increase in the HV electric field amplitude resulted in a progressive increase in electrotransfer efficacy until an optimal plateau in gene expression is reached. For higher field amplitude a decrease is observed due to increase in electrical lesions. The plateau values vary from tissue to tissue. For example in muscle plateau begins at 400V/cm, whereas in tumor the plateau starts only at 800V/cm. The comparison between the various tissues treated shows that the bigger the cells, the lower the value at which the plateau starts, which is in total agreement with the fundamentals of cell permeabilization as a result of changes in the transmembrane potential.However, in liver, contrary to all the other tissues, the HV pulse was not necessary since the LV pulses alone were sufficient to achieve efficient electrotransfer. Increase in HV amplitude did not lead to any increase in DNA transfer efficacy, even though cell toxicity was provoked when HV of high field strengths were applied. This unexpected behavior could be due to the presence of a large number of gap junctions between the hepatocytes, that electrically connect the cells.[1] S. Šatkauskas, F. André, M. F. Bureau, D. Scherman, D. Miklavcic and L. M. Mir. The electrophoretic component of the electric pulses determines the efficacy of in vivo DNA electrotransfer. Human Gene Therapy, 16, 1194-1201 The major limitation of gene therapy is the vector used for gene transfer. Viruses were used first and are still the most common approach. Due to adverse events in clinical trials, there was a need for non viral methods. Among them, DNA electrotransfer is very promising. In vivo DNA electrotransfer consist in the injection of a DNA plasmid, either locally or intravenously, followed by electric pulses directly applied to the targeted tissue. To obtain a good gene transfection in skeletal muscles, 8 short square Electric Pulses (EP) of 200 V/cm and 20ms duration delivered at a repetition frequency of 1Hz are often used. However, in our laboratory, a further increase in the efficacy of the gene transfer in skeletal muscles was obtained using combinations of short, High Voltage EP (HV; 800 V/cm, 100 μs) and long, Low Voltage EP (LV; 80 V/cm, 100 ms) [1]. The efficacy of the HV+LV combinations is due to the two different effects of the EP: the electropermeabilization of the target cells (mainly due to the HV) and the electrophoresis of the DNA (caused by the LV) towards or across the permeabilized plasma membranes. For optimal DNA transfer in the skeletal muscle and other tissues, the combinations of LV and HV have to be optimized for each tissue and a better understanding of the underlying mechanisms is required. To reach these goals, we compared DNA electrotransfer efficiency and safety in various tissues using combinations of HV and LV. We performed experiments in mice, on skeletal muscle, tumor (B16F1 melanoma), liver, kidney, and adipose tissue. We used HV and LV of different voltage to distance ratios and in some cases pulse number and duration was also varied. In all tissues low or even no transfection was obtained by means of plasmid injection only and the use of HV+LV pulses lead to an increase in transfer efficacy of up to 4 orders of magnitude compared to the injection alone. Increase in the HV electric field amplitude resulted in a progressive increase in electrotransfer efficacy until an optimal plateau in gene expression is reached. For higher field amplitude a decrease is observed due to increase in electrical lesions. The plateau values vary from tissue to tissue. For example in muscle plateau begins at 400V/cm, whereas in tumor the plateau starts only at 800V/cm. The comparison between the various tissues treated shows that the bigger the cells, the lower the value at which the plateau starts, which is in total agreement with the fundamentals of cell permeabilization as a result of changes in the transmembrane potential. However, in liver, contrary to all the other tissues, the HV pulse was not necessary since the LV pulses alone were sufficient to achieve efficient electrotransfer. Increase in HV amplitude did not lead to any increase in DNA transfer efficacy, even though cell toxicity was provoked when HV of high field strengths were applied. This unexpected behavior could be due to the presence of a large number of gap junctions between the hepatocytes, that electrically connect the cells. [1] S. Šatkauskas, F. André, M. F. Bureau, D. Scherman, D. Miklavcic and L. M. Mir. The electrophoretic component of the electric pulses determines the efficacy of in vivo DNA electrotransfer. Human Gene Therapy, 16, 1194-1201