Real time electroporation control for accurate and safe in vivo non-viral gene therapy David Cukjati a,b , Danute Batiuskaite a,c , Franck André a , Damijan Miklavčič b , Lluis M. Mir a, ⁎ a UMR 8121 CNRS, Institute Gustave–Roussy, 39 Rue Camille Desmoulins, F-94805 Villejuif Cédex, France b University of Ljubljana, Faculty of Electrical Engineering, Laboratory of Biocybernetics, Tržaška 25, SI-1000 Ljubljana, Slovenia c Vytautas Magnus University, Faculty of Natural Sciences, Department of Biology, Vileikos 8, LT-44404 Kaunas, Lithuania Received 21 August 2006; received in revised form 5 November 2006; accepted 6 November 2006 Available online 10 November 2006 Abstract In vivo cell electroporation is the basis of DNA electrotransfer, an efficient method for non-viral gene therapy using naked DNA. The electric pulses have two roles, to permeabilize the target cell plasma membrane and to transport the DNA towards or across the permeabilized membrane by electrophoresis. For efficient electrotransfer, reversible undamaging target cell permeabilization is mandatory. We report the possibility to monitor in vivo cell electroporation during pulse delivery, and to adjust the electric field strength on real time, within a few microseconds after the beginning of the pulse, to ensure efficacy and safety of the procedure. A control algorithm was elaborated, implemented in a prototype device and tested in luciferase gene electrotransfer to mice muscles. Controlled pulses resulted in protection of the tissue and high levels of luciferase in gene transfer experiments where uncorrected excessive applied voltages lead to intense muscle damage and consecutive loss of luciferase gene expression. © 2006 Elsevier B.V. All rights reserved. Keywords: DNA electrotransfer; Gene therapy; Electropermeabilization; Electroporation; Electrochemotherapy; Finite element modeling 1. Introduction Biotechnological and biomedical applications of in vivo deli- very of short high voltage pulses, like in vivo DNA electro- transfer, also termed electrogenetherapy, are rapidly developing [1–5]. For efficient in vivo gene transfer, it is necessary to inject DNA into the tissue and to achieve cell plasma membrane per- meabilization [6]. Increased membrane permeability results from supraphysiological transmembrane voltages induced by external electric pulses [7–9]. Mechanisms of DNA electrotransfer in vivo have recently been described [6,10]. The two key steps are the permeabilization of the target cells plasma membrane by electro- poration and the electrophoresis of the DNA within the tissue. These two effects can be obtained separately using the appropriate sequence of electric pulses: short (100 μs) square-wave high voltage pulses (HV) that permeabilize the cells without subs- tantial DNA transport to the cells and long (100 ms) low voltage pulses (LV), that are instrumental in facilitating the DNA transfer into the cells [6]. Even though gene transfer efficacy, measured by gene expression level, depends on the characteristics of the elec- trophoretic long low voltage pulse, target cell permeabilization is mandatory for efficient gene transfer. Moreover, for a safe gene transfer, electropermeabilization, also termed electroporation, must be reversible, that is not excessive, in order to avoid per- manent cell damage. Optimal parameters for in vivo electropora- tion can be determined using in vivo tests for cell permeabilization [11] after the pulse, like the one based on 51Cr-EDTA uptake [12], and by using mathematical modeling to determine electric field distribution [13,14]. However, it would be much better to control cell permeabilization during the pulse delivery in order to ascer- tain that (reversible) cell permeabilization will be actually achie- ved at the end of the pulse, as well as to prevent excessive (irreversible) permeabilization [11,13,15]. Real time control of electroporation appears thus critical for this non-viral gene trans- fection method that has many advantages with respect to viral methods. Here, we report that in vivo electroporation can be precisely computer-controlled to ascertain that permeabilization will be achieved at the end of the pulse, while at the same time permanent cell damage is prevented. We demonstrate that the temporal progression of tissue electroporation can be detected in real time Bioelectrochemistry 70 (2007) 501 – 507 www.elsevier.com/locate/bioelechem ⁎ Corresponding author. Tel.: +33 1 42114792; fax: +33 1 42115276. E-mail address: luismir@igr.fr (L.M. Mir). 1567-5394/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bioelechem.2006.11.001