The Role of Electrophoresis in Gene Electrotransfer

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase the permeability of a cell membrane and enables transfer of genes. Poor plasmid mobility in tissues is one of the major barriers for the successful use of gene electrotransfer in gene therapy. Therefore, we analyzed the effect of electrophoresis on increasing gene electrotransfer efficiency using different combinations of high-voltage (HV) and low-voltage (LV) pulses in vitro on CHO cells. We designed a special prototype of electroporator, which enabled us to use only HV pulses or combinations of LV + HV and HV + LV pulses. We used optimal plasmid concentrations used in in vitro conditions as well as lower suboptimal concentrations in order to mimic in vivo conditions. Only for the lowest plasmid concentration did the electrophoretic force of the LV pulse added to the HV pulse increase the transfection efficiency compared to using only HV. The effect of the LV pulse was more pronounced for HV + LV, while for the reversed sequence, LV + HV, there was only a minor effect of the LV pulse. For the highest plasmid concentrations no added effect of LV pulses were observed. Our results suggest that there are different contributing effects of LV pulses: electrophoretically increased contact of DNA with the membrane and increased insertion of DNA into permeabilized cell membrane and/or translocation due to electrophoretic force, which appears to be the dominant effect.     

Influence of Plasmid Concentration on DNA Electrotransfer In Vitro Using High-Voltage and Low-Voltage Pulses

DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer experiments 50 μl of CHO cell suspension containing 100, 10 or 1 μg/ml of the plasmid were placed between plate electrodes and subjected to various combinations of HV and LV pulses. The results showed that at 100 μg/ml plasmid concentration LV pulse delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 μg/ml) of the plasmid. In comparison to HV (1,200 V/cm, 100 μs) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold at 10 μg/ml and fivefold at 1 μg/ml. At 10 μg/ml concentration of plasmid, application of four LV pulses after HV pulse increased transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer can be investigated in vitro using HV and LV pulses.     

Calcium influx determines the muscular response to electrotransfer

Cell membrane permeabilization by electric pulses (electropermeabilization), results in free exchange of ions across the cell membrane. The role of electrotransfer-mediated Ca(2+)-influx on muscle signaling pathways involved in degeneration (β-actin and MurF), inflammation (IL-6 and TNF-α), and regeneration (MyoD1, myogenin, and Myf5) was investigated, using pulse parameters of both electrochemotherapy (8 HV) and DNA delivery (HVLV). Three pulsing conditions were used: 8 high-voltage pulses (8 HV), resulting in large permeabilization and ion flux, and a combination of one high-voltage pulse and one low-voltage pulse (HVLV), either alone or in combination with injection of DNA. Mice and rats were anesthetized before pulsing. At the times given, animals were killed, and intact tibialis cranialis muscles were excised for analysis. Uptake of Ca(2+) was assessed using (45)Ca as a tracer. Using gene expression analyses and histology, we showed a clear association between Ca(2+) influx and muscular response. Moderate Ca(2+) influx induced by HVLV pulses results in activation of pathways involved in immediate repair and hypertrophy. This response could be attenuated by intramuscular injection of EGTA reducing Ca(2+) influx. Larger Ca(2+) influx as induced by 8-HV pulses leads to muscle damage and muscle fiber regeneration through recruitment of satellite cells. The extent of Ca(2+) influx determines the muscular response to electrotransfer and, thus, the success of a given application. In the case of electrochemotherapy, in which the objective is cell death, a large influx of Ca(2+) may be beneficial, whereas for DNA electrotransfer, muscle recovery should occur without myofiber loss to ensure preservation of plasmid DNA.     

Hollow Microneedle Arrays for Intradermal Drug Delivery and DNA Electroporation

The association of microneedles with electric pulses causing electroporation could result in an efficient and less painful delivery of drugs and DNA into the skin. Hollow conductive microneedles were used for (1) needle-free intradermal injection and (2) electric pulse application in order to achieve electric field in the superficial layers of the skin sufficient for electroporation. Microneedle array was used in combination with a vibratory inserter to disrupt the stratum corneum, thus piercing the skin. Effective injection of proteins into the skin was achieved, resulting in an immune response directed to the model antigen ovalbumin. However, when used both as microneedles to inject and as electrodes to apply the electric pulses, the setup showed several limitations for DNA electrotransfer. This could be due to the distribution of the electric field in the skin as shown by numerical calculations and/or the low dose of DNA injected. Further investigation of these parameters is needed in order to optimize minimally invasive DNA electrotransfer in the skin.     

Use of Collagen Gel as a Three-Dimensional In Vitro Model to Study Electropermeabilization and Gene Electrotransfer

Gene electrotransfer is a promising nonviral method that enables transfer of plasmid DNA into cells with electric pulses. Although many in vitro and in vivo studies have been performed, the question of the implied gene electrotransfer mechanisms is largely open. The main obstacle toward efficient gene electrotransfer in vivo is relatively poor mobility of DNA in tissues. Since cells are mechanically coupled to their extracellular environment and act differently compared to standard in vitro conditions, we developed a three-dimensional (3-D) in vitro model of CHO cells embedded in collagen gel as an ex vivo model of tissue to study electropermeabilization and different parameters of gene electrotransfer. For this purpose, we first used propidium iodide to detect electropermeabilization of CHO cells embedded in collagen gel. Then, we analyzed the influence of different concentrations of plasmid DNA and pulse duration on gene electrotransfer efficiency. Our results revealed that even if cells in collagen gel can be efficiently electropermeabilized, gene expression is significantly lower. Gene electrotransfer efficiency in our 3-D in vitro model had similar dependence on concentration of plasmid DNA and pulse duration comparable to in vivo studies, where longer (millisecond) pulses were shown to be more optimal compared to shorter (microsecond) pulses. The presented results demonstrate that our 3-D in vitro model resembles the in vivo situation more closely than conventional 2-D cell cultures and, thus, provides an environment closer to in vivo conditions to study mechanisms of gene electrotransfer.     

Nanosecond Electric Pulse Effects on Gene Expression

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.     

New anti angiogenesis developments through electro-immunization: optimization by in vivo optical imaging of intradermal electro gene transfer

Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called "electro-immunization". In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.     

In vivo gene electrotransfer into skeletal muscle: effects of plasmid DNA on the occurrence and extent of muscle damage

BACKGROUND: Understanding the mechanisms underlying gene electrotransfer muscle damage can help to design more effective gene electrotransfer strategies for physiological and therapeutical applications. The present study investigates the factors involved in gene electrotransfer associated muscle damage. METHODS: Histochemical analyses were used to determine the extent of transfection efficiency and muscle damage in the Tibialis anterior muscles of Sprague-Dawley male rats after gene electrotransfer. RESULTS: Five days after gene electrotransfer, features of muscle degeneration and regeneration were consistently observed, thus limiting the extent of transfection efficiency. Signs of muscle degeneration/regeneration were no longer evident 21 days after gene electrotransfer except for the presence of central myonuclei. Neither the application of electrical pulses per se nor the extracellular presence of plasmid DNA per se contributed significantly to muscle damage (2.9 +/- 1.0 and 2.1 +/- 0.7% of the whole muscle cross-sectional area, respectively). Gene electrotransfer of a plasmid DNA, which does not support gene expression, increased significantly muscle damage (8.7 +/- 1.2%). When plasmid DNA expression was permitted (gene electrotransfer of pCMV-beta-galactosidase), muscle damage was further increased to 19.7 +/- 4.5%. Optimization of cumulated pulse duration and current intensity dramatically reduced gene electrotransfer associated muscle damage. Finally, mathematical modeling of gene electrotransfer associated muscle damage as a function of the number of electrons delivered to the tissue indicated that pulse length critically determined the extent of muscle damage. CONCLUSION: Our data suggest that neither the extracellular presence of plasmid DNA per se nor the application of electric pulses per se contributes significantly to muscle damage. Gene electrotransfer associated muscle damage mainly arises from the intracellular presence and expression of plasmid DNA.     

Evaluation of a novel non-penetrating electrode for use in DNA vaccination

Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use with a wide variety of diseases. Alone, this technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. In this study we evaluated the MEA for its use in DNA vaccination using Hepatitis B virus as the infectious model. We utilized the guinea pig model because their skin is similar in thickness and morphology to humans. The plasmid encoding Hepatitis B surface antigen (HBsAg) was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to injection only groups. While this electrode requires further study, our results suggest that the MEA has potential for use in electrically mediated intradermal DNA vaccination.     

In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells

The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and gamma-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.     

New anti angiogenesis developments through electro-immunization: Optimization by in vivo optical imaging of intradermal electrogenetransfer

Direct application of high voltage electric pulses of milliseconds duration to the skin of a mouse enhances in vivo intradermal delivery of injected therapeutic molecules such as DNA. The efficacy of gene transfer and expression is dependent on electrical parameters. DNA electrotransfer in tissues increases the associated DNA expression vaccine potency. This protocol is called “electro-immunization”. In the present study, we report a new strategy for optimizing electro-immunization. In vivo fluorescence imaging was used to detect the expression of a fluorescent protein (DsRed) and therefore allowed rapid optimization of the protocol. In vivo electrogenetransfer in the skin was well tolerated and DsRed expression was followed for over 2 weeks. Expression was voltage dependent under our conditions. Parameters were selected giving the highest level of expression. Under these optimized conditions, electrotransfer of a plasmid encoding VEGF was evaluated for its immune response as a gene therapy of interest involved in anti-angiogenic strategies. Anti VEGF 165 antibodies in sera of mice were evaluated by ELISA and compared to those obtained after conventional immunization. Comparable titres of antibodies were obtained in both groups. An IgG2a predominance was found in mice immunized with the plasmid whereas a IgG1 predominance was observed in mice immunized classically. Skin electro-immunization is therefore shown as a good route for DNA immunization for anti-angiogenesis concern.     

Topical Gene Electrotransfer to the Epidermis of Hairless Guinea Pig by Non-Invasive Multielectrode Array

Topical gene delivery to the epidermis has the potential to be an effective therapy for skin disorders, cutaneous cancers, vaccinations and systemic metabolic diseases. Previously, we reported on a non-invasive multielectrode array (MEA) that efficiently delivered plasmid DNA and enhanced expression to the skin of several animal models by in vivo gene electrotransfer. Here, we characterized plasmid DNA delivery with the MEA in a hairless guinea pig model, which has a similar histology and structure to human skin. Significant elevation of gene expression up to 4 logs was achieved with intradermal DNA administration followed by topical non-invasive skin gene electrotransfer. This delivery produced gene expression in the skin of hairless guinea pig up to 12 to 15 days. Gene expression was observed exclusively in the epidermis. Skin gene electrotransfer with the MEA resulted in only minimal and mild skin changes. A low level of human Factor IX was detected in the plasma of hairless guinea pig after gene electrotransfer with the MEA, although a significant increase of Factor IX was obtained in the skin of animals. These results suggest gene electrotransfer with the MEA can be a safe, efficient, non-invasive skin delivery method for skin disorders, vaccinations and potential systemic diseases where low levels of gene products are sufficient.     

Electroporator with automatic change of electric field direction improves gene electrotransfer <Emphasis Type="Italic">in-vitro</Emphasis>

Background  

Gene electrotransfer is a non-viral method used to transfer genes into living cells by means of high-voltage electric pulses. An exposure of a cell to an adequate amplitude and duration of electric pulses leads to a temporary increase of cell membrane permeability. This phenomenon, termed electroporation or electropermeabilization, allows various otherwise non-permeant molecules, including DNA, to cross the membrane and enter the cell. The aim of our research was to develop and test a new system and protocol that would improve gene electrotransfer by automatic change of electric field direction between electrical pulses.     

In Vitro Targeted Gene Electrotransfer to Endothelial Cells with Plasmid DNA Containing Human Endothelin-1 Promoter

Development of recombinant DNA technologies has allowed us to create new delivery systems that target specific cell types and that can be used in gene therapy. One of these targets is vascular endothelium because of its important role in tumor angiogenesis. For tumor endothelium-specific targeting, we prepared plasmid DNA encoding green fluorescent protein under the control of human endothelin-1 promoter (pENDO-EGFP), which is specific for endothelial cells. First we determined gene electrotransfer parameters for improved transfection of endothelial cells evaluating different osmolarity of electroporation buffer, voltages of applied electric pulses, and addition of fetal bovine serum immediately after electroporation to the cells for improved transfection and survival. Transfection efficacy of pENDO-EGFP in different endothelial and nonendothelial cell lines was determined next. Gene electrotransfer efficacy was evaluated using three different methods: fluorescence microscopy, fluorescence microplate reader, and flow cytometry. Our results showed that transfection efficacy was higher when cells were prepared in hypoosmolar compared to isoosmolar electroporation buffer. Furthermore, immediate addition of fetal bovine serum to the cells after pulsing also improved gene electrotransfer into target cells. We proved expression of EGFP under the control of human endothelin-1 promoter in endothelial cells, which was also significantly higher compared to nonendothelial cells. Taken together, we successfully constructed pENDO-EGFP, which was specifically expressed in endothelial cells using improved gene electrotransfer parameters.     

Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation

In vivo electroporation (EP) has been shown to augment the immunogenicity of plasmid DNA vaccines, but its mechanism of action has not been fully characterized. In this study, we show that in vivo EP augmented cellular and humoral immune responses to a human immunodeficiency virus type 1 Env DNA vaccine in mice and allowed a 10-fold reduction in vaccine dose. This enhancement was durable for over 6 months, and re-exposure to antigen resulted in anamnestic effector and central memory CD8(+) T-lymphocyte responses. Interestingly, in vivo EP also recruited large mixed cellular inflammatory infiltrates to the site of inoculation. These infiltrates contained 45-fold-increased numbers of macrophages and 77-fold-increased numbers of dendritic cells as well as 2- to 6-fold-increased numbers of B and T lymphocytes compared to infiltrates following DNA vaccination alone. These data suggest that recruiting inflammatory cells, including antigen-presenting cells (APCs), to the site of antigen production substantially improves the immunogenicity of DNA vaccines. Combining in vivo EP with plasmid chemokine adjuvants that similarly recruited APCs to the injection site, however, did not result in synergy.     

Long-term,high level in vivo gene expression after electric pulse-mediated gene transfer into skeletal muscle

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the local or systemic secretion of therapeutic proteins. However, current DNA delivery technologies have to be improved. We report very efficient luciferase gene transfer into muscle fibres obtained through the delivery of squarewave electric pulses of moderate field strength (100–200 V/cm) and of long duration (20 ms) to muscle previously injected with plasmid DNA. This intramuscular ‘electrotransfer’ method increases reporter gene expression by more than 100 times. It is noteworthy that this expression remains high and stable for at least 9 months. Moreover, intramuscular electrotransfer strongly decreases the interindividual variability usually observed after plasmid DNA injection into muscle fibres. Therefore, DNA electrotransfer in muscle possesses broad potential applications in gene therapy and for physiological, pharmacological and developmental studies.     

Numerical optimization of gene electrotransfer into muscle tissue

Background  

Electroporation-based gene therapy and DNA vaccination are promising medical applications that depend on transfer of pDNA into target tissues with use of electric pulses. Gene electrotransfer efficiency depends on electrode configuration and electric pulse parameters, which determine the electric field distribution. Numerical modeling represents a fast and convenient method for optimization of gene electrotransfer parameters. We used numerical modeling, parameterization and numerical optimization to determine the optimum parameters for gene electrotransfer in muscle tissue.     

Nucleic Acids Electrotransfer-Based Gene Therapy (Electrogenetherapy): Past,Current, and Future

About 25 years after the publication of the first report on gene transfer in vitro in cultured cells by the means of electric pulses delivery, reversible cell electroporation for gene transfer and gene therapy (DNA electrotransfer) is at a cross in its development. Present knowledge on the effects of cell exposure to appropriate electric field pulses, particularly at the level of the cell membrane, is reported here. The importance of the models of electric field distribution in tissues and of the correct choice of electrodes and applied voltages is highlighted. The mechanisms involved in DNA electrotransfer, which include cell electropermeabilization and DNA electrophoresis, are also surveyed. This knowledge has allowed developing new nucleic acids electrotransfer conditions using combinations of permeabilizing pulses of high voltage and short duration, and of electrophoretic pulses of low voltage and long duration, which are very efficient and safer. Feasibility of electric pulses delivery for gene transfer in humans is discussed taking into account that electric pulses delivery is already regularly used for localized drug delivery in the treatment of cutaneous and subcutaneous solid tumors by electrochemotherapy. Because recent technological developments made DNA electrotransfer more and more efficient and safer, this non-viral gene therapy approach is now ready to reach the clinical stage. A good understanding of DNA electrotransfer principles and the respect of safe procedures will be key elements for a successful future transfer DNA electrotransfer into the clinics.     

High quality long-term CD4+ and CD8+ effector memory populations stimulated by DNA-LACK/MVA-LACK regimen in Leishmania major BALB/c model of infection

Heterologous vaccination based on priming with a plasmid DNA vector and boosting with an attenuated vaccinia virus MVA recombinant, with both vectors expressing the Leishmania infantum LACK antigen (DNA-LACK and MVA-LACK), has shown efficacy conferring protection in murine and canine models against cutaneus and visceral leishmaniasis, but the immune parameters of protection remain ill defined. Here we performed by flow cytometry an in depth analysis of the T cell populations induced in BALB/c mice during the vaccination protocol DNA-LACK/MVA-LACK, as well as after challenge with L. major parasites. In the adaptive response, there is a polyfunctional CD4(+) and CD8(+) T cell activation against LACK antigen. At the memory phase the heterologous vaccination induces high quality LACK-specific long-term CD4(+) and CD8(+) effector memory cells. After parasite challenge, there is a moderate boosting of LACK-specific CD4(+) and CD8(+) T cells. Anti-vector responses were largely CD8(+)-mediated. The immune parameters induced against LACK and triggered by the combined vaccination DNA/MVA protocol, like polyfunctionality of CD4(+) and CD8(+) T cells with an effector phenotype, could be relevant in protection against leishmaniasis.