Real time electroporation control for accurate and safe in vivo non-viral gene therapy

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.     

Mechanisms involved in gene electrotransfer using high- and low-voltage pulses--an in vitro study

Gene electrotransfer is an established method for gene delivery which uses high-voltage pulses to increase permeability of cell membrane and thus enables transfer of genes. Currently, majority of research is focused on improving in vivo transfection efficiency, while mechanisms involved in gene electrotransfer are not completely understood. In this paper we analyze the mechanisms of gene electrotransfer by using combinations of high-voltage (HV) and low-voltage pulses (LV) in vitro. We applied different combinations of HV and LV pulses to CHO cells and determined the transfection efficiency. We obtained that short HV pulses alone were sufficient to deliver DNA into cells for optimal plasmid concentrations and that LV pulse did not increase transfection efficiency, in contrast to reported studies in vivo. However, for sub-optimal plasmid concentrations combining HV and LV pulses increased transfection rate. Our results suggest that low-voltage pulses increase transfection in conditions where plasmid concentration is low, typically in vivo where mobility of DNA is limited by the extracellular matrix. LV pulses provide additional electrophoretic force which drags DNA toward the cell membrane and consequently increase transfection efficiency, while for sufficiently high concentrations of the plasmid (usually used in vitro) electrophoretic LV pulses do not have an important role.     

Optimal salt concentration of vehicle for plasmid DNA enhances gene transfer mediated by electroporation

In vivo electroporation has emerged as a leading technology for developing nonviral gene therapies, and the various technical parameters governing electroporation efficiency have been optimized by both theoretical and experimental analysis. However, most electroporation parameters focused on the electric conditions and the preferred vehicle for plasmid DNA injections has been normal saline. We hypothesized that salts in vehicle for plasmid DNA must affect the efficiency of DNA transfer because cations would alter ionic atmosphere, ionic strength, and conductivity of their medium. Here, we show that half saline (71 mM) is an optimal vehicle for in vivo electroporation of naked DNA in skeletal muscle. With various salt concentrations, two reporter genes, luciferase and beta-galactosidase were injected intramuscularly under our optimal electric condition (125 V/cm, 4 pulses x 2 times, 50 ms, 1 Hz). Exact salt concentrations of DNA vehicle were measured by the inductively coupled plasma-atomic emission spectrometer (ICP-AES) and the conductivity change in the tissue induced by the salt in the medium was measured by Low-Frequency (LF) Impedance Analyzer. Luciferase expression increased as cation concentration of vehicle decreased and this result can be visualized by X-Gal staining. However, at lower salt concentration, transfection efficiency was diminished because the hypoosmotic stress and electrical injury by low conductivity induced myofiber damage. At optimal salt concentration (71 mM), we observed a 3-fold average increase in luciferase expression in comparison with the normal saline condition (p < 0.01). These results provide a valuable experimental parameter for in vivo gene therapy mediated by electroporation.     

Improved expression by cytomegalovirus promoter/enhancer and behavior of vascular endothelial growth factor gene after myocardial injection of naked DNA

Direct injection of the vascular endothelial growth factor (VEGF) gene plasmid DNA into the myocardium was shown to induce development of new blood vessels to increase the circulation in the heart of patients with coronary artery diseases. However, such angiogenic gene therapy (via naked DNA) was limited by low level of gene expression. Furthermore, the temporal and spatial characteristics of VEGF gene transfer in the heart are not known. In this study, we demonstrated that a plasmid vector, containing the human cytomegalovirus immediate early (HCMV IE) promoter and enhancer, induces greater expression of gene in the rat heart monitored by gene fused to the chloramphenicol acetyl transferase (CAT) reporter, than four different viral and cellular promoters. Interestingly, expression of VEGF121 protein showed an earlier peak, a shorter duration, and a wider distribution than that of CAT only. Therefore, a plasmid vector with an HCMV IE promoter/enhancer provides clear advantages over other previously developed plasmids. Furthermore, expression profile of VEGF121 gene may provide useful information in the design of angiogenic gene therapy in the heart.     

Efficacy of transgene expression in porcine skin as a function of electrode choice

Gene electrotransfer is a non-viral technique using electroporation for gene transfection. The method is widely used in the preclinical setting and results from the first clinical study in tumours have been published. However, the preclinical studies, which form the basis for the clinical trials, have mainly been performed in rodents and the body of evidence on electrode choice and optimal pulsing conditions is limited.We therefore tested plate and needle electrodes in vivo in porcine skin, which resembles human skin in structure. The luciferase (pCMV-Luc) gene was injected intradermally and subsequently electroporated. Simultaneously, studies with gene electrotransfer to porcine skin using plasmids coding for green fluorescent protein (GFP) and betagalactosidase were performed.Interestingly, we found needle electrodes to be more efficient than plate electrodes (p < 0.001) and electric field calculations showed that penetration of the stratum corneum led to much more homogenous field distribution at the DNA injection site. Furthermore, we have optimised the electric pulse regimens for both plate and needle electrodes using a range of high voltage and low voltage pulse combinations.In conclusion, our data support that needle electrodes should be used in human clinical studies of gene electrotransfer to skin for improved expression.     

Improvement of the quality of self assembled bilayer lipid membranes by using a negative potential

Cell electropermeabilization (also termed cell electroporation) is nowadays a routine technique used in biochemical and pharmacological studies for the in vitro introduction of nonpermeant molecules into living cells. But electric pulses can be used as well in vivo for the delivery of drugs or DNA into cells of tissues. This review then gives an updated overview of the therapeutic perspectives of cell electropermeabilization in vivo, in particular of the antitumour electrochemotherapy (i.e., the combination of a cytotoxic nonpermeant drug with permeabilizing electric pulses delivered to the tumours) and of in vivo DNA electrotransfer for gene therapy. After a short summary of the present knowledge on cell electropermeabilization (particularly in vivo), the basis, the present achievements, and the challenges of electrochemotherapy are described and discussed, which includes an overview of still open questions and an update on recent clinical trials. DNA electrotransfer for gene therapy is an emerging field in which results are rapidly accumulating. Present knowledge on DNA electrotransfer mechanisms, as wel as the potentialities of DNA electrotransfer to become an efficient non-viral approach for gene therapy, are reviewed.     

Effects of pulse strength and pulse duration on in vitro DNA electromobility

Interstitial transport of DNA is a rate-limiting step in electric field-mediated gene delivery in vivo. Interstitial transport of macromolecules, such as plasmid DNA, over a distance of several cell layers, is inefficient due to small diffusion coefficient and inadequate convection. Therefore, we explored electric field as a novel driving force for interstitial transport of plasmid DNA. In this study, agarose gels were used to mimic the interstitium in tissues as they had been well characterized and could be prepared reproducibly. We measured the electrophoretic movements of fluorescently labeled plasmid DNA in agarose gels with three different concentrations (1.0%, 2.0% and 3.0%) subjected to electric pulses at three different field strengths (100, 200 and 400 V/cm) and four different pulse durations (10, 50, 75, 99 ms). We observed that: (1) shorter pulses (10 ms) were not as efficient as longer pulses in facilitating plasmid transport through agarose gels; (2) plasmid electromobility reached a plateau at longer pulse durations; and (3) plasmid electromobility increased with applied electric energy, up to a threshold, in all three gels. These data suggested that both pulse strength and duration needed to be adequately high for efficient plasmid transport through extracellular matrix. We also found that electric field was better than concentration gradient of DNA as a driving force for interstitial transport of plasmid DNA.     

Interfacial ternary complex DNA/Ca/lipids at anionic vesicle surfaces

The electroporative transfer of gene DNA and other bioactive substances into tissue cells by electric pulses gains increasing importance in the new disciplines of electrochemotherapy and electrogenetherapy. The efficiency of the electrotransfer depends crucially on the adsorption of the gene DNA and oligonucleotides to the plasma cell membranes. Here it is shown that the adsorption of larger oligonucleotides such as fragments (ca. 300 bp) of sonicated calf-thymus DNA, to anionic lipids of unilamellar vesicles (diameter Phi=300+/-90 nm) is greatly enhanced by divalent cations such as Ca(2+)-ions. Applying centrifugation, bound and free DNA are monitored optically at the wavelength lambda=260 nm. Using arsenazo III as a Ca(2+)-indicator and atomic absorption spectroscopy (AAS), Ca(2+)-titrations of DNA and vesicles yield the individual equilibrium constants of Ca(2+)- and DNA-binding not only for the binary complexes: Ca/lipids, Ca/DNA and DNA/lipids, respectively, but also for the various processes to form the ternary complex DNA/Ca/lipids. The data provide the basis for goal-directed optimization protocols for the adsorption and thus efficient electrotransfer of oligonucleotides and polynucleotides into cells.     

Vascular endothelial growth factor-induced angiogenic gene therapy in patients with peripheral artery disease

This phase 1 clinical trial tested the safety of intramuscular gene transfer by using naked plasmid DNA encoding the gene for VEGF, and analyzed the potential therapeutic benefits in patients with severe peripheral arterial disease (PAD). This study was an open-labeled, dose- escalating, single-center trial on nine male patients with severe debilitating PAD who had not responded to conventional therapy. Seven had Buerger's disease and two had arteriosclerosis obliterans. Plasmid DNA (pCK) containing human VEGF165 was given by eight intramuscular injections in and around the area in need of new blood vessels. The study evaluated three escalating total doses (2, 4, and 8 mug of pCK- VEGF165), with half of each total dose given four weeks apart. The follow-up duration was nine months. The gene injections were well tolerated without significant side effects or laboratory abnormalities related to gene transfer. Three patients showed transient edema in their extremities. Ischemic pain of the affected limb was relieved or improved markedly in six of seven patients. Ischemic ulcers healed or improved in four of six patients. The mean ankle-brachial index (ABI) improved significantly. Six of nine patients showed an increase in collateral vessels around the injection sites demonstrated by digital subtraction angiography. However, there was no relationship between the degree of ABI improvement and the dose given. Mean plasma levels of VEGF did not increase significantly. In conclusion, intramuscular injections of pCK- VEGF165 can be performed safely to induce therapeutic angiogenesis in patients with severe PAD.     

Binding and condensation of plasmid DNA onto functionalized carbon nanotubes: toward the construction of nanotube-based gene delivery vectors

Carbon nanotubes (CNTs) constitute a class of nanomaterials that possess characteristics suitable for a variety of possible applications. Their compatibility with aqueous environments has been made possible by the chemical functionalization of their surface, allowing for exploration of their interactions with biological components including mammalian cells. Functionalized CNTs (f-CNTs) are being intensively explored in advanced biotechnological applications ranging from molecular biosensors to cellular growth substrates. We have been exploring the potential of f-CNTs as delivery vehicles of biologically active molecules in view of possible biomedical applications, including vaccination and gene delivery. Recently we reported the capability of ammonium-functionalized single-walled CNTs to penetrate human and murine cells and facilitate the delivery of plasmid DNA leading to expression of marker genes. To optimize f-CNTs as gene delivery vehicles, it is essential to characterize their interactions with DNA. In the present report, we study the interactions of three types of f-CNTs, ammonium-functionalized single-walled and multiwalled carbon nanotubes (SWNT-NH3+; MWNT-NH3+), and lysine-functionalized single-walled carbon nanotubes (SWNT-Lys-NH3+), with plasmid DNA. Nanotube-DNA complexes were analyzed by scanning electron microscopy, surface plasmon resonance, PicoGreen dye exclusion, and agarose gel shift assay. The results indicate that all three types of cationic carbon nanotubes are able to condense DNA to varying degrees, indicating that both nanotube surface area and charge density are critical parameters that determine the interaction and electrostatic complex formation between f-CNTs with DNA. All three different f-CNT types in this study exhibited upregulation of marker gene expression over naked DNA using a mammalian (human) cell line. Differences in the levels of gene expression were correlated with the structural and biophysical data obtained for the f-CNT:DNA complexes to suggest that large surface area leading to very efficient DNA condensation is not necessary for effective gene transfer. However, it will require further investigation to determine whether the degree of binding and tight association between DNA and nanotubes is a desirable trait to increase gene expression efficiency in vitro or in vivo. This study constitutes the first thorough investigation into the physicochemical interactions between cationic functionalized carbon nanotubes and DNA toward construction of carbon nanotube-based gene transfer vector systems.     

Visualizing Gene Expression in Living Mammals Using a Bioluminescent Reporter

Abstract— Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.     

INHIBITION OF TRANSIENT GENE EXPRESSION IN CHINESE, HAMSTER OVARY CELLS BY TRIPLET-SENSITIZED UV-B IRRADIATION OF TRANSFECTED DNA

The biological effectiveness of thymine-thymine cyclobutane dimers specifically induced by photosensitized ultraviolet-B irradiation was analyzed by host-cell reactivation of triplet-sensitized, UV-B irradiated plasmid pRSV beta gal DNA transfected into normal and repair-deficient Chinese hamster ovary cells. For comparison, pRSV beta gal DNA was also UV-C irradiated and transfected into the same cell lines. Ultraviolet endonuclease-sensitive site induction was determined after UV-C irradiation or acetophenone-sensitized UV-B irradiation of plasmid pRSV beta gal DNA. These data were used to calculate the number of cyclobutane pyrimidine dimers required to inactivate expression of the lacZ reporter gene in each irradiation condition. Transfection with UV-C-irradiated plasmid DNA resulted in a significantly greater reduction of reporter gene expression than did transfection with acetophenone-sensitized UV-B-irradiated pRSV beta gal DNA at equivalent induction of enzyme-sensitive sites. Since only a fraction of the inhibition could be accounted for by noncyclobutane dimer photoproducts, these results suggest that cytosine-containing pyrimidine cyclobutane dimers may be more effective than thymine-thymine dimers in inhibiting transient gene expression as measured in such host-cell reactivation experiments in mammalian cells.     

New estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore

We have designed and synthesized estrogen antagonists bearing dicarba-closo-dodecaborane (carborane) as a hydrophobic pharmacophore based on the structure of 1-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane, a potent estrogen agonist that we reported previously. Compounds with a long alkyl chain bearing an amide moiety on the carborane skeleton (6, 7) showed estrogen antagonistic activity in a luciferase reporter gene assay using COS-1 cells transfected with a rat ER alpha-expression plasmid and as an appropriate reporter plasmid.     

The Role of Formamidine Groups in Dextran Based Nonviral Vectors for Gene Delivery on Their Physicochemical and Biological Characteristics

Dextran‐formamidine esters (dextran‐N‐[(dimethylamino)methylene]‐β‐alanine ester) with different degrees of substitution (0.45–0.92) are synthesized in an one‐pot reaction. Dextran (Mw 60 000 g mol^?1) is allowed to react with unprotected beta‐alanine and iminium chloride and investigated regarding the potential as gene delivery system for the transfer of plasmid DNA. With degrees of substitution ≥ 0.63 improved DNA binding with formation of enzymatically stable complexes of about 130–160 nm with negative surface charges are obtained. These physicochemical characteristics correlated with increasing transfection rates in CHO‐K1 cells determined by a luciferase reporter gene assay in dependency of the number of formamidine residues, N/P ratios and amount of DNA. The role of the number of formamidine groups is also highlighted by in vitro cyto‐ and hemotoxicity tests under the chosen conditions. These results indicate that dextran‐formamidine esters are a very promising material for the safe and efficient gene delivery.     

Layer-by-layer assembly of poly(ethyleneimine) and plasmid DNA onto transparent indium-tin oxide electrodes for temporally and spatially specific gene transfer

The layer-by-layer assembly technique was used to adsorb alternately poly(ethyleneimine) and plasmid DNA onto the surface of a transparent electrode made of indium-tin oxide. The surface with adsorbed poly(ethyleneimine) and DNA was characterized by X-ray photoelectron spectroscopy, attenuated total reflectance Fourier transform infrared spectroscopy, and contact angle measurements. These analyses revealed that the alternate adsorption process generated a multilayered assembly of cationic poly(ethyleneimine) and anionic DNA. For the spatially and temporally specific gene transfer, cells were cultured on the plasmid-loaded electrode and then a short electric pulse was applied to the cell-electrode system. It was shown that, upon electric pulsing, the plasmid was released from the electrode and transferred into the cells, resulting in efficient gene expression even in primary cultured cells. Transfection could be effected for hippocampal neurons after 3-day culture on the plasmid-loaded electrode, which indicated the feasibility of selecting the time of transfection. Our results also showed that electroporation could be performed in a spatially specific manner by using a plasmid-arrayed electrode, demonstrating the feasibility of the method for the fabrication of transfected cell microarrays.     

Development of an efficient endothelial cell specific vector using promoter and 5' untranslated sequences from the human preproendothelin-1 gene

We report here, that a vector constructed based on ppET-1 gene promoter and 5' untranslated region induced a high level of gene expression in endothelial cells and the specificity is even further enhanced under hypoxia-mimic conditions due to a natural hypoxia responsive element within the promoter region. A naked DNA vector that confers endothelial cell specific gene expression as well as efficient levels of gene expression was constructed with an endothelial cell specific naked DNA vector, pETlong, by using the full length promoter of the preproendothelin-1 gene and the entire 5' untranslated region upstream from the start codon. Inclusion of the entire 5' untranslated region in pETlong increased gene expression 2.96 fold as compared with that from pETshort, which contains only the promoter sequences. Reporter gene expression from pETlong was 7.9 fold higher as compared with that from CMV-driven promoter based vector in calf pulmonary endothelial cells. However, in nonendothelial COS cells, luciferase activity from pETlong was only 0.3 fold as compared with that of CMV-based vector. Similar results were observed in other nonendothelial cells. These results demonstrate that the pETlong drives gene expression in endothelial cells with high efficacy and specificity. We have examined hypoxia responsiveness of pETlong as the promoter region of the preproendothelin-1 gene contains hypoxia responsive elements. The activity of the pETlong vector was increased 1.6 fold under hypoxia-mimic conditions using cobalt chloride. The high levels of hypoxia-inducible expression in endothelial cells relative to the low levels of background expression in other cells shows that pETlong could be a useful tool for vascular targeting of vascular disease and cancer gene therapy.     

Gene transfection into adherent cells using electroporation on a dendrimer-modified gold electrode

Gene transfection into adherent cells from plasmid DNA (pDNA)-arrayed substrates known as gene transfection arrays appears to be a promising tool for the high-throughput analysis of gene functions and protein-protein interaction networks. We tested the ability of electric pulse-stimulated gene transfection from a substrate to overcome low expression efficiency and cross contamination between spots on arrays. We prepared the electrodes used for electric pulse-stimulated gene transfection by sequentially loading a gold thin layer, a self-assembled monolayer of a carboxylic acid-terminated alkanethiol (COOH-SAM), and poly(amidoamine) (PAMAM) dendrimers, either through electrostatic interactions or by covalent linkage to COOH-SAM and then to pDNA. When dendrimers were loaded onto the electrode using electrostatic interactions, the gene-expression efficiency of adherent cells increased as the generation numbers of the dendrimers that we used increased. Gene expression was rarely observed in adherent cells when dendrimers were covalently immobilized onto the electrode. Additionally, we successfully demonstrated site-specific gene transfer using a dendrimer-array electrode with no cross contamination between spots on the electrode.     

Simulation and experimental demonstration of the electric field assisted electroporation microchip for in vitro gene delivery enhancement

Simulation and experimental demonstration of the in vitro gene delivery enhancement using electrostatic forces and electroporation (EP) microchips were conducted. Electroporation is a technique with which DNA molecules can be delivered into cells using electric field pulses. This study demonstrates that plasmid DNA can be attracted to the cell surfaces at the specific regions using an electrostatic force. Therefore, the DNA concentration on the cell surface is dramatically increased, which highly enhances the gene transfection efficiency compared to that without an attracting-electric field. The electrostatic force can be designed into specific regions, where the DNA plasmids are attracted to, to provide the region-targeting function. In this micro-device, the top electrode and the interdigitated electrodes provided the DNA attracting-electric field, and the interdigitated electrodes provided adequate electric fields for the electroporation process on the chip surface. Using the EP microchip, cells could be manipulated in situ without detachment if adherent cells were used for electroporation. Five different cells of two different types, primary cell and cell line, were successfully transfected under multi-pulse or single pulse electric field stimulation without applying an attracting-electric field. This study simulated and analyzed the electric field distributions at the DNA attracting and electroporation processes, and successfully demonstrated that the electrostatic force attracted DNA plasmids to specific regions and highly enhanced the gene delivery. In summary, this EP microchip should provide many potential applications for gene therapy.