Current Gene Therapy - Volume 10, Issue 4, 2010

Delivery of genetic materials into target cells and tissues has been steadily improving over the years and is starting to fulfil its long awaited promises. Safety concerns are now answered by various non-viral approaches among them being the method of electrotransfer. This physical approach, referred as electroporation or electropermeabilization, relies on the application of electric pulses that transiently and locally permeabilize cells and tissues allowing therapeutic molecules such as anticancer drugs and nucleic acids to be transferred into them. Nearly 30 years after the pioneering work of Prof Eberhard Neumann, electric fields are now used in routine in different countries as a palliative treatment of cancer, a modality called electrochemotherapy. Nowadays, electrotransfer represents one of the most widespread techniques used in molecular genetics. In vivo electrotransfer is of special interest since it is the most efficient non-viral strategy for gene delivery and also because of its low cost, easiness of realization and safety. One of the most widely targeted tissues is skeletal muscle. The strategy is not only promising for the treatment of muscle disorders, but also for the systemic secretion of proteins. Vaccination and oncology gene therapy are the other main fields of application. This, together with the capacity to deliver large DNA constructs, greatly expands the research and clinical applications of in vivo DNA electrotransfer. The mechanisms underlying cell membrane permeabilization and associated gene transfer are not completely understood. Their elucidations are of high importance for improving the current protocols. The purpose of this special issue is to give to the specialist as well as the non-specialist reader current work coming from different complementary research groups from basic science to clinics. These six reviews not only provide extensive insight into our (lack of) current knowledge on gene electrotransfer, but also indicate the gap to transfer them from the cells in Petri dish to the organ in a patient. The first review, by Golzio et al., gives an account of the mechanisms of electropermeabilization in a number of contexts of varying complexity, starting with giant vesicles, discussing single cell level and finishing with the more complex relevant situation that is tissues. Practical aspects of in vivo nucleic acids electrotransfer are described in the second review by Andre et al. Different strategies are presented for each limiting step of the procedure to improve its outcome, with their advantages and drawbacks. The next four reviews provide examples of the use of DNA electrotransfer in pre-clinical and clinical purposes. Chiarella et al. report recent literature on electrotransfer as an effective strategy to improve DNA-based vaccination protocols. The most relevant patents studies developed for the applications of electrotransfer against infectious diseases and tumours in preclinical and clinical studies are given. An overview of the clinical perspectives of DNA electrotransfer to skin is presented by Gothelf et al. The skin is a very interesting tissue, not only due to its accessibility but also to its capability to produce transgenes and elicit immunological responses. The two last reviews by Cemazar et al., and Heller et al., wrap-up studies on electrogene therapy with Il-12 that culminated in the first published clinical study for treatment of subcutaneous melanoma metastases by the group of Prof. Richard Heller and show promise to further translate this therapy into human and veterinary oncology. They discuss preclinically tested gene electrotransfer therapies for melanoma and the conversion of these therapies to clinical trials. My hope for this issue is that the reader obtains an overview of this very active field, showing that this efficient process is more complex than simply pushing DNA through electropores, and inspiring novel applications in nucleic acid delivery. As the development of electrotransfer moves forward, I am highly confident that in the next future several new clinical trials for cancer but also other severe diseases will be initiated. Finally, I want to thank all authors for their insightfulness and dedication and extend my special thanks to the reviewers, to all the students I had the pleasure to supervise, to Dr Jean-Michel Escoffre for his advices and to Brenda and Yves for their hard work and Support.

We discuss experimental observations of DNA uptake induced by electropermeabilisation. First we describe how experiments on giant unilamellar vesicles can be used to understand the effect of electric fields on lipid membranes and the associated transfer of DNA across the plasma membrane. We then discuss how DNA interacts with electropermeabilised cells in culture and how gene expression is correlated to observations of interactions between DNA and the cell membrane. Finally we discuss how DNA uptake occurs and can be optimised for the most medically relevant case of tissues.

Nucleic acids transfer has been steadily improving over the years and is slowly starting to fulfill its long awaited promises. In the beginning, viral approaches raised strong safety concerns that are now answered by various nonviral techniques. Among the physical approaches developed, nucleic acids electrotransfer is probably the one with the highest momentum. Here we review the present knowledge on the mechanistic and practical aspects of in vivo nucleic acids electrotransfer. For each step of this procedure we present different strategies that are used, with their advantages and drawbacks. As we report here, practical solutions have been found to overcome each limiting step in the procedure and to improve its outcome. Some crucial issues are beyond the application of the electric pulses itself, like the administration (i.e., in almost all of the cases, the injection) of the nucleic acids to the tissue or the body. High efficiency and safety are at reach if all the present knowledge and strategies are put to use. Electrotransfer is now a mature technique as proven by the fact that clinical trials using nucleic acids electrotransfer have already started within the past few years.

Vaccination is historically one of the most important methods for preventing infectious diseases in humans and animals. Due to recent advances in understanding the biology of the immune system, a more rational design of vaccines and vaccination strategies such as those based on gene transfer has been proposed. In particular, naked DNA vaccination is emerging as a promising approach for introducing foreign antigens into the host, inducing protective immunity against infectious diseases and malignant tumours. Plasmid DNA vaccines offer several advantages in comparison to traditional vaccines such as safety, tolerability and feasibility in manufacture. Nevertheless, because of their poor immunogenicity, plasmid DNA vaccines need further implementation. Recent data suggest electroporation as a useful strategy to improve DNA-based vaccination protocols, being able to stimulate both the humoural and cellular immune responses. In preclinical trials, electroporation is successfully used in prime-boost combination protocols and its efficacy and tolerability have been demonstrated in Phase I clinical trials. Since these initial results appear promising, in the next future we will assist further developments of naked DNA vaccination associated to the electroporation technology. This approach not only provides the basis for human studies but also a practical application to veterinary medicine.

Gene electrotransfer, which designates the combination of gene transfer and electroporation, is a non-viral means for transfecting genes into cells and tissues. It is a safe and efficient method and reports regarding the use of this technique in a variety of animal models and organs have been published in the literature. We find that gene electrotransfer to skin is of particular interest; not only due to the easy accessibility of this organ, which renders both treatment and evaluation feasible, but also the capability of the skin to produce transgenes and elicit immunological responses. Up to now more than 40 papers have been published in which gene electrotransfer was the technique used for gene transfection to skin in vivo. The aim of this review is to summarize which plasmids were injected and the electrical parameters applied. Furthermore an overview of the clinical perspectives of gene electrotransfer to skin will be presented.

Electrogene therapy combines administration of plasmid DNA into tissue followed by local application of electric pulses. In electrogene therapy with interleukin-12 (IL-12), different routes of administration, different doses of plasmid DNA and different protocols for delivery of electric pulses were evaluated in numerous preclinical studies. Antitumor effectiveness was tested in different types of primary tumors, distantly growing tumors and induced metastases. Intratumoral IL-12 electrogene therapy has been proved to be very effective in local tumor control, having also a systemic effect. Intramuscular and peritumoral IL-12 electrogene therapy had also a pronounced systemic effect and when combined with other treatment strategies resulted in tumor cures. Antitumor effectiveness of IL-12 electrogene therapy is due to the induction of adaptive immunity and innate resistance and anti-angiogenic action. Translation of preclinical studies into clinical trials in human and veterinary oncology has started with encouraging results that would hopefully lead to further investigation of this therapy, also in combination with other cancer treatment modalities.

In vivo electroporation (EP) is a versatile delivery method for gene transfer which can be applied to any accessible tissue. Delivery of plasmid DNA encoding therapeutic genes or cDNAs with in vivo EP has been tested extensively in preclinical melanoma models. Direct delivery to the tumor has been shown to generate a direct antitumor effect. Delivery to alternative sites may generate additional therapeutic options, for example the production of cancer vaccines, the reduction of tumor angiogenesis, or the induction of tumor cell apoptosis. Several of the preclinical therapies tested have a demonstrated therapeutic effect against melanomas. Two immunotherapies have advanced to melanoma clinical trials. Delivery of a plasmid DNA encoding interleukin-12 (IL-12) or interleukin-2 (IL-2) using electroporation was demonstrated to be safe with no grade 3 or 4 toxicities reported. Delivery of IL-12 with electroporation resulted in significant necrosis of melanoma cells in the majority of treated tumors and significant lymphocytic infiltrate in biopsies from patients in several cohorts. In addition, clinical evidence of responses in untreated lesions suggested the induction of a systemic response following therapy. This review discusses preclinically tested electroporation gene therapies for melanoma with clinical potential and the conversion of these therapies to clinical trials.