Current Gene Therapy (v.16, #2)

Editorial (Thematic Issue: Gene Transfer by Electric Fields) by Marie Pierre Rols, Damijan Miklavcic (73-74).

Gene Electrotransfer in 3D Reconstructed Human Dermal Tissue by Moinecha Madi, Marie-Pierre Rols, Laure Gibot (75-82).
Gene electrotransfer into the skin is of particular interest for the development of medical applications including DNA vaccination, cancer treatment, wound healing or treatment of local skin disorders. However, such clinical applications are currently limited due to poor understanding of the mechanisms governing DNA electrotransfer within human tissue. Nowadays, most studies are carried out in rodent models but rodent skin varies from human skin in terms of cell composition and architecture. We used a tissue-engineering approach to study gene electrotransfer mechanisms in a human tissue context. Primary human dermal fibroblasts were cultured according to the self-assembly method to produce 3D reconstructed human dermal tissue. In this study, we showed that cells of the reconstructed cutaneous tissue were efficiently electropermeabilized by applying millisecond electric pulses, without affecting their viability. A reporter gene was successfully electrotransferred into this human tissue and gene expression was detected for up to 48h. Interestingly, the transfected cells were solely located on the upper surface of the tissue, where they were in close contact with plasmid DNA solution. Furthermore, we report evidences that electrotransfection success depends on plasmid mobility within tissue- rich in collagens, but not on cell proliferation status. In conclusion, in addition to proposing a reliable alternative to animal experiments, tissue engineering produces valid biological tool for the in vitro study of gene electrotransfer mechanisms in human tissue.

Thermal Assisted In Vivo Gene Electrotransfer by Amy Donate, Anna Bulysheva, Chelsea Edelblute, Derrick Jung, Mohammad A. Malik, Siqi Guo, Niculina Burcus, Karl Schoenbach, Richard Heller (83-89).
Gene electrotransfer is an effective approach for delivering plasmid DNA to a variety of tissues. Delivery of molecules with electric pulses requires control of the electrical parameters to achieve effective delivery. Since discomfort or tissue damage may occur with high applied voltage, the reduction of the applied voltage while achieving the desired expression may be an important improvement. One possible approach is to combine electrotransfer with exogenously applied heat. Previous work performed in vitro demonstrated that increasing temperature before pulsing can enhance gene expression and made it possible to reduce electric fields while maintaining expression levels. In the study reported here, this combination was evaluated in vivo using a novel electrode device designed with an inserted laser for application of heat. The results obtained in this study demonstrated that increased temperature during electrotransfer increased expression or maintained expression with a reduction in applied voltage. With further optimization this approach may provide the basis for both a novel method and a novel instrument that may greatly enhance translation of gene electrotransfer.

Visualization of Nonspecific Antitumor Effectiveness and Vascular Effects of Gene Electro-Transfer to Tumors by Urska Kamensek, Marie-Pierre Rols, Maja Cemazar, Muriel Golzio (90-97).
Background: Gene Electro Transfer (GET) is a promising method for therapeutic purposes. Intratumoral GET has reached clinical evaluation for antitumor treatment. An increasing number of studies suggests that antitumor effectiveness not only depends on the transfection efficiency, but also on the induction of immune responses and vascular effects that result in the nonspecific induction of cell death. Real time noninvasive optical imaging methods allow longitudinal studies of these dynamic biological processes. Objective: In the present study, a noninvasive bioluminescence technology was used to further explore the phenomena associated with GET to tumors by a real time monitoring of the transfection efficiency as well as cell death following the treatment. Method: By using transgenic light-producing tumors, tumor growth was visualized, and since dead cells stop producing light, effectiveness of the treatment or the emergence of necrotic areas in the tumors was followed visually. The transfection efficiency of reporter genes (iRFP protein and luciferase) in the subcutaneous tumors was also evaluated. Results: Our results showed that the GET of a reporter gene can lead to nonspecific antitumor effectiveness and even complete regression of tumors. Using light-producing tumors, we were also able to indirectly visualize the previously described vascular effects of electroporation. Additionally, using the intratumoral GET of a luciferase encoding plasmid, we localized the source of the expression mainly in the peritumoral and not in the tumoral region. Conclusion: The data obtained provide new insights into some of the phenomena associated with GET to tumors, which should be taken into account when designing improved and more effective cancer gene therapy, in order to accelerate the transfer of the technology into clinical trials.

Gene Electrotransfer: A Mechanistic Perspective by Christelle Rosazza, Sasa Haberl Meglic, Andreas Zumbusch, Marie-Pierre Rols, Damijan Miklavcic (98-129).
Gene electrotransfer is a powerful method of DNA delivery offering several medical applications, among the most promising of which are DNA vaccination and gene therapy for cancer treatment. Electroporation entails the application of electric fields to cells which then experience a local and transient change of membrane permeability. Although gene electrotransfer has been extensively studied in in vitro and in vivo environments, the mechanisms by which DNA enters and navigates through cells are not fully understood. Here we present a comprehensive review of the body of knowledge concerning gene electrotransfer that has been accumulated over the last three decades. For that purpose, after briefly reviewing the medical applications that gene electrotransfer can provide, we outline membrane electropermeabilization, a key process for the delivery of DNA and smaller molecules. Since gene electrotransfer is a multipart process, we proceed our review in describing step by step our current understanding, with particular emphasis on DNA internalization and intracellular trafficking. Finally, we turn our attention to in vivo testing and methodology for gene electrotransfer.

Oncotarget Strategies For Herpes Simplex Virus-1 by Lumin Zhang, Tsurumi Tatsuya, Yukihiro Nishiyama (130-143).
The high level of manipulability of viral genome has set up HSV-1 to be an ideal viral vector for oncolytic virotherapy. In the past two decades, several oncolytic HSV-1 viruses have been successfully developed and assessed in animal studies. Accumulated evidences show that oncolytic HSV- 1 can efficiently infect many tumor cells and augment anti-tumor effect by induction of systemic innate and adaptive immune responses. Inspiring results have been accomplished in several phase I clinical trials for glioma, head and neck squeous cells carcinoma and Melanoma using oncolytic HSV- 1 viruses. More recently, oncovey, one of oncolytic HSV-1 viruses has been approved by FDA for the comprehensive evolution of its anti-tumor effects in phase III clinical trials. These promising studies encourage more efforts to be devoted to craft the new generation of oncolytic HSV-1. Herein, we will review and summarize the basic strategies to construct oncolytic HSV-1 viruses and their applications in cancer therapy.

Administration of DNA Plasmid Coding Protein Aggregating Domain Induces Inflammatory Bone Loss by Dimitrios Agas, Fabio Concetti, Melania Capitani, Giovanna Lacava, Antonio Concetti, Luigi Marchetti, Fulvio Laus, Andrea Marchegiani, Vasco Azevedo, Maria Giovanna Sabbieti, Franco Maria Venanzi (144-152).
Background: Plasmids coding protein aggregation polypeptides from different sources have been proposed as genetic adjuvants for DNA vaccines. We reported that a plasmid (pATRex), encompassing the DNA sequence for the von Willebrand A (vWA/A) domain of the Anthrax Toxin Receptor-1 (ANTXR-1, alias TEM8, Tumor Endothelial Marker 8), acts as strong immune adjuvant by inducing formation of insoluble intracellular aggregates and subsequent cell death. Objective: In the present study we addressed the question of whether there is any substantial immunotoxicity associated with the use of self-aggregating proteins as genetic adjuvants. Methods & Results: Here we report, by mean of histology, X-ray and molecular examinations of bone specimens, the unexpected finding that intramuscular injection of pATRex in mice triggers, per se, severe bone loss (osteoporosis) independently from the sex and genotype of the treated animals. Conclusion: Even though the study suggests that proteinaceous “sticky “ adjuvants are unlikely to find their way into practical vaccination, the information gained is of value as ATRex injections could provide an additional, simplified, mouse model of osteoporosis. Moreover, our results provide experimental support to the hypothesis that proteotoxic aggregates chronically activate the innate immune system in amyloid and aggregosome associated disorders.