Search the repository

A+ | A- | | SLO | ENG

Keywords: phase I clinical trial, phIL12 plasmid, interleukin-12, gene electrotransfer, basal cell carcinoma
Published in DiRROS: 13.04.2026; Views: 261; Downloads: 256
Full text (5,35 MB)
This document has many files! More...

Abstract: Gene therapy has emerged as a transformative biomedical approach, offering new therapeutic possibilities from many so far uncurable diseases through the introduction of recombinant nucleic acids into target cells. Among non-viral delivery techniques, gene electrotransfer (GET) has become one of the frequently applied methods in clinical trials. It is based on the application of short, high-intensity electric pulses that transiently permeabilize cell membranes and enable the efficient transfer of plasmid DNA or other types of recombinant nucleic acids into various cell types. Beyond its role in gene delivery, GET can trigger complex cellular responses, as the introduced DNA interacts with intracellular DNA sensing pathways involved in innate immunity and inflammation. These responses can influence the therapeutic outcome – either by enhancing antitumour and vaccine-related immune activation or by reducing transfection efficiency when excessive inflammation or cell death occur. Our experimental findings in tumour, muscle, and skin models have shown that even non-coding plasmid DNA delivered by GET can induce local immune stimulation and tissue-specific inflammatory signaling, suggesting that the delivered DNA itself contributes to therapeutic efficacy. Conclusions The dual nature of cellular responses following plasmid DNA GET represents both an opportunity and a challenge. Controlled activation of innate immunity can be harnessed to amplify antitumour or vaccine efficacy, while excessive responses may hinder applications requiring cell survival and sustained expression. Understanding these mechanisms enables the rational optimization of GET parameters and plasmid vector design to fully exploit the adjuvant effect or reduce the off-target effect of DNA sensing after GET, based on the desired application.
Keywords: gene electrotransfer, DNA sensors, gene therapy
Published in DiRROS: 16.01.2026; Views: 657; Downloads: 257
Full text (747,72 KB)

Abstract: Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.
Keywords: electropermeabilization, gene electrotransfer, muscle, tempol, reactive oxygen species
Published in DiRROS: 26.02.2025; Views: 1243; Downloads: 661
Full text (1,48 MB)
This document has many files! More...

Abstract: Tumor irradiation combined with adjuvant treatments, either vascular targeted or immunomodulatory, is under intense investigation. Gene electrotransfer of therapeutic genes is one of these approaches. The aim of this study was to determine, whether gene electrotransfer of plasmid encoding shRNA for silencing endoglin, with vascular targeted effectiveness, can radiosensitize melanoma B16F10 tumors. Materials and methods. The murine melanoma B16F10 tumors, growing on the back of C57Bl/6 mice, were treated by triple gene electrotransfer and irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice. Furthermore, histological analysis of tumors (necrosis, apoptosis, proliferation, vascularization, presence of hypoxia and infiltration of immune cells,) was used to evaluate the therapeutic mechanisms. Results. Gene electrotransfer of plasmid silencing endoglin predominantly indicated vascular targeted effects of the therapy, since significant tumor growth delay and 44% of tumor free mice were obtained. In addition, irradiation had minor effects on radioresistant melanoma, with 11% of mice tumor free. The combined treatment resulted in excellent effectiveness with 88% of mice tumor free, with more than half resistant to secondary tumor challenge, which was observed also with the plasmid devoid of the therapeutic gene. Histological analysis of tumors in the combined treatment group, demonstrated similar mode of action of the gene electrotransfer of plasmid encoding shRNA for silencing endoglin and devoid of it, both through the induction of an immune response. Conclusions. The results of this study indicate that irradiation can in radioresistant melanoma tumors, by release of tumor associated antigens, serve as activator of the immune response, besides directly affecting tumor cells and vasculature. The primed antitumor immune response can be further boosted by gene electrotransfer of plasmid, regardless of presence of the therapeutic gene, which was confirmed by the high radiosensitization, resulting in prolonged tumor growth delay and 89% of tumor free mice that were up to 63% resistant to secondary challenge of tumor. In addition, gene electrotransfer of therapeutic plasmid for silencing endoglin has also a direct effect on tumor vasculature and tumors cells; however in combination with radiotherapy this effect was masked by pronounced immune response.
Keywords: gene therapy, electrotransfer, plasmid, irradiation, immune response, melanoma
Published in DiRROS: 24.05.2024; Views: 2181; Downloads: 688
Full text (1,13 MB)

Abstract: Gene immunotherapy has become an important approach in the treatment of cancer. One example is the introduction of genes encoding immunostimulatory cytokines, such as interleukin 2 and interleukin 12, which stimulate immune cells in tumours. The aim of our study was to determine the effects of gene electrotransfer of plasmids encoding interleukin 2 and interleukin 12 individually and in combination in the CT26 murine colon carcinoma cell line in mice. In the in vitro experiment, the pulse protocol that resulted in the highest expression of IL-2 and IL-12 mRNA and proteins was used for the in vivo part. In vivo, tumour growth delay and also complete response were observed in the group treated with the plasmid combination. Compared to the control group, the highest levels of various immunostimulatory cytokines and increased immune infiltration were observed in the combination group. Long-term anti-tumour immunity was observed in the combination group after tumour re-challenge. In conclusion, our combination therapy efficiently eradicated CT26 colon carcinoma in mice and also generated strong anti-tumour immune memory.
Keywords: colon carcinoma, gene electrotransfer, gene immunotherapy
Published in DiRROS: 21.03.2024; Views: 1920; Downloads: 960
Full text (6,92 MB)
This document has many files! More...