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.

Targeting the tumor vasculature through specific endothelial cell markers involved in different signaling pathways represents a promising tool for tumor radiosensitization. Two prominent targets are endoglin (CD105), a transforming growth factor β co-receptor, and the melanoma cell adhesion molecule (CD1046), present also on many tumors. In our recent in vitro study, we constructed and evaluated a plasmid for simultaneous silencing of these two targets. In the current study, our aim was to explore the therapeutic potential of gene electrotransfer-mediated delivery of this new plasmid in vivo, and to elucidate the effects of combined therapy with tumor irradiation. The antitumor effect was evaluated by determination of tumor growth delay and proportion of tumor free mice in the syngeneic murine mammary adenocarcinoma tumor model TS/A. Histological analysis of tumors (vascularization, proliferation, hypoxia, necrosis, apoptosis and infiltration of immune cells) was performed to evaluate the therapeutic mechanisms. Additionally, potential activation of the immune response was evaluated by determining the induction of DNA sensor STING and selected pro-inflammatory cytokines using qRT-PCR. The results point to a significant radiosensitization and a good therapeutic potential of this gene therapy approach in an otherwise radioresistant and immunologically cold TS/A tumor model, making it a promising novel treatment modality for a wide range of tumors.

DNA vaccination is one of the emerging approaches for a wide range of applications, including prophylactic vaccination against infectious diseases and therapeutic vaccination against cancer. The aim of this study was to evaluate the feasibility of our previously optimized protocols for gene electrotransfer (GET)-mediated delivery of plasmid DNA into skin and muscle tissues on a model of COVID-19 vaccine. Plasmids encoding the SARS-CoV-2 proteins spike (S) and nucleocapsid (N) were used as the antigen source, and a plasmid encoding interleukin 12 (IL-12) was used as an adjuvant. Vaccination was performed in the skin or muscle tissue of C57BL/6J mice on days 0 and 14 (boost). Two weeks after the boost, blood, spleen, and transfected tissues were collected to determine the expression of S, N, IL-12, serum interferon-γ, the induction of antigen-specific IgG antibodies, and cytotoxic T-cells. In accordance with prior in vitro experiments that indicated problems with proper expression of the S protein, vaccination with S did not induce S-specific antibodies, whereas significant induction of N-specific antibodies was detected after vaccination with N. Intramuscular vaccination outperformed skin vaccination and resulted in significant induction of humoral and cell-mediated immunity. Moreover, both boost and adjuvant were found to be redundant for the induction of an immune response. Overall, the study confirmed the feasibility of the GET for DNA vaccination and provided valuable insights into this approach.