Abstract 3480: Gene electrotransfer in cancer: different immunomodulatory profiles and cell death pathways depend on different electric pulse protocols

Abstract Gene electrotransfer (GET) is an excellent strategy for delivering therapeutic genes and vaccines into the target cell using electric pulses. In view of the insights gained during the last COVID-19 pandemic, it is widely acknowledged that genetic vaccines represent a safe and affordable option with considerable promise for addressing a range of diseases, including infectious diseases and cancer. Growing evidence from preclinical studies and clinical trials, which include both intramuscular and intratumoral immunotherapy, suggests that GET-based methods could be adapted for application in veterinary and clinical treatment protocols. At present, a key focus of research is on enhancing plasmid DNA (pDNA) electrotransfer techniques to reduce tissue damage and improve gene transfection efficiency. However, the molecular effects of electroporation (EP)-assisted DNA delivery in vivo are still not well understood. We assessed two GET protocols for the intratumoral administration of pDNA: Short and High Voltage (SHV) electric pulses or an adapted High Voltage - Low Voltage (HV-LV) pulses protocol, both of which are used for reversible electroporation. We investigated the ability of these two electric pulse conditions to trigger immune and tissue responses in murine CT26 colorectal tumors treated with intratumoral non-coding pDNA EP, instead of using an antigen or immunostimulatory agent. Immune cell infiltration in the tumors was assessed after both treatments, and a transcriptomic analysis through RNA sequencing was conducted to examine the gene expression changes induced by GET, with a particular focus on immune and cellular signaling pathways. We observed no significant differences between the two GET protocols in counteracting tumor development in the absence of immunomodulatory molecules. However, important variations in intratumoral macrophage, CD4+ T cells and even higher CD8+ T cells recruitment were reported between the two treatment methods. Transcriptomic analysis revealed that apoptosis is a predominant mode of cell death after GET by SHV pulses, whereas, GET by HV-LV pulses is associated with immunogenic necrotic pathways as well as a prominent activation of both innate and adaptive immune response. We showed that varying pulse parameters can trigger different immunomodulatory profiles within the tumor microenvironment, emphasizing the significance of choosing the right pulse settings in EP-based therapies. This analysis could provide valuable insights for selecting the most effective EP method or for developing new EP-based strategies to enhance the success of GET protocols used for vaccination or gene therapy. Citation Format: Mariangela De Robertis, Tim Bozic, Iva Santek, Flaviana Marzano, Bostjan Markelc, Domenico Alessandro Silvestris, Apollonia Tullo, Graziano Pesole, Maja Cemazar, Emanuela Signori. Gene electrotransfer in cancer: different immunomodulatory profiles and cell death pathways depend on different electric pulse protocols [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3480.