1:Division of Gene Therapy and Regulation of Gene Expression, Cima Universidad de Navarra, Av. Pio XII 55, 31008, Pamplona, Spain; 2:Instituto de Investigación Sanitaria de Navarra (IdISNA), Pamplona, Spain; 3:Program of Immunology and Immunotherapy, Cima Universidad de Navarra, Av. Pio XII 55, 31008, Pamplona, Spain

Vectors based on alphavirus self-amplifying RNA (saRNA) generate high and transient levels of transgene expression and induce strong innate immune responses, constituting an interesting tool for antitumor therapy. Administration of naked saRNA in vivo could have advantages compared to viral particles, but the high RNA instability makes this approach poorly efficient. A quick and simple strategy to increase RNA uptake by cells is electroporation. Here, we used Semliki Forest Virus (SFV) saRNA for local treatment of murine colorectal MC38 subcutaneous tumors. First, we optimized conditions for SFV saRNA electroporation in tumors using a saRNA coding for luciferase and evaluating in vivo expression by bioluminiscence. As proof-of-concept, the therapeutic potential of this approach was evaluated using saRNA coding for interleukin 12 (IL-12), a proinflammatory cytokine with potent antitumor effects. The delivery of SFV-IL-12 saRNA by electroporation led to an improvement in tumor control and higher survival compared to mice treated with electroporation or SFV-IL-12 saRNA as single therapies. Our results suggest that this combinatorial therapy enhances the overall antitumor effect by promoting antitumor specific immune responses and tumor cell death. Combination of local SFV-IL-12 saRNA electroporation with systemic PD-1 blockade led to an increase in antitumor effects, indicating that saRNA electroporation could benefit from already approved therapies. This therapy, which was also validated in a hepatic tumor model, suggest that local delivery of saRNA by electroporation could represent an attractive strategy for cancer immunotherapy. This approach could have an easy translation to the clinical practice, especially for percutaneously accessible tumors.