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Aim: Gene-based immunotherapy against cancer is limited by low gene transfer efficiency. In the literature, interleukin-12 (IL-12) encoding plasmid associated with sonoporation has been shown to enhance antitumoral activity. Moreover, non-viral carriers and high-frequency ultrasound have both been shown to promote immune response activation. Here, IL-12 encoding plasmid, non-viral carrier stimulating the immune response and focused ultrasound were combined in order to improve the antitumoral efficiency., Methods: In order to enhance a gene-based antitumoral immune response, home-made lipids Toll-like receptor 2 (TLR2) agonists and plasmid free of antibiotic resistance version 4 (pFAR4), a mini-plasmid, encoding the IL-12 cytokine were combined with high-intensity focused ultrasound (HIFU). The lipid composition and the combination conditions were selected following in vitro and in vivo preliminary studies. The expression of IL-12 from our plasmid construct was measured in vitro and in vivo . The combination strategy was evaluated in mice bearing colon carcinoma cells (CT26) tumors following their weight, tumor volume, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) levels in the serum and produced by splenocytes exposed to CT26 tumor cells., Results: Lipid-mediated cell transfection and intratumoral injection into CT26 tumor mice using pFAR4-IL-12 led to the secretion of the IL-12 cytokine into cell supernatant and mice sera, respectively. Conditions of thermal deposition using HIFU were optimized. The plasmid encoding pFAR4-IL-12 or TLR2 agonist alone had no impact on tumor growth compared with control mice, whereas the complete treatment consisting of pFAR4-IL-12, TLR2 lipid agonist, and HIFU limited tumor growth. Moreover, only the complete treatment increased significantly mice survival and provided an abscopal effect on a metastatic CT26 model., Conclusions: The HIFU condition was highly efficient to stop tumor growth. The combined therapy was the most efficient in terms of IL-12 and IFN-γ production and mice survival. The study showed the feasibility and the limits of this combined therapy which has the potential to be improved., Competing Interests: The authors declare that they have no conflicts of interest., (© The Author(s) 2022.)