Gene Therapy (2018) 25:93–103 https://doi.org/10.1038/s41434-018-0006-y BRIEF COMMUNICATION Improving therapeutic efficacy of IL-12 intratumoral gene electrotransfer through novel plasmid design and modified parameters C. Burkart 1 ● A. Mukhopadhyay 1 ● S. A. Shirley 1 ● R. J. Connolly 2 ● J. H. Wright 1 ● A. Bahrami 1 ● J. S. Campbell 2 ● R. H. Pierce 2 ● D. A. Canton 1 Received: 8 August 2017 / Revised: 14 December 2017 / Accepted: 1 February 2018 / Published online: 9 March 2018 © Macmillan Publishers Ltd., part of Springer Nature 2018 Abstract The use of immunomodulatory cytokines has been shown effective in regressing a wide range of tumors. However, systemic delivery of recombinant cytokines results in serious, potentially life-threatening, adverse effects. By contrast, nucleic acid transfer via electroporation (EP) is a safe and effective method of delivering plasmid-encoded cytokines to tumors. Intratumoral delivery of IL-12 plasmid DNA by electroporation (IT-pIL12-EP) produced objective response rates in Phase 2 clinical trials in metastatic melanoma. However, only 17.9% of patients receiving IT-pIL12-EP show a complete therapeutic response. Here, we sought to improve the antitumor efficacy of our clinical IT-pIL12-EP plasmid electroporation platform. We evaluated multiple plasmid designs for IL-12 expression. IL-12 expression from a plasmid incorporating a picornavirus- derived co-translational P2A site was the most effective in expressing IL-12p70. In addition, modifying the electroporation parameters improved transfection efficiency and expression of plasmid-derived IL-12p70, as well as its downstream effector IFN-γ in vivo. Finally, using a murine melanoma model that is representative of the intended target patient population, we show that combining modified electroporation conditions with the pIL12-P2A plasmid expression enhances the systemic antitumor response. These improvements to the IT-pIL12-EP platform may improve patient clinical response rates and survival when translated to clinical trials. Introduction Gene electrotransfer (GET), a non-viral method to deliver nucleic acids to target cells via application of pulsed elec- trical fields, was first described in the early eighties using mouse lyoma cells [1]. Since then, electroporation (EP)- based technologies have expanded into biotechnological and medical applications [2, 3]. In the medical field, the delivery of plasmid DNA coding for therapeutic genes into solid tumors has shown promise both in preclinical and clinical settings. However, the therapeutic use of EP- mediated transfer of plasmid DNA to tumor tissues has been hampered by inefficient gene transfer leading to low protein expression. Interleukin-12 (IL-12) is an immunomodulatory cytokine mainly produced by antigen-presenting cells (APCs) upon bacterial or viral infection [4, 5]. The IL-12-specific hetero- dimeric receptor complex IL-12R is found on natural killer (NK) cells, NK T cells and activated T cells [6]. IL-12 enhances cytotoxicity of effector T lymphocytes and NK cells, and induces proliferation and interferon-γ (IFN-γ) production [7, 8]. IFN-γ has long been associated with potent antitumor and angiostatic properties [9, 10]. In addition, IL- 12 induces maturation of type 1T helper cells and enhances antigen-specific CD8 + T-cell responses [8, 11]. Several approaches to deliver IL-12 intratumorally have been shown to invoke a strong localized immune response leading to regression of a wide range of established tumors while producing low or no systemic toxicity [12–15]. One method to deliver therapeutic levels of IL-12 is EP of * D. A. Canton dcanton@oncosec.com 1 Oncosec Medical Inc., 5820 Nancy Ridge Drive, San Diego, CA 92121, USA 2 Fred Hutchinson Cancer Center, 1100 Fairview Avenue N., Seattle, WA 98109, USA Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41434-018-0006-y) contains supplementary material, which is available to authorized users. 1234567890();,: