Intratumoral mRNA electrotransfer: establishment and preclinical proof of concept

Intratumoral immunotherapies are promising strategies to increase in situ bioavailability of therapeutics while minimizing systemic side effects. Among state-of-the-art therapeutics, mRNA molecules have been challenged for treatment of cancer. Comparatively, mRNA molecules confer several advantages over viral vectored therapeutics, DNA gene therapies and proteins. However, the successful clinical translation of in vivo mRNA gene therapies has been limited due to inadequate understanding of the expression profile of mRNA therapeutics and relatively short duration of protein production, which necessitates repeated administrations. To enhance the protein expression, trans-amplifying mRNAs have been employed and proved safe in clinical studies. They achieved comparable effect as mRNA, but required much less RNA. The expression profile of the mRNA therapeutics also vary based on the delivery strategy. Among the technologies used to vehicle mRNA, the delivery by lipid nanoparticles and mRNA electroporation have been shown to be safe and effective. Contrary to widely used electroporation of cells, intratumoral naked mRNA electrotransfer (electroporation of tumors in vivo) was investigated in one study only. Therefore, we found there is a lot of room for improvement and believe that electroporation could be exploited as a delivery method for intratumoral mRNA immunotherapy. This is the first time, to our knowledge, that the intratumoral electrotransfer of naked trans-amplifying mRNAs will be investigated in vivo. The project will bridge the gap between two technologies; mRNA and in vivo electroporation of tumors. The three objectives are: 1) To optimize intratumoral mRNA electrotransfer for delivery of mRNA molecules in vivo; 2) to investigate the pharmacokinetics, toxicity, tolerance and immunogenicity of in vivo mRNA electrotransfer with or without mRNA amplification; 3) to investigate the local and systemic immunological antitumor effects of dual in vivo mRNA electrotransfer. Specifically, we will design state-of-the-art reporter mRNA molecules, optimize electric pulse parameters and investigate transfection efficacy in vitro and in vivo, pharmacokinetics, toxicity, tolerance as well as immunogenicity. We expect superior efficiency and safety of the mRNA electrotransfer compared to plasmid electrotransfer, evaluated using state-of-the-art methods as in vivo imaging and droplet digital PCR. Additionally, warming-up immunologically cold B16F10 melanoma by intratumoral electrotransfer of MHC-I mRNA and aPD-L1 mRNA alone or in combination with replicase encoding mRNA would serve as a preclinical proof of principle. The project results will thus pave the way for the translation of intratumoral mRNA electrotransfer in clinical testing and will expand the armamentarium of mRNA-based therapeutic approaches in oncology.