Gene immuno-therapy of solid tumors based on mRNA encoding interleukin-12: So-rIL

Gene therapy is a therapeutic strategy that uses genes as therapeutic factors. During gene therapy, a therapeutic gene is inserted into target cells for a variety of purposes, including stimulating an immune response. The most commonly used vectors for nucleic acid delivery are viral, but much of the focus in has been on the development of plasmid DNA, which is delivered into cells by a variety of means, including electroporation - gene electrotransfer (GET). Plasmid vectors are considered to be safer than viral vectors and can be delivered more than once. In addition, they can contain a long genetic information and are relatively easy and inexpensive to produce. However, there are also certain concerns about them, such as integration into genomic DNA, low transfection efficiency, and horizontal transfer of the antibiotic resistance gene into the gut microbiome, when a plasmid carrying the antibiotic resistance gene is used. mRNA-based gene therapy is alternative to plasmid DNA-based therapies. In contrast to DNA-based drugs, mRNA transcripts have relatively high transfection efficiency and low toxicity, as they do not need to enter the nucleus to function. mRNA-based therapeutics also have the advantage of a relatively short half-life, which allows for a transient and more controlled expression of the encoded therapeutic protein, and can be produced in a cell-free environment by in vitro transcription (IVT), thus avoiding the use of microbes or cell culturing for production. This allows for easy downstream purification and rapid and cost-effective production, leading to a reduction in the cancer treatment cost in the long term. The aim of the proposed project will be to determine the antitumor efficacy of GET mRNAs encoding interleukin-12 (IL-12; mRIL12), and to determine its effect on the activation of PRRs. IL-12 was chosen because we are currently conducting a Phase I clinical study of gene therapy with GET of plasmid DNA encoding IL-12 and we wish to expand the range of potential drug applications or to develop an even more accessible therapy. In this study, we will perform GET and lipofection of mRIL12 and GET of plasmid DNA in two murine cell lines, the melanoma cell line B16F10 and the colorectal carcinoma cell line CT26. At the in vitro level, we will monitor the mRNA transfection rate, the survival of individual cell lines at different time points after GET and lipofection of mRIL12 with different chemical modifications affecting mRNA immunogenicity. In addition, we will monitor the protein level of IL-12, the level of biological activity of IL-12 after the therapies, and the gene expression level of different pattern recognition receptors, as the therapeutic mRNA represents a foreign nucleic acid in the cells. The same parameters will then be monitored by GET with plasmid DNA and compared with each other. In the next steps, we will evaluate all the above-mentioned therapies under in vivo conditions. By measuring tumor growth delay, animal survival, determining the effect on the growth of secondary tumors (abscopal effect), we will determine the antitumor efficacy. Toxicity and immunogenicity will be determined by monitoring clinical signs, blood analysis and presence of mRNA encoding IL-12 in different tissues. We will determine the presence of different immune cells in the tumor microenvironment by immunofluorescence staining of frozen tumor sections. We expect that the antitumor effects of GET of mRNA will be greater than the antitumor effects of lipofection with the mRNA and comparable to the antitumor effect of GET of plasmid DNA. The results of the proposed project will thus open a new avenue for mRNA-based immune gene therapy.