The Effect of mRNA Modifications on Target Protein Translation and Immune Response

The introduction of uridine modifications to improve the efficiency of protein translation and immune activation is a key area of ​​development in mRNA technologies, determining their therapeutic potential in the treatment of various diseases. This research was aimed to study the impact of certain uridine modifications on target protein translation and the main pathways of the immune response at cellular level. Uridine-modified mRNA-GFP variants were obtained in a single-step in vitro transcription reaction and transfected into HEK293 and THP-1 cells. Transfection efficiency and GFP-fluorescence intensity were measured using flow cytometry and a live-cell imaging system. The number of GFP copies for both cell cultures was also determined with RT-qPCR for each mRNA preparation studied. GFP protein translation was assessed with Western blotting. Interferon response gene levels were estimated with multiplex cytokine analysis and RT-qPCR. The data obtained show that complete substitution of the uracyl with pseudouridine was associated with the highest translation levels and more significant IRF7-mediated interferon response. On the contrary, complete substitution of the uracyl with 5'-methoxyuridine significantly reduces the immunogenicity of the drug. Meanwhile, 50% substitution with pseudo- or 5'-methoxyuridine produces approximately the same results in terms of GFP expression levels and cellular immune response. Pseudouridine modifications may be potentially useful for minimizing the use of adjuvants in the production of oncotherapeutic mRNA vaccines, while 5'-methoxyuridine modifications help reduce the intrinsic immunogenicity of the mRNA, which may be of potential use in treatment of infectious diseases. However, it makes sense to explore the possibility of gently modulating the immunogenic potential of target drugs by altering the ratio of uridine modifications.