ИСТИНА

mRNA transfection is considered to be a promising approach for gene delivery due to minimal hazards of genome modification and no dependence on cell proliferation status. It may be successfully applied for stem cell reprogramming, vaccination and substitutional therapy. However, in vitro transcribed mRNAs activate the innate immune response, complicating transfection of primary cells. Incorporation of modified nucleotides into a transcript can both decrease the immune response and improve mRNA stability. However, published data on the effects of nucleotide modifications on protein production are controversial. Here, we examined the effects of two modified nucleosides, N1-methylpseudouridine and 5-methylcytidine, within a luciferase transcript, as well as three 5’ cap variants, on reporter mRNA expression in several in vitro and in vivo systems. We compared efficiency and kinetics of regular and modified transcript translation in cell extracts, immortalized cultured cells, and mouse primary hepatocytes and fibroblasts. All mRNAs bearing m7G-cap, both with and without m5C and N1mΨ, efficiently produced luciferase for up to 10-12 hours in living cells and for 1-2 hours in cell-free systems. The effects of mRNA modifications on translation efficiency differed for the systems used. For modified mRNAs, we documented a delay in product appearance, indicating a decrease in elongation rate that was especially pronounced in the case of m5C containing transcripts. By using reporter mRNAs with various 5’ untranslated regions, we showed that the modified nucleotides within mRNA leader negatively affect translation initiation, presumably due to interference with ribosomal scanning. Thus, 5’ leaders with poor CU content may be preferred for therapeutic applications. The work was supported by the grant of the Russian Federation government №14.W03.31.0012.