Effects of Combinations of Untranslated-Region Sequences on Translation of mRNA

Abstract

mRNA-based therapeutics have been found to be a promising treatment strategy in immunotherapy, gene therapy, and cancer treatments. Effectiveness of mRNA therapeutics depends on the level and duration of a desired protein’s expression, which is determined by various cis- and trans-regulatory elements of the mRNA. Sequences of 5′ and 3′ untranslated regions (UTRs) are responsible for translational efficiency and stability of mRNA. An optimal combination of the regulatory sequences allows researchers to significantly increase the target protein’s expression. Using both literature data and previously obtained experimental data, we chose six sequences of 5′UTRs (adenoviral tripartite leader [TPL], HBB, rabbit β-globin [Rabb], H4C2, Moderna, and Neo2) and five sequences of 3′UTRs (mtRNR-EMCV, mtRNR-AES, mtRNR-mtRNR, BioNTech, and Moderna). By combining them, we constructed 30 in vitro transcribed RNAs encoding firefly luciferase with various combinations of 5′- and 3′UTRs, and the resultant bioluminescence was assessed in the DC2.4 cell line at 4, 8, 24, and 72 h after transfection. The cellular data enabled us to identify the best seven combinations of 5′- and 3′UTRs, whose translational efficiency was then assessed in BALB/c mice. Two combinations of 5′- and 3′UTRs (5′Rabb-3′mtRNR-EMCV and 5′TPL-3′Biontech) led to the most pronounced increase in the luciferase amount in the in vivo experiment in mice. Subsequent analysis of the stability of the mRNA indicated that the increase in luciferase expression is explained primarily by the efficiency of translation, not by the number of RNA molecules. Altogether, these findings suggest that 5′UTR-and-3′UTR combinations 5′Rabb-3′mtRNR- EMCV and 5′TPL-3′Biontech lead to high expression of target proteins and may be considered for use in preventive and therapeutic modalities based on mRNA.