Incorporation of a Human-Derived CU-Rich Element in the 3′ UTR Improves Synthetic mRNA Stability and Expression In Vivo

Synthetic mRNA provides a powerful platform to transfer genetic information encoding therapeutic proteins in vivo. However, their applications are limited by their intrinsic instability and insufficient protein yield. Here we report a 40-nt RNA sequence (ARF6.40) that markedly improves mRNA stability and protein bioavailability of synthetic mRNA. ARF6.40, identified from the 3' UTR of the human ARF6 mRNA, contains a novel CU-rich element that interacts with the protein U2AF2. Compared with the broadly used globin 3' UTR, fusing ARF6.40 to the 3' UTR of EGFP mRNA results in markedly increased mRNA half-life and protein level; transfection of cells with SARS-CoV-2 RBD-encoding mRNA or administration of mice with LNP-encapsulated firefly luciferase mRNA fused with ARF6.40 leads to significantly higher and more sustained RBD secretion or luciferase expression in vivo. Together, our study demonstrates the potential of ARF6.40 in mRNA therapeutics and provides new insights into how the expression of ARF6 is regulated.