The impact of the chemical modifications N1-methylpseudouridine and N4-acetylcytidine on messenger RNA translation, kinetics and cell viability

Abstract

The concept of mRNA-based protein supplementation relies on introducing synthetic mRNA as a transient carrier of genetic information into an organism, where it is translated into the desired therapeutic protein using the cell's machinery. mRNA-based approaches have become increasingly popular as an alternative to DNA-based gene therapy for replacing absent or faulty protein. The landmark discovery that RNA immunogenicity can be reduced by incorporating modified nucleotides initiated the advancement of mRNA from an underappreciated molecule to a promising new class of therapeutics. This thesis investigates the impact of the chemically modified nucleotides N4- acetylcytidine (ac4C) and N1-methylpseudouridine (N1Ψ) incorporated in IVT mRNA on the kinetic profile of protein translation. mKate2-encoding synthetic mRNA modified with N1Ψ and ac4C was transfected into A549 cells in vitro. For comparison, mRNA modified with Ψ and m5C as well as mRNA modified with N1Ψ and m5C, which represent the former and current state-of-the-art modifications to enhance translational capacity and cell viability, were included in the investigations in addition to unmodified mRNA. Subsequently, their translational kinetics, mRNA decay, and their influence on cell viability were examined over time. mRNA modified with N1Ψ and ac4C demonstrated superior translational capacity that lasted for at least 5 days and resulted in a higher yield of reporter protein mKate2. Additionally, N1Ψ- and ac4C-modified mRNA caused only marginal cytotoxicity, resulting in minor losses in terms of cell viability. These results indicate the potential of modifying IVT mRNA with N1Ψ and ac4C as a promising tool for mRNA-based protein supplementation.