5-Ethynyluridine: a bio-orthogonal uridine variant for mRNA-based therapies and vaccines

Abstract

The identification of pseudo- and N1-methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues propelled the development of mRNA-based vaccines and therapeutics. Here, we characterised another uridine analog, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it did not induce interleukin-6 secretion nor expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity as the other uridine analogs. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent from the RNA structure. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics.