Incorporation of noncanonical base Z yields modified mRNA with minimal immunogenicity and improved translational capacity in mammalian cells

Chemically modified mRNAs hold great potential for therapeutic applications in vivo. Currently, the base modification scheme largely preserves the canonical Watson-Crick base pairing, thus missing one mode of mRNA modulation by altering its secondary structure. Here we report the incorporation of base Z (2-aminoadenine) into mRNA to create Z-mRNA with improved translational capacity, decreased cytotoxicity, and drastically reduced immunogenicity compared to the unmodified mRNA in mammalian cells. In particular, the A-to-Z substitution renders modified mRNAs less immunogenic than the state-of-the-art base modification N¹-methylpseudouridine (m1ψ) in mouse embryonic fibroblast cells. As a proof of concept, we developed a Z-mRNA-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Antigen-encoding Z-mRNA elicited substantial humoral and cellular immune responses in vivo in mice, albeit with relatively lower efficacy than the state-of-the-art m1ψ-mRNA. Z-mRNA expands the scope of mRNA base modifications toward noncanonical bases and could offer an advantageous platform for mRNA-based therapeutics where minimal immunogenicity is desired.