Internal cap-initiated translation provides efficient protein production from circular mRNA

Circular mRNA, emerging as a groundbreaking RNA therapeutic strategy, faces challenges in enhancing its translation potential. Here, we introduce two innovative molecular designs that bolster circular mRNA translation through an internal cap-initiated mechanism. The first design involved a circular mRNA with a covalently attached N⁷-methylguanosine (m⁷G) cap through a branching structure (cap-circ mRNA). This modification allows circular mRNA to recruit translation machinery and produce proteins more efficiently than IRES-containing circular mRNAs. Combining N¹-methylpseudouridine (m¹Ψ) modification, cap-circ mRNA exhibits a lower acute immunostimulatory effect, maintaining high translation ability, in mice. The second design features the non-covalent attachment of an m⁷G cap to a circular mRNA through hybridization with an m⁷G cap-containing oligonucleotide, significantly enhancing translation by more than 50-fold. This setup allows the design of circular mRNAs to synthesize reporter proteins upon hybridizing with capped mRNAs or long non-coding RNAs and to undergo rolling circle-type translation. These advancements have broadened the therapeutic applications of circular mRNA by minimizing their molecular size, elevating translation efficiency, and facilitating cell-type selective translation.