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

Circular mRNA faces challenges in enhancing its translation potential as an RNA therapeutic. Here we introduce two molecular designs that bolster circular mRNA translation through an internal cap-initiated mechanism. The first consists of 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 internal ribosome entry site (IRES)-containing circular mRNAs. Combining with an N¹-methylpseudouridine (m¹Ψ) modification, cap-circ mRNA exhibits a lower acute immunostimulatory effect, maintaining high translation 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, enhancing translation by more than 50-fold. This setup allows 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 broaden the therapeutic applications of circular mRNAs by minimizing their molecular size, elevating translation efficiency and facilitating cell-type-selective translation.