Highly efficient cellular expression of circular mRNA enables prolonged protein expression

SUMMARY A major problem with mRNA therapeutics is the limited duration of protein expression due to the short half-life of mRNA. New approaches for generating highly stable circular mRNA in vitro have allowed increased duration of protein expression. However, it remains difficult to genetically encode circular mRNAs in mammalian cells, which limits the use of circular mRNA in cell-derived therapeutics. Here we describe the adaptation of the Tornado (Twister-optimized RNA for durable overexpression) system to achieve in-cell synthesis of circular mRNAs. We identify the promoter and internal ribosomal entry site (IRES) that result in high levels of protein expression in cells. We then show that these circular mRNAs can be packaged into virus-like particles (VLPs) thus enabling prolonged protein expression. Overall, these data describe a platform for synthesis of circular mRNAs and how these circular mRNAs can markedly enhance the ability of VLPs to function as a mRNA delivery tool.