Highly efficient cellular expression of circular mRNA enables prolonged protein expression

Abstract

A major problem with mRNA therapeutics is that the mRNA is usually degraded within a few hours after entering the cytosol. This limits the duration and total amount of protein expression. New approaches for in vitro synthesis of circular mRNA have allowed increased levels and duration of protein synthesis from mRNA therapeutics due to the long half-life of circular mRNA. However, it remains difficult to genetically encode circular mRNAs in mammalian cells, which makes it difficult to use 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 screen different promoters and internal ribosomal entry sites (IRESs), and identify combinations that result in high levels of circular mRNA and protein expression in cells. We 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.