Messenger RNA (mRNA) has broad potential for application in biological systems. However, one fundamental limitation to its use is its relatively short half-life in biological systems. Here we develop exogenous circular RNA (circRNA) to extend the duration of protein expression from full-length RNA messages. First, we engineer a self-splicing intron to efficiently circularize a wide range of RNAs up to 5 kb in length in vitro by rationally designing ubiquitous accessory sequences that aid in splicing. We maximize translation of functional protein from these circRNAs in eukaryotic cells, and we find that engineered circRNA purified by high performance liquid chromatography displays exceptional protein production qualities in terms of both quantity of protein produced and stability of production. This study pioneers the use of exogenous circRNA for robust and stable protein expression in eukaryotic cells and demonstrates that circRNA is a promising alternative to linear mRNA.

Circular RNAs (circRNAs) are a class of singlestranded RNAs with a contiguous structure that have enhanced stability and a lack of end motifs necessary for interaction with various cellular proteins. Here, we show that unmodified exogenous circRNA is able to bypass cellular RNA sensors and thereby avoid provoking an immune response in RIG-I and Toll-like receptor (TLR) competent cells and in mice. The immunogenicity and protein expression stability of circRNA preparations are found to be dependent on purity, with small amounts of contaminating linear RNA leading to robust cellular immune responses. Unmodified circRNA is less immunogenic than unmodified linear mRNA in vitro, in part due to the evasion of TLR sensing. Finally, we provide the first demonstration to our knowledge of exogenous circRNA delivery and translation in vivo, and we show that circRNA translation is extended in adipose tissue in comparison to unmodified and uridine-modified linear mRNAs.

The heterogeneity of cellular states in cancer has been linked to drug resistance, cancer progression and presence of cancer cells with properties of normal tissue stem cells1,2. Secreted Wnt signals maintain stem cells in various epithelial tissues, including in lung development and regeneration3–5. Here we report that murine and human lung adenocarcinomas display hierarchical features with two distinct subpopulations, one with high Wnt signaling activity and another forming a niche that provides the Wnt ligand. The Wnt responder cells showed increased tumour propagation ability, suggesting that they have features of normal tissue stem cells. Genetic perturbation of Wnt production or signaling suppressed tumour progression. Small molecule inhibitors targeting essential post-translational modification of Wnt reduced tumour growth and dramatically decreased proliferative potential of the lung cancer cells, leading to improved survival of tumour-bearing mice. These results indicate that strategies for disrupting pathways that maintain stem-like and niche cell phenotypes can translate into effective anti-cancer therapies.

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