Messenger RNA (mRNA) has recently emerged as a new drug modality with great therapeutic potential. However, linear mRNAs are relatively unstable and also require base modification to reduce their immunogenicity, imposing a limitation to the broad application. With improved stability, the circular RNA (circRNA) presents a better alternative for prolonged expression of the proteins, however the in vitro circularization of RNA at a large scale is technically challenging. Here we developed a new self-catalyzed system to efficiently produce circRNAs in a co-transcriptional fashion. By rational sequence design, we can efficiently produce scarless circRNAs that do not contain foreign sequences. The resulting circRNAs are very stable and have low immunogenicity, enabling prolonged protein translation in different cells without cellular toxicity. The circRNAs generated from this platform can be encapsulated in lipid nanoparticles and efficiently delivered into mice to direct robust protein expression. Finally, the circRNAs encoding RBD of SARS-CoV-2 S protein induced strong antibody productions, with neutralization antibody titers higher than the preclinical data from the linear mRNAs. Collectively, this study provided a general platform for efficient production of circRNAs, demonstrating the potential of circRNAs as the new generation of mRNA therapy.
Programmable RNA editing enables rewriting gene expression without changing genome sequences. Current tools for specific RNA editing dependent on the assembly of guide RNA into an RNA/protein complex, causing delivery barrier and low editing efficiency. We report a new gRNA-free system, RNA editing with individual RNA-binding enzyme (REWIRE), to perform precise base editing with a single engineered protein. This artificial enzyme contains a human-originated programmable PUF domain to specifically recognize RNAs and different deaminase domains to achieve efficient A-to-I or C-to-U editing, which achieved 60–80% editing rate in human cells, with a few non-specific editing sites in the targeted region and a low level off-target effect globally. The RNA-binding domain in REWIREs was further optimized to improve editing efficiency and minimize off-target effects. We applied the REWIREs to correct disease-associated mutations and achieve both types of base editing in mice. As a single-component system originated from human proteins, REWIRE presents a precise and efficient RNA editing platform with broad applicability.
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