UC San Diego

RNAs are a powerful therapeutic class, but their inherent transience limits their activity. Circularization of RNA can improve their persistence, but simple and scalable approaches to achieve this are lacking. Toward this, we leveraged the ubiquitous expression of the RtcB protein to engineer in situ circularized RNAs (icRNAs), a strategy that enables simple linear RNA to be efficiently circularized upon delivery into the cell. We optimized icRNA protein translation by screening different internal ribosome entry sites (IRESs) and 3’ untranslated regions (UTRs), and we demonstrated icRNA persistence in vitro in cell lines and stem cell derived cells, and in vivo in mice. We showed that addition of modified nucleotides and urea can tune the immunogenicity of icRNAs, and we explored therapeutically relevant icRNA applications in genome and epigenome engineering with zinc finger nucleases (ZFNs) and zinc finger-KRAB fusions (ZF-KRABs). Although other groups have previously developed RNA circularization methods, we conclude that the simplicity and scalability of the icRNA platform make it a promising venture for future optimization.