Circular RNA: Design Criteria for Optimal Therapeutical Utility

Abstract

A growing body of work has shown mRNA-based therapeutics are a promising new potential class of drugs, despite the longstanding belief that RNA was too unstable to be efficiently utilized for such applications. Previous studies have established several RNA modifications (e.g., modified nucleotides, cap analogs, polyadenylation, and codon optimization) that enable production of therapeutic levels of protein. Although the established modifications function, in part, by enhancing the stability of RNA, none does so by providing protection against the large family of exonucleases that are primarily responsible for RNA degradation. Thus, we examined whether exonuclease-resistant, circular RNA (circRNA) could be utilized as a superior alternative to conventional, linear RNA. As there are significant differences between linear and circular RNA there was a need to establish appropriate design criteria specific to circRNA. Therefore, we concentrated our work on three areas of focus, the synthesis, immunity, and translation efficiency specifications of circRNA. Regarding synthesis, we have developed a methodology in which large, circRNAs can be efficiently and reliably generated. We showed that the incorporation of a complement-reverse complement (CRC) motif into circRNA and HPLC-mediated purification respectively improve ligation efficiency and drastically reduce immune activation. Additionally, we have identified optimal regulatory motifs that, when incorporated into circRNA, yield enhanced RNA stability, robust protein translation, and minimal immune stimulation both in vitro and in vivo. Lastly, our proof-of-concept in vivo studies using circRNA encoding erythropoietin (EPO) showed significantly longer protein expression and elevated reticulocyte levels compared to standard, modified linear mRNA. Together, this work offers details regarding the design criteria necessary for circRNA to function well in the therapeutic setting and provides the first in vivo-based evidence for the utility of circRNA as a beneficial modification for enhancing the stability and overall efficacy of mRNA therapeutics.