Synthetic biology approaches for bio-orthogonal labeling of circular RNAs

SUMMARY The key challenges for RNA therapeutics are delivery and stability inside of cells. Circular RNAs that lack a 5’ and 3’ end are resistant to intracellular degradation, which allows them to persist for much longer than linear RNAs. Meanwhile lipid nanoparticle encapsulation of RNA has improved stability in serum and delivery of RNAs to cells. However, all RNA-based therapies are currently limited by inefficient escape from endosomes. Even when encapsulated in LNPs, less than 1% of RNAs are able to efficiently escape from endosomes. Yet, evidence exists that endosomal escape of siRNAs can be improved by conjugating RNA with hydrophobic lipids. These groups are thought to destabilize the endosome membrane, facilitating escape to the cytoplasm, where they can exert their therapeutic effect. While it is possible to add hydrophobic lipids to chemically synthesized small RNAs, this is more difficult for mRNAs and circular RNAs that are typically produced by in vitro transcription. Chimerna scientists have developed a highly efficient method of producing circular RNAs in bacteria. Chimerna has also developed a proprietary affinity tag to purify circular RNAs from crude lysates. The resulting workflow is rapid, efficient, and scalable, while generating >90% pure circular RNA. Chimerna scientists have also shown that the ligation junction of these circular RNA can be mutated so that they are site-specifically amino functionalized by an endogenous tRNA-guanine transglycosylase. This amino functional group can be conjugated lipids that are widely available or are simple to activate for conjugation using a routine coupling method. In this way, Chimerna is able to produce a variety of site-specifically lipid-conjugated circular RNAs that may accelerate endosomal escape. In this project, we aim to 1) optimize a bacterial strain and expression conditions for high efficiency amino functionalization. This will involve knocking out genes downstream of the transglycosylase and knocking in additional copies of upstream genes. 2) improve the endosomal escape of RNAs by site-specific lipid conjugation. We will test a variety of lipid conjugates to a smaller circular RNA that is a miRNA sponge, and a larger circular mRNA that is translated by cap-independent translation. Both RNAs will be encapsulated using standard methods and we will test efficiency of endosomal escape. Taken together, we will generate a scalable and low-cost method for semi-synthesis of site-specifically labeled circular RNAs. We will leverage this approach to solve the endosomal escape challenges of RNA therapeutics delivery.