Engineering circular ribonucleic acids (circRNAs) for improved protein production

Circular ribonucleic acids (circRNAs) are a promising platform for gene expression studies as a stable and prevalent ribonucleic acid in eukaryotic cells, which arise from back-splicing. In a new report now published in Nature Biotechnology, Robert Chen and a team of interdisciplinary researchers at Stanford University, California, U.S., developed a systematic approach to rapidly assemble and test features affecting protein production based on synthetic circular RNAs. The team maximized translation of the circRNA by optimizing fine elements to implement design principles to improve circular RNA yield by several hundred-fold. The outcomes facilitated an increased translation of the RNA of interest, when compared to messenger RNA (mRNA) levels, to provide durable translation in vivo.

Developing circular RNA (circRNA) in the lab

Therapeutics based on ribonucleic acids span across messenger RNA (mRNA), small interfering RNAs (siRNA) and microRNAs (miRNA) with expansion into modern medicine including small molecules, biologics and cell therapeutics. For example, the lately popular mRNA vaccines can be designed in the lab and developed at a rapid pace to respond to evolving and urgent medical crises. Coding RNAs can be circularized into circRNAs to extend the duration of protein translation, based on RNA molecules that covalently join head-to-tail. Bioengineers have also advanced the synthesis of circular long transcripts into circRNAs. However, the fundamental mechanisms of initiating translation to form circular RNA or messenger RNA differ due to the lack of a 7-methylguanylate (M⁷G) cap on the circular RNAs. As a result of this, researchers need to thoroughly examine the principles of circular RNA translation to build better therapies and potentially surpass the translational capacities of mRNA. To examine this aspect, the team developed a modular high-throughput platform to build and test synthetic circular RNAs for optimized translation and improved protein yields.

A modular circRNA assembly platform

The scientists developed a modular cloning platform made of a set of parts compatible with Golden Gate and Gibson cloning to allow higher-throughput testing of circRNAs. Using the platform, they determined how specific aspects of circular RNA design affected its translation. For instance, the team had previously shown how circular RNA triggered immune responses can be avoided in vivo by modifying the molecules with m⁶A. However, researchers must still understand the impact of this step on circular RNA translation. To address this, Chen and the team used their cloning platform and incorporated m⁶A. When compared to unmodified circRNAs, those containing 5% m⁶A showed equal translation after transfection or electroporation in vitro. The scientists thus experimentally gauged the impact of the modification on circular RNA stability.

Uncovering the dynamics of circRNA for strong translation outcomes

To uncover the principles underlying circRNA vector topology necessary for strong translation, the researchers began synthesizing circRNAs to generate variants with peptides encoded by the process. Based on the outcomes, the team showed that increasing the spacer-length was non-beneficial for translation. Next, they showed how the 5′ and 3′ untranslated regions could improve circRNA translation. The researchers also conducted a series of experiments to examine circRNA optimization and then compared them in a single experiment. They showed how the changes progressively increased the expression of circRNA without compromising the RNA yield or efficiency of circularization. They also showed how the kinetics of circRNA and mRNA translation significantly differed, where circRNA took more than 24-hours to reach its maximum translation length, far exceeding the translation duration of mRNA. They then combined the series of circRNA optimizations to test their expression in vivo. To deliver the RNAs, the team formulated them with charge-altering releasable transporters (CARTs) or cationic molecules mediating mRNA expression in mouse models. The outcomes showed how the engineered circRNAs could be expressed at strengths similar to modified RNAs in vivo, albeit with greater duration.

Outlook

In this way, Robert Chen and colleagues showed how RNA circularization has great potential to transform RNA-based medicines by extending the durability of relatively highly transient molecules. Given the fundamental differences between the mechanisms of circRNA and mRNA translation, the existing knowledge of maximizing mRNA translation did not necessarily translate to circRNAs. To facilitate this study, the team created a circRNA modular cloning platform to test numerous sequence variations and optimizations of multiple parameters. Using the platform, they identified several approaches to improve protein translation from circRNAs, with applications to broadly engineer RNAs, to produce more protein than mRNAs in vitro, and exhibit greater durability of its translation in vivo and in vitro. The scientists systematically dissected the elements regulating circRNA translation to then optimize the regions of interest for increased circRNA protein yields for durable protein production in vivo.

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