SUMMARY: Circular RNAs (circRNAs) are a novel class of RNAs that hold immense potential as therapeutics due to their unique cellular functions. However, the challenges associated with their synthesis and purification have impeded their research and development. Chimerna proposes to develop a platform technology using yeast to manufacture high-quality circRNAs for use in research, therapeutics, and diagnostics. The proposed technology builds upon Chimerna's Tornado technology for expressing RNAs as a circle in cells. When used in E. coli, the Tornado technology results in the production of RNA at high levels not previously seen with conventional linear RNAs. The stability of circular RNA allows it to accumulate to high levels in E. coli, and also confers high stability in vitro and when transfected into mammalian cells, thus mitigating the persistent degradation problem of conventional linear RNAs. The project also utilizes a proprietary affinity tag that is encoded into the circRNA making downstream purification easier. This technology resolves key problems associated with current circular RNA synthesis methods that use in vitro transcription for production. However, production of circRNA in E. coli results in high levels of LPS, which is difficult to remove and not acceptable for therapeutic indications. The current proposal aims to engineer a yeast strain capable of producing circRNAs at high yields. Unlike bacteria, yeast do not contain LPS, which is difficult to remove in downstream purification applications. Moreover, yeast has been extensively used for manufacturing many bioactive compounds such as proteins and lipids. This makes yeast an ideal organism in which to develop a circRNA manufacturing platform. The specific aims of the project are: (1) - To engineer a yeast strain that can be induced to express circular RNA at high yields: This aim involves engineering multiple yeast strains to produce circRNAs and testing their yield under various inducible promoters. The yeast strain will be further optimized by introducing RNA processing enzymes required for maximum circularization efficiency; (2) To benchmark circRNA manufacturing to existing manufacturing technologies: The second aim of the project involves comparing the yield and cost per gram of RNA produced in yeast versus traditional IVT. The researchers will also examine protein expression and RNA half-life from a circular mRNA versus a linear mRNA for total protein output in mammalian cells. Finally, they will measure inflammatory responses to transfected circRNAs versus linear mRNAs. Taken together, the development of a yeast manufacturing platform for synthesizing circRNAs holds immense promise for the production of this highly important type of RNA. The proposed technology will greatly reduce the cost of producing circRNAs while also simplifying the purification and downstream processing. This new manufacturing platform will accelerate the entry of circRNAs for medical use, thereby...