Scalable production of immune-silent circular RNAs for efficient protein expression

Liu, X.; Xiao, H.; Whitehead, M.; Varshney, D.; Du, Y.

doi:10.1101/2025.11.19.689249

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bioRxiv · 10.1101/2025.11.19.689249

Scalable production of immune-silent circular RNAs for efficient protein expression

Liu, X.·Xiao, H.·Whitehead, M.·Varshney, D.·Du, Y.

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Abstract

Messenger RNA-based therapeutics enable transient protein expression but are limited by its instability. Circular RNA (circRNA) offers enhanced stability, yet scalable production of high-purity circRNA remains challenging. Here, we report an oligo (dT) matrix-based purification strategy for tunable, scalable circRNA production at room temperature. By adjusting poly (A) length, circRNAs are positioned at the midpoint of the oligo (dT) elution profile, enabling one-step separation from both weakly and strongly bound RNA contaminants. We demonstrate that this approach scales linearly with comparable yields for small- and large-scale purifications. In scale-up experiments, 331 mg of highly pure circRNA was recovered in a single run. Incorporating alkaline phosphatase and RNase R digestion prior to chromatography efficiently removes immunogenic contaminants, yielding circRNAs with undetectable immune activation and robust translation in human cells. This workflow provides a practical platform for scalable production of high-quality circRNAs.

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Liu, X., Xiao, H., Whitehead, M., Varshney, D., Du, Y.. 2025-11-19. Scalable production of immune-silent circular RNAs for efficient protein expression. https://doi.org/10.1101/2025.11.19.689249

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