Purifying Circular RNA Therapeutics

Author

Karen Guillen-CuevasFollow

Date of Award

12-2025

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical and Biomolecular Engineering

Committee Chair/Advisor

Scott Husson

Committee Member

Marc Birtwistle

Committee Member

Eric Davis

Committee Member

Igor Luzinov

Abstract

Circular RNA (circRNA) has emerged as a promising therapeutic modality due to its enhanced stability and resistance to exonuclease degradation compared with linear mRNA. However, its stability and functionality depend strongly on achieving high purity, and current methods such as size-exclusion chromatography suffer from peak overlap, low yields, and poor scalability. This dissertation addresses these limitations by developing ultrafiltration-based strategies to purify circRNA directly from in vitro transcription (IVT) and self-splicing (circularization) reactions. First, the feasibility of ultrafiltration was demonstrated using polyethersulfone membranes, where sieving coefficients and critical flux values of circRNA, precursor RNA, and nicked RNA were measured to establish separation conditions. Ultrafiltration experiments under these conditions achieved an 86% purity of circRNA with yields exceeding 50%, outperforming size-exclusion high-performance liquid chromatography. Building on these findings, we employed polycarbonate track-etched membranes with highly uniform pore diameters precisely tailored by gold plating to enhance the separation of RNA conformers. Sieving coefficients and critical flux values of circRNA and linear RNA were again measured to establish separation conditions. Continuous diafiltration under these conditions achieved 94% purity and 59% yield after six diavolumes. Importantly, purified circRNA retained gene expression in HEK293T cells, demonstrating comparable functionality to enzymatically purified circRNA. Together, these studies establish ultrafiltration with well-defined membranes as a scalable, non-chromatographic platform for circRNA purification and provide a foundation for future therapeutic manufacturing.

Recommended Citation

Guillen-Cuevas, Karen, "Purifying Circular RNA Therapeutics" (2025). All Dissertations. 4142.
https://open.clemson.edu/all_dissertations/4142