(11b) Purification of Circular RNA By Ultrafiltration

Scott M. Husson, Karen Guillen-Cuevas, Xiaoming Lu, Marc Birtwistle

Department of Chemical and Biomolecular Engineering, Clemson University, Clemson, SC 29634, USA

Messenger RNA (mRNA) therapies reached an important milestone in 2020 when the U.S. FDA issued the first emergency use authorization for an mRNA vaccine for the prevention of COVID-19. Despite their high efficiency, the linear structure of mRNA molecules makes them susceptible to enzymatic degradation at the free ends, which shortens their half-life in vivo and limits the duration of therapeutic protein expression. Circularization of RNA is a promising strategy for increasing the duration and quantity of therapeutic protein production and decreasing immunogenicity relative to linear mRNA. Unfortunately, the synthesis of circular RNA produces solutions that contain contaminating linear RNA and nicked RNA that cause cellular immune responses; and no effective purification method has been demonstrated. In this presentation, I will describe a novel approach to purify circular RNA. I will show how ultrafiltration can be used to separate RNA based on shape. During the ultrafiltration process, RNA transmission begins to occur at a critical permeate flux. The critical flux for transmission depends on the RNA shape. Thus, by careful control of the permeate flux, it is possible to separate circular RNA from linear and nicked RNA impurities. Findings from this research have the potential to significantly impact the development of and access to circular RNA therapies by demonstrating a high yield approach for their purification that does not exist today. Research reported in this presentation was supported by the National Institute of General Medical Sciences of the National Institutes of Health under the award R21GM145980.