(156g) Purification of Circular RNA By Ultrafiltration

The development of messenger RNA (mRNA) vaccines against COVID-19 renewed the interest in using mRNA to treat existing, emerging, and rare diseases. Despite their high efficiency, the linear structure of the mRNA molecule makes it susceptible to enzymatic degradation at the free ends, which shortens their half-life in vivo and limits the duration of therapeutic protein expression. Circular RNA has increased therapeutic potential relative to standard mRNA due to the elimination of the free ends associated with exonuclease-mediated degradation. The immunogenicity and protein expression stability of circRNA depend strongly on purity. The current purification method is high-performance liquid chromatography (HPLC), which has low recovery yield (about 50% for 90% purity) due to peaks overlapping. This method does not scale well and is not an option for commercial therapeutic circRNA purification. Thus, there is a pressing need for innovative, easy to implement, scalable purification methods to recover circRNA from self-splicing reaction solutions with high purity and yield. The difference in critical flux has been used to separate linear and circular DNA (plasmid) with the same molecular weight using ultrafiltration membranes and varying pressure. The objectives of this proof-of-concept study is to investigate whether ultrafiltration can be used to purify circRNA from self-splicing reaction solutions with high purity (>95%) and high yield (>70%). We hypothesized that ultrafiltration (UF) can be used to separate RNA based on size and form; differences in UF critical fluxes provide a basis for the purification of circRNA from precursor, and nicked RNA. We purified fractions of the different RNA conformers by gel electrophoresis purification and quantified their critical fluxes by varying pressure. While the critical fluxes for precursor and nicked RNA were around 200 µm/s, the critical flux for circRNA was above 457 µm/s.Therefore, we are using the differences in these critical fluxes to purify circRNA from binary mixtures. These data ultimately will be used to establish a protocol for purifying circRNA directly from self-splicing reaction solutions using UF, which is expected to increase purity and yield significantly and make it an attractive option for commercial circRNA purification.

.