A complete approach for circRNA therapeutics from purification to lyophilized delivery using novel ionizable lipids

Circular RNA (circRNA) has gained significant attention as a potential therapeutic tool due to its remarkable stability and resistance to degradation by exonucleases. However, scalable and efficient methods for purification and delivery remain critical challenges that must be addressed. In this study, we developed and evaluated an optimized affinity chromatography method using Oligo(dT) columns for the purification of circRNA, achieving high yield and purity comparing with high-performance liquid chromatography. Additionally, we investigated the in vivo efficacy of circRNA-Oligo(dT) encapsulated in lipid nanoparticles (LNPs) formulated with emerging STAAR ionizable lipids, including CP-LC-0867 and CP-LC-0729. Our results showed that LNPs formulated with CP-LC-0867 consistently produced higher protein expression compared to SM-102, with sustained luciferase activity observed over a 14-day period. Furthermore, we assessed the lyophilization potential of LNP-circRNA-Oligo(dT) using CP-LC-0729 to extend shelf life and eliminate the need for ultra-low temperature storage. Remarkably, the lyophilized LNPs exhibited no significant differences in protein expression compared to their non-lyophilized counterparts, demonstrating that lyophilization is a viable strategy for extending the storage and transport of circRNA therapies. These findings underscore the potential of optimized new ionizable lipids, improved purification strategies, and lyophilization techniques to enhance the scalability, stability, and practical application of circRNA therapies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=91 SRC="FIGDIR/small/620632v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@8bb7eeorg.highwire.dtl.DTLVardef@d8a1eeorg.highwire.dtl.DTLVardef@6f359forg.highwire.dtl.DTLVardef@1295d05_HPS_FORMAT_FIGEXP M_FIG C_FIG