A Complete Approach for circRNA Therapeutics from Purification to Lyophilized Delivery Using Novel Ionizable Lipids

Area of Science:

• Biotechnology
• RNA Therapeutics
• Drug Delivery

Background:

• Circular RNA (circRNA) shows therapeutic promise due to its stability.
• Scalable purification and efficient delivery methods for circRNA are critical challenges.
• Existing methods require optimization for clinical translation.

Purpose of the Study:

• To develop and evaluate an optimized affinity chromatography method for circRNA purification.
• To assess the in vivo efficacy of circRNA delivered via lipid nanoparticles (LNPs) using novel ionizable lipids.
• To investigate the potential of lyophilization for stabilizing LNP-circRNA formulations.

Main Methods:

• Affinity chromatography using Oligo (dT) columns for circRNA purification.
• Formulation of circRNA-Oligo (dT) in lipid nanoparticles (LNPs) with ionizable lipids CP-LC-0867 and CP-LC-0729.
• In vivo evaluation of protein expression and luciferase activity in LNP-treated models.
• Lyophilization of LNP-circRNA-Oligo (dT) formulations.

Main Results:

• Oligo (dT) affinity chromatography yielded high purity and yield of circRNA.
• LNPs formulated with CP-LC-0867 demonstrated superior protein expression and sustained activity over 14 days compared to SM-102.
• Lyophilized LNP-circRNA-Oligo (dT) formulations showed comparable protein expression to non-lyophilized counterparts.
• Lyophilization proved effective for extending shelf life and enabling ambient temperature storage.

Conclusions:

• Optimized Oligo (dT) affinity chromatography provides an efficient circRNA purification strategy.
• Novel ionizable lipids like CP-LC-0867 enhance LNP-mediated circRNA delivery and expression.
• Lyophilization is a viable method for stabilizing circRNA-loaded LNPs, improving storage and transport.
• These advancements collectively enhance the scalability, stability, and practical application of circRNA therapeutics.