The success of mRNA vaccines against SARS-CoV-2 has prompted interest in mRNA-based pharmaceuticals due to their rapid production, adaptability, and safety. Despite these advantages, the inherent instability of mRNA and its rapid degradation in vivo underscores the need for an encapsulation system for the administration and delivery of RNA-based therapeutics. Lipid nanoparticles (LNPs) have proven the most robust and safest option for in vivo applications. However, the mid- to long-term storage of mRNA-LNPs still requires sub-zero temperatures along the entire chain of supply, highlighting the need to develop alternatives to improve mRNA vaccine stability under non-freezing conditions to facilitate logistics and distribution. Lyophilization presents itself as an effective alternative to prolong the shelf life of mRNA vaccines under refrigeration conditions, although a complex optimization of the process parameters is needed to maintain the integrity of the mRNA-LNPs. Recent studies have demonstrated the feasibility of freeze-drying LNPs, showing that lyophilized mRNA-LNPs retain activity and stability. However, long-term functional data remain limited. Herein, we focus on obtaining an optimized lyophilizable mRNA-LNP formulation through the careful selection of an optimal buffer and cryoprotectant and by tuning freeze-drying parameters. The results demonstrate that our optimized lyophilization process maintains LNP characteristics and functionality for over a year at refrigerated temperatures, offering a viable solution to the logistical hurdles of mRNA vaccine distribution.

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 compared with high-performance liquid chromatography. Additionally, we investigated the in vivo efficacy of circRNA-Oligo (dT) encapsulated in lipid nanoparticles (LNPs) formulated with emerging 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 the 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.

Background/Objectives: Lipid nanoparticles (LNPs) containing ionizable lipids represent the most advanced non-viral delivery vehicles and have become state-of-the-art carriers for RNA therapeutics. However, further improvements in endosomal escape efficiency and biodegradability are still needed, especially for nucleic acids with transient activity such as messenger RNA (mRNA) and transfer RNA (tRNA). Methods: In this study, a novel library of highly biodegradable ionizable lipids featuring thioester groups within the linker region was designed and synthesized, thereby expanding the chemical linker toolbox for future ionizable lipid development. Results: Comprehensive in vivo structure–activity relationship studies led to the identification of CP-LC-1272 as a lead candidate that markedly enhances endosomal escape and exhibits superior in vivo biodegradability, attributed to the high acid-lability of thioester bonds. LNPs containing CP-LC-1272 maintained in vivo activity after six months of storage in lyophilized form and demonstrated superior in vivo efficiency compared to SM-102 in mRNA expression studies, as well as similar protein restoration in a tRNA delivery model targeting premature stop-codon mutations. Conclusions: The rapid biodegradability of these thioester-activated ionizable lipids (TAILs) suggests a reduced risk of accumulation, with the potential to enable safe repeated dosing or high-dosage RNA therapies, positioning TAILs as a versatile and safe platform for next-generation RNA therapeutics.