Search bioRxiv⌕ Search

Biology subjects

Casabona, D.

Publications and source records attributed to Casabona, D..

2 recordsLinked to original sources
bioRxiv2024

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

molecular biology↗
bioRxiv2024

Multicomponent Thiolactone-Based Ionizable Lipid Screening Platform for an Efficient and Tunable mRNA Delivery to the Lungs

Ionizable lipids are an essential component of lipid nanoparticles (LNPs) for an efficient mRNA delivery. However, optimizing their chemical structures for high protein expression, efficient endosomal escape, and selective organ targeting remains challenging due to complex structure-activity relationships and multistep synthesis. In this study, we introduce a rapid, high-throughput platform for screening ionizable lipids using a two-step, scalable synthesis involving a one-pot 3-component click-like reaction. This method, herein known as the STAAR approach, standing for Sequential Thiolactone Amine Acrylate Reaction, allowed for the combinatorial synthesis and in vivo screening of 91 novel lipids, followed by a structure-activity study. This led to the development of CP-LC-0729, an ionizable lipid that significantly surpasses the benchmark in protein expression while showing no in vivo toxicity. Additionally, the STAAR lipid platform was further validated by incorporating a one-step strategy to yield a permanently cationic lipid which was tested following a fifth-lipid formulation strategy. The in vivo results showed a highly selective lung delivery with a 32-fold increase in protein expression, outperforming current endogenous targeting strategies. All these findings underscore the potential of lipid CP-LC-0729 and the STAAR lipid platform in advancing the efficiency and specificity of mRNA delivery systems, while also advancing the development of new ionizable lipids.

biochemistry↗