Optimization of ionizable lipids for aerosolizable mRNA lipid nanoparticles

Abstract

Although mRNA lipid nanoparticles (LNPs) are highly effective as vaccines, their efficacy for pulmonary delivery has not yet fully been established. A major barrier to this therapeutic goal is their instability during aerosolization for local delivery. This imparts a shear force that degrades the mRNA cargo and therefore reduces cell transfection. In addition to remaining stable upon aerosolization, mRNA LNPs must also possess the aerodynamic properties to achieve deposition in clinically relevant areas of the lungs. We addressed these challenges by formulating mRNA LNPs with SM‐102, the clinically approved ionizable lipid in the Spikevax COVID‐19 vaccine. Our lead candidate, B‐1, had the highest mRNA expression in both a physiologically relevant air–liquid interface (ALI) human lung cell model and in healthy mice lungs upon aerosolization. Further, B‐1 showed selective transfection in vivo of lung epithelial cells compared to immune cells and endothelial cells. These results show that the formulation can target therapeutically relevant cells in pulmonary diseases such as cystic fibrosis. Morphological studies of B‐1 revealed differences in the surface structure compared to LNPs with lower transfection efficiency. Importantly, the formulation maintained critical aerodynamic properties in simulated human airways upon next generation impaction. Finally, structure–function analysis of SM‐102 revealed that small changes in the number of carbons can improve upon mRNA delivery in ALI human lung cells. Overall, our study expands the application of SM‐102 and its analogs to aerosolized pulmonary delivery and identifies a potent lead candidate for future therapeutically active mRNA therapies.

Discovery of peptides for targeted delivery of mRNA lipid nanoparticles to cystic fibrosis lung epithelia

“…Multiple groups have recently used NLuc (and modified NLuc) mRNA to optimize LNP formulations, even for respiratory delivery. (68–73) For instance, Lokugamage et al, optimized LNPs for nebulization using a modified NLuc reporter gene and demonstrated that their lead LNP could successfully deliver therapeutic mRNA as well. This highlighted that it is possible to replace NLuc reporter mRNA with a more clinically relevant cargo and still produce significant results.…”

“…We prepared nanoluciferase mRNA (NLuc) as previously published. (73) Briefly, we created a template for NLuc mRNA encoding sequences for a T7 promoter, 5’ UTR, codon-optimized NLuc, and 3’ UTR. We synthesized mRNA using AmpliScribe™ T7-Flash Transcription Kit (Lucigen, catalog #ASF-3507) as described previously.…”

Applying machine learning to identify ionizable lipids for nanoparticle-mediated delivery of mRNA

“…Starting with 43 papers that report on luciferase mRNA LNPs as of April 2023, we excluded 29 papers that either (i) added a fifth component (either for stability or targeting purposes), (ii) modified other lipid components (such as helper lipid or cholesterol), or (iii) tested only in vivo. With these exclusions, the final dataset included 2,332 LNPs from 14 different papers across 10 different labs [10][11][12][13][14][15][35][36][37][38][39][40][41][42] .…”

Inhalable Gene Therapy and the Lung Surfactant Problem

“…However, inhalable delivery comes with its own challenges as well, particularly relating to the aerosolization of the RNA-carrying LNPs and the successful passage of the nanostructures through the lungs' protective mucociliary clearance system. 17,18 Thus, there has been substantial research focused on designing LNPs that are capable of overcoming these obstacles and successfully transfecting the cells that make up the lung epithelium.…”

“…Because directing LNPs into the lung epithelium via IV injection has proven to be difficult in the past, the administration of LNPs via inhalation has often been explored as a more direct alternative route of delivery. , This introduces the benefits of avoiding rapid hepatic clearance, minimizing delivery to other off-target organs and tissues, and bypassing the need for LNPs to cross the pulmonary endothelial barrier. However, inhalable delivery comes with its own challenges as well, particularly relating to the aerosolization of the RNA-carrying LNPs and the successful passage of the nanostructures through the lungs’ protective mucociliary clearance system. , Thus, there has been substantial research focused on designing LNPs that are capable of overcoming these obstacles and successfully transfecting the cells that make up the lung epithelium.…”