(447f) Optimized Lipid Nanoparticles and Delivery Conditions for State-of-the-Art Nebulized mRNA Delivery to the Lungs

Safe, efficient nebulized delivery of mRNA using lipid nanoparticles (LNPs) has the potential to treat, cure, or prevent a wide variety of diseases. While nebulized mRNA delivery has excellent potential, safe and effective delivery of mRNA to the lung epithelium remains a challenge. This is primarily due to two barriers: first, nebulization itself induces powerful shear forces that can disrupt nanoparticle structure and induce aggregation and second, the lung epithelium itself is a challenging target because NP uptake is most efficient on the basolateral side of polarized lung epithelial cells, but the basolateral side is rendered inaccessible to apically delivered LNPs by tight junctions. Here, we addressed each of these barriers to yield an optimized delivery approach. We first found that incorporation of cationic helper lipid into LNPs improved stability to nebulization and subsequent in vivo delivery. Next, we further enhanced stability by altering the nebulization buffer and adding additional excipients to prevent nebulization-induced aggregation which increased nebulized delivery efficiency in vivo. Lastly, we used fully differentiated air-liquid interface cultured primary lung epithelial cells as an ex vivo model for the lung to screen a large, novel library of biodegradable ionizable lipids to identify several excellent lung-transfecting hits. Our final combination of novel ionizable lipid, charge-stabilized formulation, and stability-enhancing excipient yielded a significant improvement over state-of-the-art delivery vehicles for nebulization in terms of nebulized stability and in vivo delivery efficacy. Furthermore, these optimized, nebulized LNPs also drive protein expression for multiple days, allow for multiple dosing, and have no apparent toxicity. Thus, these LNP formulations are excellent candidates for applications from pulmonary vaccination to protein replacement therapy.