(562c) Combinatorial Development of Nebulized Formulations for Pulmonary mRNA Delivery

Inhaled delivery of mRNA using lipid nanoparticles (LNPs) has the potential to treat a wide variety of diseases. Nebulized delivery of mRNA to the lungs faces several unique physical and biological challenges. This includes stability during nebulization, where shear forces imparted on the formulation cause LNPs both to release encapsulated mRNA as well as to aggregate. Additionally, the lung epithelial microenvironment poses substantial cellular and extracellular barriers to delivery. The dense mucus matrix covering the lung epithelium represents a significant transport barrier to reaching epithelial cells. Following transport across the mucus layer, epithelial cells in the lungs are extremely difficult to transfect apically owing to evolutionary pressures to resist endocytosing potential pathogens inhaled into the lungs. Here, we systematically addressed both the physical and biological barriers to inhaled mRNA-LNP delivery to yield a highly effective inhaled mRNA delivery approach for the lungs.

To address the challenge of nebulizer instability, we first utilized a design of experiment screening methodology to identify incorporation of a cationic helper lipid which promoted greater electrostatic complexation of mRNA within the LNP to prevent nebulizer-induced mRNA loss. We further enhanced nebulizer stability by rationally designing the LNP nebulization buffer composition. By reducing the pH of the nebulization buffer to protonate ionizable lipids within the LNP formulation, we were able to increase LNP surface charge and improve colloidal stability through electrostatic repulsion. The addition of branched polymeric excipients into the LNP formulation further prevented aggregation during nebulization through steric hindrance. Having developed a generalizable LNP formulation that was stable to nebulization, we next addressed the challenge of intracellular delivery to the lung epithelium. We synthesized and screened a large, novel library of biodegradable ionizable lipids using fully differentiated air-liquid interface cultured primary lung epithelial cells to identify several excellent lung-transfecting hits. Our final combination of novel ionizable lipid, charge-stabilized formulation, and stability-enhancing excipient yielded a state-of-the-art formulation for mRNA delivery to the lung epithelium in mice. Furthermore, repeat dosing of our optimized LNP formulation was well tolerated and did not result in loss of efficacy. By addressing the physical and biological bottlenecks to nebulized mRNA delivery, we developed LNP formulations that are excellent candidates for applications from pulmonary vaccination to protein replacement therapy.