Designing Lipid Nanoparticle Formulations for Pulmonary Delivery of Nucleic Acids

With the breakthrough success of SARS-CoV-2 mRNA vaccines, lipid nanoparticles (LNPs) have emerged as transformative non-viral formulations to deliver nucleic acids as genetic medicines. To target genetic diseases in extrahepatic tissues, it is necessary to develop new LNP formulations and/or alternate routes of delivery that circumvent endogenous liver targeting of LNPs traditionally observed with most systemically delivered LNPs. In particular for diseases that affect the respiratory tract, such as cystic fibrosis (CF), local, pulmonary delivery of nucleic acid therapeutics to the airways is attractive to achieve higher dosing with less off-tissue uptake compared to systemic delivery. However, for effective pulmonary delivery of LNPs, they need to (1) be sufficiently physically stable after aerosolization, (2) possess the aerodynamic properties for deposition in clinically relevant areas of the lungs, and (3) penetrate the mucus barrier and target the underlying airway epithelia harboring disease-causing mutations. To address these issues, we have focused on two areas – to develop stable, aerosolized LNPs for delivery in the airways and to identify ligands that facilitate mucus penetration and target LNP delivery to airway epithelia. First, through design of experiments screening of aerosolized LNPs, we identified a lead LNP candidate that delivered and exhibited highest reporter mRNA activity in both a physiologically relevant air-liquid interface (ALI) human lung cell model and in healthy mice lungs compared to control compositions used in FDA-approved mRNA LNPs Comirnaty and Spikevax. Using next generation impaction, we confirmed that our formulations exhibited desired aerodynamic properties needed for effective deposition in the airways. In our second aim, we developed and used phage display technology with principles of directed evolution for unbiased identification of select peptide ligands that not only facilitate mucus penetration but also achieve targeted uptake into primary airway epithelial cells from CF patients. Subsequently, we have demonstrated that our lead peptide ligand can improve uptake of LNPs ~8-fold in ALI and animal models. Importantly, our peptide-functionalized LNPs demonstrate selective targeting to airway epithelia compared to immune and endothelial cells in mouse lungs. Collectively, these findings suggest that LNPs can be formulated and administered to target the airways towards effective nucleic acid delivery into the pulmonary tract.