(I-350) Combinatorial engineering of nebulized mRNA formulations for inhaled delivery to the lungs

Introduction:: Safe and efficient nebulized delivery of mRNA using lipid nanoparticles (LNPs) holds great potential for treating or curing a wide variety of pulmonary diseases including cystic fibrosis (CF). While nebulized mRNA delivery is promising as a non-invasive and localized route of lung administration, 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 highly inefficient on the apical side accessible to inhalation. Here, we addressed each of these barriers to yield an optimized, inhaled delivery system.

Materials and Methods:: LNP formulations were first screened using a design of experiment (DOE) approach and through excipient and buffer optimization to identify formulations which maintained their stability during nebulization but also demonstrated high transfection efficiency in vitro and in vivo. Simultaneously, a library of biodegradable, ionizable lipids were screened in air-liquid interface (ALI) cultures which model the lung epithelium microenvironment to identify highly transfecting lipids. The top ionizible lipids from the ALI screen were combined with the optimized nebulization formulation and evaluated in multiple mouse models to characterize their ability to deliver mRNA via nebulization to the lung epithelium.

Results, Conclusions, and Discussions:: DOE screening of LNP formulations yielded a cationic helper lipid-containing formulation which demonstrated no loss of mRNA encapsulation efficacy or in vitro transfection efficiency following nebulization. LNP stability was further enhanced by altering the nebulization buffer and adding additional excipients to prevent nebulization-induced aggregation which increased nebulized delivery efficiency in vivo by over an order of magnitude. Screening of a large, novel library of biodegradable ionizable lipids in ALI cultures resulted in the identification of multiple lipids demonstrating excellent lung transfection. The 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 while also being well tolerated. The optimized, combinatorial nebulization formulation demonstrated excellent transfection in the airways of healthy mice as well as transfection in the lungs of diseased, CF phenotypic mice. These results demonstrate that the optimized nebulization formulation identified in this study holds significant promise for the inhaled delivery of mRNA therapies to the lung. The development of optimized nebulization formulations for mRNA delivery to the lungs, as presented in this study, hold several implications for the fields of nucleic acid delivery and pulmonary delivery. Our optimized nebulization formulation demonstrated the ability to improve nebulizer stability and in vivo, inhaled delivery efficiency for LNPs incorporating ionizable lipids of diverse structures and this formulation is expected to be widely applicable for improving the nebulized performance of LNP formulations in general. Additionally, we have shown that ALI cultures can be effectively used to screen large lipid libraries to identify high transfecting lipids for use in the lung epithelium. Finally, the optimized nebulization formulation is capable of efficiently transfecting the lungs of diseased mice with abnormally thick mucus layers which is the common phenotype in patients with CF, demonstrating the potential of this formulation for delivering inhaled mRNA therapies to potentially treat or even cure CF.

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