Inhaled dry powders of mRNA polyplexes prepared by thin-film freeze-drying

Localized delivery of messenger RNA (mRNA) therapeutics into the lungs presents a promising approach to prevent or treat respiratory diseases. Previously, we have shown that polyplexes comprised of a thiol-containing poly-β-amino-thio-ester and mRNA are safe and effective in treating viral infections in the lungs after being nebulized in animal models. Herein, we report the development of a stable, inhalable dry powder of mRNA-polymer polyplexes utilizing thin-film freeze-drying (TFFD). After systematic screening and optimization of the formulation composition and process parameters using design-of-experiments (DOE), we developed a TFFD mRNA-polyplex powder, EF85, comprised of mRNA encoding glycosylphosphatidylinositol-anchored nanoluciferase (aNLuc)-P81 polyplexes, trehalose, mannitol, and leucine that preserved the polyplex particle size and polydispersity index, mRNA encapsulation efficiency and integrity, as well as the transfection ability of the polyplexes in cell culture and in a mouse model. The EF85 powder was amorphous with a high glass transition temperature (Tg ∼ 114°C), rendering it stable for months when stored at room temperatures. Furthermore, the powder showed desirable aerosol properties (e.g., fine particle fraction (FPF_{≤5 μm}) as high as 71 ± 5%) for oral inhalation delivery into the lungs. TFFD represents an enabling technology for producing stable, highly aerosolizable mRNA-polyplexes for pulmonary delivery.