During the 18-month project duration of RNhale, we were able to establish a reliable platform technology for the production of NEM formulations that can be administered pulmonarily as dry powder. A semi-automated process was developed, culminating in a continuous spray drying procedure. It was successfully demonstrated that a variety of LNP formulations with different lipid compositions, containing diverse RNA cargos (siRNA and mRNA), were successfully embedded in increasing concentrations in excipient matrices with excellent properties for pulmonary delivery. The goal of reaching a human dose concentration of 10% RNA in dry powder was reached.
Consistent gene silencing capability was demonstrated across various in vitro and ex vivo models compared with fresh LNP suspensions (Figure 2). Additionally, in the ex vivo model using human precision-cut lung slices (hPCLS), no toxic side effects were observed.
Furthermore, LNPs containing mRNA were successfully spray-dried. This poses a significantly greater challenge compared to siRNA, as mRNA is extremely sensitive to higher temperatures and stress that may occur during the process. All in vito and ex vivo models were carefully chosen for the sole purpose of mimicking pulmonary conditions such as cell types and pulmonary disease models.
Additionally, formulations were developed that exhibit optimal powder properties for inhalation, with a mass median diameter (MMD) ranging from 1 to 5 µm and a high fine particle fraction (FPF) exceeding 60 %. A residual moisture content of less than 7% is crucial for both good storage stability and low susceptibility to microbial contamination of the formulations. This threshold was significantly undershot in both fresh spray dried formulations and after storage at room temperature and 4 °C for 3 and 18 months (Figures 3 and 4).
In vivo and ex vivo experiments using spray dried powders confirmed therapeutically relevant GATA-3 knockdown with subsequent downregulation of Th2 cytokines and reduced eosinophilia. In human precision-cut lung slices (hPCLS), T cells were activated for three days and transfected for two days. GATA3 mRNA levels were assessed using RT-qPCR. Additionally, a multiplex ELISA was performed on the supernatants of hPCLS to evaluate IL-6, TNF-α, IL-1ß, and IL-10 activation.
