Firefly Luciferase mRNA (no modification)

Noninvasive aerosol inhalation is an established method for pulmonary drug delivery and remains a promising approach for nucleic acid therapeutics. In vitro transcribed (IVT) mRNA has broad therapeutic applicability as it allows for time- and dose-dependent control of the expression of the encoded protein. Inhalation delivery of IVT-mRNA is unproven and requires the development of safe and effective materials. To address this need, hyperbranched poly(β-amino ester) (hPBAE) was synthesized to enable inhalation administration of stable and concentrated nanocomplexes. This strategy resulted in uniform distribution of luciferase mRNA in the five lobes of the lung and production of 101.2 ng g-1 of luciferase protein 24 h after inhalation of the hPBAE complex. Importantly, delivery was confined to the lungs, with no luminescence observed in other tissues. Furthermore, using an Ai14 reporter mouse model, the researchers identified that 24.6% of the total lung epithelial cell population was transfected after a single administration. Repeated inhalation of hPBAE-mRNA resulted in stable protein production in the lungs without local or systemic toxicity. These results suggest that hPBAE vector-assisted IVT-mRNA aerosol delivery may provide a clinically relevant lung epithelial delivery system.

Here, the researchers used a vibrating mesh nebulizer connected to a whole-body chamber to nebulize stable hyperbranched PBAE and bPEI vectors into mice at a concentration of 0.5 mg mL−1 of firefly luciferase mRNA (Figure 1A). Electron microscopy confirmed that the hDD90-118 complex remained stable before and after nebulization. Samples were taken 24 hours after nebulization and bioluminescence was observed to be confined to the lungs, which is different from intravenous administration, when translation may also be observed in the spleen and liver. The luminescence brightness of the lungs transfected with hDD90-118 complexes was significantly increased compared with hC32-118 and bPEI (Figure 1C). Quantification of luciferase protein in lung tissue homogenates again demonstrated significantly higher translation of luciferase encoding mRNA with hDD90-118 polyplexes producing 101.2 ng g−1 (±15.3) of luciferase protein relative to total protein compared to hC32-118 (35.9 ng g−1, ± 8.9) and bPEI (17.4 ng g−1, ± 9/7) (Figure 1B). These data confirm that the chemical composition of PBAE has a significant effect on protein production from IVT-mRNA.

Figure 1. Inhalation of mRNA polyplexes. (Patel A K, et al., 2019)