Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA

Schrom, Eva; Huber, Maja; Aneja, Manish; Dohmen, Christian; Emrich, Daniela; Geiger, Johannes; Hasenpusch, Günther; Herrmann-Janson, Annika; Kretzschmann, Verena; Mykhailyk, Olga; Pasewald, Tamara; Oak, Prajakta; Hilgendorff, Anne; Wohlleber, Dirk; Hoymann, Heinz-Gerd; Schaudien, Dirk; Plank, Christian; Rudolph, Carsten; Kubisch-Dohmen, Rebekka

For the purpose of pulmonary cmRNA delivery, we developed a novel proprietary lipid formulation especially designed for lung-targeted cmRNA delivery by intravenous injection (referred to as the pulmonary lipid formulation [PLF] in the following). First, the lung targeting of the formulation was evaluated by intravenous application of luciferase cmRNA in PLF in mice at a dose of 1 mg/kg. 6 hr after injection, a strong luciferase signal was detected selectively in the lungs, whereas other organs did not show any signal ( Figure 5A ). After we identified PLF as a selective delivery agent for lung application, we formulated a mixture of 90% ACE2 cmRNA or 90% control cmRNA and 10% luciferase cmRNA in PLF. A 1-mg/kg dose of this formulation was administered intravenously to mice, while sham-treated animals received a single injection of PBS. After 6 hr, lung-specific delivery was confirmed by luciferase activity, which showed high levels in the lungs compared to other organs for all animals that received PLF formulations ( Figure 5B ). In situ hybridization of ACE2 cmRNA-treated animals showed a homogeneous distribution of positive-stained cells in the alveolar walls, which were interpreted to be type II and type I AECs ( Figure 5C , left panel). Furthermore, single macrophages with cmRNA were found. Immunohistochemical stainings for ACE2 protein in ACE2-treated animals revealed multifocal membranous positive staining of cells located predominantly in the alveolar angles, most likely type II AECs or macrophages (Figure 5C , middle panel). In addition, ACE2 cmRNA-treated animals www.moleculartherapy.org showed positive cells in the alveolar walls with strong membrane and moderate cytoplasmic staining. The morphology of these cells is indicative of type I AECs. ACE2 protein abundance was increased in the ACE2 cmRNA-treated group, while control cmRNA and sham samples showed only endogenous ACE2 protein levels detected by western blot ( Figure 5D ). Overall, the application of cmRNA resulted in mild to moderate focally disseminated free alveolar erythrocytes ( Figure 5C , right panel). Taken together, we have shown that intravenous administration of lipoplexes of a codon-optimized haG ACE2 cmRNA in lipidoid nanoparticles (LLF) or PLF is able to strongly induce ACE2 protein translation selectively in the liver or lung.