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

During Post-translational Modification, ACE2 Is Glycosylated and Integrated into the Plasma Membrane ACE2 is a typical type I integral membrane protein with the core domain located at the extracellular surface. The extracellular domain is flanked by a signal peptide followed by the catalytic domain, which has several glycosylation sites. 37 It was crucial for our in vivo studies to guarantee a locally expressed membranebound version of ACE2 protein; therefore, we specifically investigated post-translational modifications (e.g., glycosylation and intramolecular disulfide bonds) with regard to these biological properties. N-linked glycosylation is pivotal for proper folding, assembly, and trafficking of membrane proteins. Therefore, we investigated whether cmRNA-derived ACE2 protein is glycosylated and correctly integrated into the plasma membrane. Cells transfected with ACE2 cmRNA produced an ACE2 protein with a size of 120 kDa, corresponding to the mature, fully glycosylated form of the protein. 38 The glycosylation process was successfully inhibited by treating cells with tunicamycin, an inhibitor of N-linked glycosylation. 39 Retrospective deglycosylation of mature ACE2 protein by enzymatic deglycosylation resulted in the same protein size as inhibition of glycosylation, confirming full protein glycosylation ( Figure 2A) . Additionally, the formation of disulfide bonds is shown in Figure S8 . To verify correct protein integration and expression on the cell surface, ACE2 cmRNA-transfected cells were stained with anti-ACE2 antibody recognizing the ACE2 core domain located on the cell surface. Cells were then analyzed by flow cytometry (Figures 2B and 2C) . In all three cell lines, ACE2 cmRNA-transfected samples showed a clear increase in ACE2 translation relative to untransfected samples. To localize cmRNA-derived protein, A549 and HepG2 cells were transfected with ACE2 cmRNA and ACE2 protein was visualized by fluorescence staining (HepG2 and A549 cells in Figures 2D and S3 , respectively). In both cell lines, samples transfected with ACE2 cmRNA stained positive for ACE2 protein, while transfection with control cmRNA showed only a weak background signal in HepG2 cells. In light of potential future therapeutic applications for lung and liver fibrosis, endogenous levels of ACE2 protein are not sufficient to prevent disease onset and progress; hence, strong ACE2 translation is pivotal. The immunocytochemical images revealed ACE2 protein localization throughout the cytoplasm and on the plasma membrane. The accumulations found throughout the cytoplasm showed a dotted pattern, indicating protein enrichment in vesicular structures probably involved in protein maturation or trafficking to the plasma membrane. The presence of ACE2 protein at the plasma membrane was indicated by co-localization of ACE2 with wheat germ agglutinin, a plasma membrane marker (white staining patterns in overlay images). Taken together, we could prove that ACE2 cmRNA-derived protein undergoes biological post-translational modifications leading to correct integration into the plasma membrane.