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

First, all cells were screened for their endogenous levels of ACE2 mRNA ( Figure S1 ). Endogenous levels in pulmonary cells were either not detectable (as in the case of lung fibroblasts) or were at detection limit (in the case of A549 cells). All other cells showed moderate levels of ACE2 mRNA relative to three reference genes. Cellular uptake of cmRNA after transfection was analyzed by real-time PCR ( Figure 1A ). cmRNA uptake was quantified against a set of reference genes (which were not affected by the experimental conditions; data not shown). 24 hr after transfection, ACE2 cmRNA was successfully taken up in all ACE2 cmRNAtreated samples, while no ACE2 cmRNA could be detected in control cmRNA or untransfected samples. In a next step, we aimed at verifying whether ACE2 cmRNA is successfully translated into ACE2 protein. First, ACE2 protein abundance after transfection was analyzed by western blot. For this purpose, A549, HepG2, and HEK293 cells were transfected with two doses of ACE2 cmRNA, respectively. All three cell lines showed clear dose-dependent expression levels for ACE2 protein ( Figure 1B ). Likewise, transfection of primary liver and lung cells with ACE2 cmRNA also led to clearly detectable ACE2 protein levels. Second, enzymatic activity of ACE2 protein was analyzed by an ACE2 activity assay ( Figure 1C ). All ACE2 cmRNA-transfected samples showed a significant induction in ACE2 activity relative to untransfected samples. These findings demonstrate that ACE2 cmRNA transfection leads to translation of an enzymatically active protein in liver and lung cells.