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

The RAS is a signaling cascade producing multiple biological active intermediates. 13 Initially, drug development was mainly focused on ACE inhibitors in the context of blood pressure regulation. 47 However, with increasing knowledge about the physiologic and pathologic properties of the biological intermediates further down the proteolytic cascade, AT1 receptor blockers, AT2 receptor agonists, recombinant ACE2 or ACE2 activators, and Ang-(1-7) analogs gained momentum. 48, 49 With the exception of ACE2, the therapeutic purpose of all of these drugs was to either reduce pro-inflammatory and pro-fibrotic signaling through the AT1 receptor or to counterbalance AT1 receptor signaling by increased Mas receptor signaling. The effect of ACE2 regulators, however, is unique in the RAS, as they can achieve both. By cleaving AngII to Ang-(1-7), ACE2 reduces stimulation of AT1 receptor signaling and at the same time increases antiinflammatory and anti-fibrotic signaling through the Mas receptor. The therapeutic effects of modulating the RAS by increased ACE2 signaling as just described were repeatedly shown for both liver and lung fibrosis. However, previous studies struggled with hurdles such as organ-specific delivery, controllable protein expression, and immunogenicity, among others. Post-translational modifications such as glycosylation are indispensable for full functionality of proteins subjected to such modifications, especially for ACE2 (a membrane-integrated protein), which cannot be guaranteed by recombinant protein therapy. These obstacles and requirements could potentially be solved by RTT; hence, we aimed at investigating the potential of ACE2 RTT. Thus, we designed an in vitro-transcribed chemically modified ACE2 mRNA sequence. This sequence was suc-cessfully tested in vitro for cellular cmRNA uptake and translation into enzymatically active protein in a generic test system (HEK293 cells), as well as in human and murine pulmonary and hepatocellular cells ( Figure 1 ).