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

Cell-and tissue-specific delivery of cmRNA is currently considered a major obstacle to overcome for RTT. 26 We could show that intravenous delivery of reporter cmRNA in LLF as described by Jarzębi nska et al. 35 led to strong and liver-specific protein translation ( Figures 4A and 4B ). Immunohistochemical stainings indicate that intravenous application is a potent delivery mechanism for reaching a high number of hepatocytes, which was previously shown for delivery of small interfering RNA (siRNA) as well as mRNA. 26, [55] [56] [57] [58] The current understanding of the underlying mechanism is that lipoplexes enter the liver lobuli via fenestrated capillaries, which lack a diaphragm (unlike other types of capillaries). This makes them highly permeable for small molecules, and lipoplexes are easily taken up into the interstitium, where they are transported by ligand-based targeting or diffusion from the afferent to the efferent vessels. 56, 57, 59 This is visible in the histochemical stainings for luciferase protein showing a gradual decrease in protein abundance from the afferent to the efferent vessels ( Figure 4C ). This effect is observable throughout the whole organ, confirming the advantage of RTT over recombinant protein therapy and pDNA delivery-namely, to reach a large pool of cells even beyond physical barriers such as endothelium in both mitotic and non-mitotic cells, which was postulated previously by Matsui et al. 58 With the aim of shifting the local balance of the RAS toward resolution of fibrosis, we consider reaching hepatocytes, the most abundant cell type in the liver, a major achievement for strong translation of our target protein. In a second step, we applied the same delivery method to ACE2 cmRNA and saw markedly increased ACE2 protein translation and activity 6 hr after treatment ( Figures 4F and 4G ). Our in vitro findings (Figure 3 ) showed that ACE2 protein is detectable up to 5 days. If this expression pattern can be established in future in vivo models by RTT, ACE2 expression lies above levels reached by recombinant ACE2 therapy in humans 33 and below levels reached by adeno-associated viral ACE2 therapy in experimental models. 16 This emphasizes once more the advantages of RTT for a flexible dosing regimen for potential future clinical application.