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

Dysregulation of the local RAS contributes significantly to inflammation and fibrosis, 44,45 a process that can best be counterbalanced by specific ACE2 translation in the affected organs. For the purpose of liver-targeted cmRNA delivery, we chose lipoplexes (referred to as the liver lipidoid formulation [LLF] in the following) as described by Jarzębi nska et al. 35 The liver specificity of LLF was first confirmed in a formulation with firefly luciferase cmRNA. A 1 mg/kg dose of this formulation was administered intravenously to mice and luciferase protein activity was measured 6 hr after administration. All animals showed strong and selective cmRNA uptake in the liver, without signals in other organs ( Figures 4A and 4B ). Luciferase protein expression in the liver clearly mirrored the blood flow through the organ, thereby reflecting lipoplex distribution after intravenous injection ( Figures 4C and S7 ). Strong protein enrichment was observed close to the portal region, where afferent vessels enter the liver, with a gradual decrease toward the efferent central vein. After we successfully verified targeted and selective enrichment of LLF-complexed cmRNA and protein translation in the liver, we formulated two doses of ACE2 cmRNA (4 and 2 mg/kg) and a single dose (2 mg/kg) of control cmRNA in LLF for intravenous injection in mice. 6 hr after treatment, in situ hybridization of livers from ACE2 cmRNA-treated animals showed cmRNA uptake by hepatocytes and detection of cmRNA in liver sinusoids throughout the liver sample in a more or less homogeneous pattern ( Figures 4D and S7 ). These results were confirmed by real-time PCR, showing significant ACE2 cmRNA in liver homogenates of these animals ( Figure 4E ). Quantification of deposited ACE2 cmRNA showed a clear dose-dependent uptake of 0.032 ± 0.007 ng ACE2 cmRNA/mg total RNA for a 4-mg/kg dose and 0.016 ± 0.002 ACE2 cmRNA/mg total RNA for a 2-mg/kg dose. There was no ACE2 cmRNA detected in the control group. ACE2 cmRNA was successfully translated, as shown in western blot analysis ( Figure 4F ) by a clear increase in ACE2 protein abundance in the ACE2 treatment groups. Glycosylation was shown by enzymatic deglycosylation of liver homogenates, inducing a shift in protein size indicative for the active glycosylation process in vivo. In the ACE2 activity assay ( Figure 4G ), significantly higher protein activity was detected in ACE2-transfected liver samples than in the control group showing endogenous ACE2 baseline activity.