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

Liver and lung tissues were excised, fixed for 24 hr in 4% buffered formaldehyde solution, and embedded in paraffin for histological examination. Tissues were then sectioned into 3-to 4-mm slices, deparaffinized, and rehydrated in a graded ethanol series. For antigen retrieval, tissue sections were incubated in 10 mM citrate buffer, pH 6.0, for 30 min using a waterbath at 96 C, washed in PBS, and quenched for 5 min in 3% H 2 O 2 . After another washing step, sections for luciferase were blocked for 1 hr at RT in 2.5% horse serum in PBS (Vector Laboratories). The sections were then incubated with primary anti-luciferase antibody (1:300, G7451; Promega) in PBS supplemented with 0.3% Triton X-100 at 4 C overnight. Tissue sections were washed in PBS and incubated with ImmPRESS reagent (Vector Laboratories) for 30 min at RT. After washing again with PBS, 3,3 0 -diaminobenzidine substrate at 0.5 mg/mL in PBS was added for 1-8 min at RT. For ACE2 staining, the tissue sections were additionally subjected to an avidin/biotin block (Vector Laboratories) for 15 min at RT, followed by a brief wash. Then tissues were blocked in Vectastain serum-blocking reagent D (Vector Laboratories) for 30 min at RT, followed by an overnight incubation at 4 C with primary anti-ACE2 antibody (10 mg/mL, AF933; R&D Systems) in 10% normal goat serum (Life Technologies) in PBS supplemented with 0.2% Triton X-100. After washing with PBS, the sections were incubated with Vectastain biotinylated anti-goat immunoglobulin G (IgG) (diluted according to the manufacturer's protocol; Vector Laboratories), washed again, and incubated with Vectastain ABC reagent (Vector Laboratories), each for 30 min at RT. 3,3 0 -diaminobenzidine substrate was added at 0.5 mg/mL in PBS for 1 min at RT and the reaction was stopped by washing tissue sections in distilled water. After a counterstaining with hematoxylin (Roth), sections were evaluated under a Leica DM2000 LED microscope (Leica Microsystems).