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

Ex vivo samples were prepared as described by Joyner et al. 72 In brief, frozen liver and lung samples were homogenized in ACE2 reaction buffer (Complete; Roche Diagnostics), which contained 25 nM HEPES buffer, 125 nM NaCl, and 10 mM ZnCl 2 , pH 7.4, followed by a centrifugation at 2,500 Â g for 5 min. The supernatant was collected and spun for 10 min at 28,000 Â g. The resulting pellet www.moleculartherapy.org was re-suspended in reaction buffer before overnight incubation with 0.5% Triton X-100 at 4 C and gentle shaking. Samples were centrifuged for 5 min at 28,000 Â g, the supernatant was collected, and the total protein content was measured by the BCA assay. For the assay, 20 mg protein was incubated for 20 min at RT in ACE2 reaction buffer containing 10 mM Captopril (Santa Cruz Biotechnology), an ACE inhibitor. 1 mM MLN4760 (Exclusive Chemistry), another ACE2 inhibitor, was added, followed by another incubation for 20 min at RT. Finally, 1 mM substrate Mca-A-P-K(DnP) (Caslo) was added and samples were incubated for 1 hr at 37 C.