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

Chemically modified mRNA was synthesized as previously described. 71 In brief, the respective pDNA templates were subjected to in vitro transcription using T7 RNA Polymerase (Thermo Fisher Scientific) with a predefined mix of natural and chemically modified ribonucleotides. To enhance RNA stability, a C1-m7G cap structure was enzymatically added at the 5 0 end of the transcript, while the 3 0 end was subjected to polyadenylation of $ 200 nucleotides. The cmRNA product was purified by ammonium-acetate precipitation and was re-suspended in water at the desired concentration. Purity and quality were checked on a NanoDrop2000C spectrophotometer (Thermo Fisher Scientific) and with the Standard Sensitivity RNA Analysis Kit on a Fragment Analyzer (Advanced Analytical Technologies). The following cmRNA sequences were designed: human For liver-targeted in vivo experiments, cmRNA was formulated in LLF as previously described. 35 Lung-targeted lipoplexes (PLF) were prepared by mixing cmRNA with in-house polyethylene glycol (PEG) co-polymer in aqueous solution and applying it to a mix of preassembled lipid micelles. After incubation for 15 min at RT for self-assembly of cmRNA in this PLF, the mix was transferred into Dulbecco's PBS (Thermo Fisher Scientific) supplemented with 2% sucrose. Lipoplexes were applied in a final volume of 150 mL per animal with a cmRNA dose of 1 mg/kg. (PAN-Biotech) . After cell attachment, the medium was changed to culture medium supplemented with 1% FCS.