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

Total RNA was isolated from cell culture or tissue samples using the Nucleospin RNA Plus Kit (Machery-Nagel) and total RNA was transcribed into cDNA using the Transcriptor First-Strand cDNA Synthesis Kit (Roche Diagnostics). Real-time qPCR was performed with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a Roche Light Cycler 96 (Roche Diagnostics). The following primers were used: ACE2 cmRNA in vitro experiments (5 0 -ggccaatcactacg aggact-3 0 and 5 0 -tccactccgttcacctcata-3 0 ); native ACE2 cmRNA construct screen (5 0 -aaaaatgagatggcaagagca-3 0 and 5 0 -gccatctaccccattt acttca-3 0 ); codon-optimized ACE2 cmRNA construct screen (5 0 -aata cgtggtgctgaagaacg-3 0 and 5 0 -agtcgccgtagtcctcgtag-3 0 ); human ACE2 (5 0 -ccagtggatgaaaaagtggtg-3 0 and 5 0 -gtttcatcatggggcacag-3 0 ); human b-actin (PrimePCR SYBR Green Assay; Bio-Rad); human TATAbinding protein (PrimePCR SYBR Green Assay); human b-2-microglobulin (5 0 -ttctggcctggaggctatc-3 0 and 5 0 -tcaggaaatttgactttccattc-3 0 ); human mitochondrial ribosomal protein L19 (MRPL19) (5 0 -ggaatgtt atcgaaggacaagg-3 0 and 5 0 -caggaagggcatctcgtaag-3 0 ); human succinate dehydrogenase, subunit A (SDHA) (5 0 -tccactacatgacggagcag-3 0 and 5 0 -ccatcttcagttctgctaaacg-3 0 ); mouse ACE2 (5 0 -tgtagaacgtaccttcgc agag-3 0 and 5 0 -gtaggaagggtaggtatccatcaa-3 0 ); mouse hypoxanthineguanine phosphoribosyltransferase (Hprt) (5 0 -ggagcggtagcacctcct-3 0 and 5 0 -aacctggttcatcatcgctaa-3 0 ); mouse MRPL19 (5 0 -cgagtacagcacc tttgacg-3 0 and 5 0 -ggcttcattttaactttcagcttg-3 0 ); mouse SDHA (5 0 -ttgagat ccgtgaaggaagag-3 0 and 5 0 -tagacgtgtggccagttgc-3 0 ; and mouse glucuronidase beta (Gusb) (PrimePCR SYBR Green Assay).