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

With potential future applications of ACE2 RTT in humans, careful consideration of the biochemical properties relating to its biological implications for pharmacokinetics and pharmacodynamics is required. Therefore, we optimized the ACE2 cmRNA sequence with regard to cmRNA stability, protein abundance, enzymatic activity, and translation kinetics. Based on our and other groups' work, we decided on a set of modifications that we considered most promising for our purpose. Cell type and cell state have a strong impact on how the cell reacts to variations in artificially introduced mRNA sequences. 42, [50] [51] [52] The biological implications of these modifications need to be carefully evaluated. UTRs are key elements in the mRNA sequence for translation initiation, elongation, and termination as well as intracellular localization and mRNA stability, 53,54 all of which have a significant impact on final protein expression kinetics. Therefore, we designed sequences with three UTR modifications and a minimal 5 0 UTR known for a high level of protein translation over an extended time period. [41] [42] [43] Codon optimization is another technique that is frequently used for strong protein translation. In this technique, translation rates are markedly increased by replacing rare codons with abundant codons without modifying the amino acid sequence of the encoded protein. 29, 50 In addition, codon optimization reduces secondary structures of cmRNA, which may otherwise induce cellular immune reactions. 50 Therefore, for all four sequences, we designed one natural version and one codon-optimized version of the ORF. In our screen with A549 and HepG2 cells (Figure 3 ), we observed that codon optimization also led to an increased half-life for all cmRNA sequences in A549 cells, but not in HepG2 cells. Protein translation, however, was markedly increased (legend on next page) from 6 to 144 hr in both cell lines for codon-optimized sequences. We could further increase ACE2 protein translation and enzymatic activity by also replacing natural ACE2 UTRs with UTRs of strongly translated proteins. As the different UTRs did not affect the physical halflife of cmRNA (Table 1) , we conclude that replacing UTRs did primarily increase translational efficiency, which is in line with previous findings. 41 For the purpose of liver-and lung-targeted application of ACE2 cmRNA, we could identify codon-optimized haG ACE2 cmRNA as the best performing sequence. Furthermore, we observed that codon optimization of the haG ACE2 cmRNA sequence led to reduced immunogenicity ( Figures S4-S6) , probably leading to a prolonged half-life and strong protein translation for up to 6 days in both cell lines.