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

First, we were interested in the effect of the cmRNA modifications on intracellular ACE2 cmRNA stability. Overall, cmRNA followed a degradation pattern of an exponential one-phase decay ( Figure S2 ). The RNA of all sequences was detectable 72 hr after transfection. Overall, codon optimization slowed the rate of cmRNA degradation, leading to higher levels of cmRNA at later time points. The only exception was the codon-optimized natural ACE2 sequence, which had the highest number of non-codon-optimized nucleotides in its sequence due to the long natural 5 0 and 3 0 UTR regions. Based on these data, the half-life for each sequence was calculated for the decay phase (Table 1 ). In A549 cells, all codon-optimized sequences showed an extended half-life compared to native sequences. In HepG2 cells, codon optimization led to a prolonged half-life for haG and minimal cmRNA sequences. Looking at ACE2 translation efficiency, codon optimization led to stronger protein translation in both cell lines for up to 144 hr (A549 and HepG2 cells in Figure 3A , left and right panels, respectively). The strongest protein translation was observed for codon-optimized haG cmRNA, followed by codon-optimized minimal cmRNA. Despite the extended half-life of codon-optimized CYBA cmRNA, ACE2 protein abundance could not reach the levels of codon-optimized minimal and haG cmRNA. Data obtained by western blot were confirmed by an ACE2 activity assay (A549 and HepG2 cells in Figure 3B , left and right panels, respectively). In both cell lines, ACE2 enzymatic activity was significantly increased for samples transfected with codon-optimized haG cmRNA and codon-optimized minimal cmRNA relative to untransfected samples. Based on these results, codon-optimized minimal and codon-optimized haG cmRNA were identified as the best performing sequences with regard to cmRNA stability, protein translation, and kinetics. As the codon-optimized haG cmRNA sequence showed a slightly longer half-life than the minimal sequence, it was used in all subsequent in vivo studies.