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

Choosing the optimal administration route for lung-targeted cmRNA delivery requires careful consideration of the pathologic characteristics of the targeted disease. IPF is characterized by AEC apoptosis in response to repetitive microinjuries, and this effect is particularly evident adjacent to fibroblast foci. 60 Previous in vitro studies have shown that AECs produce more AngII in response to injury, while ACE2 mRNA is reduced at the same time. This makes AECs even more prone to injury and AngII-induced apoptosis. 20,61-63 We therefore consider delivering ACE2 cmRNA especially to these areas of epithelial cell death essential for therapeutic success. Due to scarring of the lung parenchyma, these areas are poorly ventilated, hindering the uptake of drugs via the airways. Looking at the vascularization of fibrotic lungs, it was shown that fibroblast foci themselves are poorly vascularized, while the adjacent non-fibrotic areas, where AEC apoptosis takes place, are highly vascularized. 64 ,65 Therefore, we hypothesized that it may be more effective to reach these areas via the pulmonary vasculature than via the airways. Thus, we used a lipid-based cmRNA formulation (PLF) optimized for pulmonary delivery for intravenous application in mice. This formulation showed strong and selective protein translation in the lung (Figures 5A and 5B ). Administration of PLF containing ACE2 cmRNA led to equally strong ACE2 protein translation in the lung ( Figure 5C ). Immunostainings showed that shamtreated animals expressed ACE2 in type II AECs as already observed by Wiener et al., 66 while ACE2 cmRNA-treated lungs presumably also express ACE2 in type I AECs. These findings are especially valuable for therapeutic application in IPF; unlike rodent lungs, human lungs also express ACE2 in type I AECs, 67,68 rendering them an enormous pool for locally active ACE2 protein to break the vicious circle of AngII-stimulated ACE2 downregulation and apoptosis. Establishing ACE2 translation in type I AECs could not be achieved by recombinant protein therapy 21 or lentiviral-mediated ACE2 overexpression. 23 In summary, we advanced the latest RNA technology for sustained local ACE2 translation in the liver or lung. These achievements highlight the strengths of RTT over recombinant protein therapy, where protein half-life, 33 immunogenicity, 69 and organ-or cell-targeted delivery are still challenging. Compared to non-viral gene transfer by pDNA, RTT shows strong translation efficiency without the risk of insertional mutagenesis, 25 a problem also found with retroviral, lentiviral, and adeno-associated gene transfer. 70 In addition, the time frame of viral-mediated gene expression is difficult to control, lasting up to years in the case of adeno-associated viruses. 70 Furthermore, in contrast to gene therapy, cell division is not needed for successful protein expression in RTT. In this study, we designed an ACE2 cmRNA sequence, leading to strong and stable ACE2 protein translation in vitro and in vivo. In combination with selected lipidoid and lipid nanoparticles serving as carrier systems, we were able to translate ACE2 protein selectively in the liver and lung, which may allow reestablishment of local RAS balance. Due to its self-limited translation without the risk of genomic integration, ACE2 protein translation can be fine-tuned to each patient's needs with a controllable treatment duration until the halt or resolution of fibrosis. Therefore, we consider ACE2-base