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• W2607297673 abstract "Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models. Changes in lifestyle and environmental conditions give rise to an increasing prevalence of liver and lung fibrosis, and both have a poor prognosis. Promising results have been reported for recombinant angiotensin-converting enzyme 2 (ACE2) protein administration in experimental liver and lung fibrosis. However, the full potential of ACE2 may be achieved by localized translation of a membrane-anchored form. For this purpose, we advanced the latest RNA technology for liver- and lung-targeted ACE2 translation. We demonstrated in vitro that transfection with ACE2 chemically modified messenger RNA (cmRNA) leads to robust translation of fully matured, membrane-anchored ACE2 protein. In a second step, we designed eight modified ACE2 cmRNA sequences and identified a lead sequence for in vivo application. Finally, formulation of this ACE2 cmRNA in tailor-made lipidoid nanoparticles and in lipid nanoparticles led to liver- and lung-targeted translation of significant amounts of ACE2 protein, respectively. In summary, we provide evidence that RNA transcript therapy (RTT) is a promising approach for ACE2-based treatment of liver and lung fibrosis to be tested in fibrotic disease models. Fibrotic diseases are major causes of mortality and morbidity worldwide, leading to a serious economic burden and challenges for health services.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat. Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2100) Google Scholar, 2Rosenbloom J. Mendoza F.A. Jimenez S.A. Strategies for anti-fibrotic therapies.Biochim Biophys Acta. 2013; 1832: 1088-1103Crossref PubMed Scopus (136) Google Scholar Fibrosis is caused by repetitive noxious stimuli leading to cell stress, injury, and apoptosis, causing organ dysfunction and ultimately organ failure.3Liedtke C. Luedde T. Sauerbruch T. Scholten D. Streetz K. Tacke F. Tolba R. Trautwein C. Trebicka J. Weiskirchen R. Experimental liver fibrosis research: update on animal models, legal issues and translational aspects.Fibrogenesis Tissue Repair. 2013; 6: 19Crossref PubMed Scopus (238) Google Scholar, 4Funke M. Geiser T. Idiopathic pulmonary fibrosis: the turning point is now!.Swiss Med. Wkly. 2015; 145: w14139PubMed Google Scholar Fibrosis can affect nearly every organ, with liver and lung fibrosis showing a rising prevalence due to lifestyle changes or unfavorable environmental conditions.5Wynn T.A. Cellular and molecular mechanisms of fibrosis.J. Pathol. 2008; 214: 199-210Crossref PubMed Scopus (3077) Google Scholar, 6Almeda-Valdes P. Aguilar Olivos N.E. Barranco-Fragoso B. Uribe M. Méndez-Sánchez N. The role of dendritic cells in fibrosis progression in nonalcoholic fatty liver disease.BioMed Res. Int. 2015; 2015: 768071Crossref PubMed Scopus (26) Google Scholar, 7Raghu G. Collard H.R. Egan J.J. Martinez F.J. Behr J. Brown K.K. Colby T.V. Cordier J.F. Flaherty K.R. Lasky J.A. et al.ATS/ERS/JRS/ALAT Committee on Idiopathic Pulmonary FibrosisAn official ATS/ERS/JRS/ALAT statement: idiopathic pulmonary fibrosis: evidence-based guidelines for diagnosis and management.Am. J. Respir. Crit. Care Med. 2011; 183: 788-824Crossref PubMed Scopus (5364) Google Scholar Currently, there are several treatment options under evaluation for liver and lung fibrosis; however, these have had limited therapeutic success, raising the need for new therapeutic approaches.8Margaritopoulos G.A. Vasarmidi E. Antoniou K.M. Pirfenidone in the treatment of idiopathic pulmonary fibrosis: an evidence-based review of its place in therapy.Core Evidence. 2016; 11: 11-22Crossref PubMed Scopus (45) Google Scholar, 9Rogliani P. Calzetta L. Cavalli F. Matera M.G. Cazzola M. Pirfenidone, nintedanib and N-acetylcysteine for the treatment of idiopathic pulmonary fibrosis: A systematic review and meta-analysis.Pulm. Pharmacol. Ther. 2016; 40: 95-103Crossref PubMed Scopus (94) Google Scholar, 10Koyama Y. Xu J. Liu X. Brenner D.A. New developments on the treatment of liver fibrosis.Dig. Dis. 2016; 34: 589-596Crossref PubMed Scopus (78) Google Scholar Investigations of the underlying mechanisms of fibrotic diseases showed that dysregulation of the organ-specific renin-angiotensin system (RAS) plays a critical role in disease onset by triggering excessive pro-inflammatory and pro-fibrotic signaling.1Wynn T.A. Ramalingam T.R. Mechanisms of fibrosis: therapeutic translation for fibrotic disease.Nat. Med. 2012; 18: 1028-1040Crossref PubMed Scopus (2100) Google Scholar Angiotensin-converting enzyme 2 (ACE2) is a family member of the RAS and acts as a metallo-carboxypeptidase cleaving angiotensin II (AngII) to Ang-(1-7). AngII acts on the AngII type I receptor (AT1R), leading to pro-inflammatory and pro-fibrotic signaling, while Ang-(1-7) acts on the Mas oncogenic receptor (Mas receptor), leading to anti-inflammatory and anti-fibrotic signaling. Thus, ACE2 can shift the RAS balance by reducing the amount of AngII and at the same time increasing the amount of Ang-(1-7) molecules. Therefore, ACE2 not only re-establishes the physiologic balance of the RAS, but it also clearly shifts it toward resolution of inflammation and fibrosis.11Marshall R.P. McAnulty R.J. Laurent G.J. Angiotensin II is mitogenic for human lung fibroblasts via activation of the type 1 receptor.Am. J. Respir. Crit. Care Med. 2000; 161: 1999-2004Crossref PubMed Scopus (200) Google Scholar, 12Marshall R.P. Gohlke P. Chambers R.C. Howell D.C. Bottoms S.E. Unger T. McAnulty R.J. Laurent G.J. Angiotensin II and the fibroproliferative response to acute lung injury.Am. J. Physiol. Lung Cell. Mol. Physiol. 2004; 286: L156-L164Crossref PubMed Scopus (215) Google Scholar, 13Clarke N.E. Turner A.J. Angiotensin-converting enzyme 2: the first decade.Int. J. Hypertens. 2012; 2012: 307315Crossref PubMed Scopus (158) Google Scholar ACE2 levels are markedly increased in patients with liver fibrosis as well as in experimental models, which may be due to a counter-regulatory response to RAS upregulation.14Lubel J.S. Herath C.B. Tchongue J. Grace J. Jia Z. Spencer K. Casley D. Crowley P. Sievert W. Burrell L.M. Angus P.W. Angiotensin-(1-7), an alternative metabolite of the renin-angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat.Clin. Sci. 2009; 117: 375-386Crossref PubMed Scopus (88) Google Scholar ACE2 knockout studies revealed that the loss of ACE2 leads to exacerbation of liver injury, which can be attenuated by administration of recombinant ACE215Osterreicher C.H. Taura K. De Minicis S. Seki E. Penz-Osterreicher M. Kodama Y. Kluwe J. Schuster M. Oudit G.Y. Penninger J.M. Brenner D.A. Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice.Hepatology. 2009; 50: 929-938Crossref PubMed Scopus (105) Google Scholar or by adeno-associated ACE2 gene therapy.16Mak K.Y. Chin R. Cunningham S.C. Habib M.R. Torresi J. Sharland A.F. Alexander I.E. Angus P.W. Herath C.B. ACE2 therapy using adeno-associated viral vector inhibits liver fibrosis in mice.Mol. Ther. 2015; 23: 1434-1443Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Similar therapeutic effects were shown by administration of Ang-(1-7)17Warner F.J. Lubel J.S. McCaughan G.W. Angus P.W. Liver fibrosis: a balance of ACEs?.Clin. Sci. 2007; 113: 109-118Crossref PubMed Scopus (87) Google Scholar and as a side-effect of AngII receptor blocker (ARB) therapy.18Moreno M. Gonzalo T. Kok R.J. Sancho-Bru P. van Beuge M. Swart J. Prakash J. Temming K. Fondevila C. Beljaars L. et al.Reduction of advanced liver fibrosis by short-term targeted delivery of an angiotensin receptor blocker to hepatic stellate cells in rats.Hepatology. 2010; 51: 942-952Crossref PubMed Scopus (98) Google Scholar, 19Corey K.E. Shah N. Misdraji J. Abu Dayyeh B.K. Zheng H. Bhan A.K. Chung R.T. The effect of angiotensin-blocking agents on liver fibrosis in patients with hepatitis C.Liver Int. 2009; 29: 748-753Crossref PubMed Scopus (70) Google Scholar Interestingly, unlike observations in liver fibrosis, ACE2 levels are markedly decreased in lung tissue from patients suffering from idiopathic pulmonary fibrosis (IPF).20Li X. Molina-Molina M. Abdul-Hafez A. Uhal V. Xaubet A. Uhal B.D. Angiotensin converting enzyme-2 is protective but downregulated in human and experimental lung fibrosis.Am. J. Physiol. Lung Cell. Mol. Physiol. 2008; 295: L178-L185Crossref PubMed Scopus (141) Google Scholar In murine models, two studies showed that intraperitoneal injection of exogenous ACE2 protein in bleomycin-induced lung fibrosis led to re-establishment of local ACE2 levels and reduced levels of lung injury.21Wang L. Wang Y. Yang T. Guo Y. Sun T. Angiotensin-converting enzyme 2 attenuates bleomycin-induced lung fibrosis in mice.Cell. Physiol. Biochem. 2015; 36: 697-711Crossref PubMed Scopus (41) Google Scholar, 22Rey-Parra G.J. Vadivel A. Coltan L. Hall A. Eaton F. Schuster M. Loibner H. Penninger J.M. Kassiri Z. Oudit G.Y. Thébaud B. Angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury.J. Mol. Med. (Berl.). 2012; 90: 637-647Crossref PubMed Scopus (78) Google Scholar The same protective effect of ACE2 could be shown in a bleomycin-induced mouse model by intratracheal administration of lentiviral packaged Ang-(1–7) fusion gene or ACE2 cDNA.23Shenoy V. Ferreira A.J. Qi Y. Fraga-Silva R.A. Díez-Freire C. Dooies A. Jun J.Y. Sriramula S. Mariappan N. Pourang D. et al.The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension.Am. J. Respir. Crit. Care Med. 2010; 182: 1065-1072Crossref PubMed Scopus (201) Google Scholar RNA transcript therapy (RTT) has gained substantial attention as a newly evolving therapeutic approach. mRNA exerts its function in the cytoplasm, leading to high and reliable transfection efficiency in proliferating as well as quiescent cells without the risk of insertional mutagenesis faced by viral vectors or plasmid DNA (pDNA). In comparison to recombinant protein therapy, RTT is not limited to secreted proteins, making it an interesting alternative for translation of intracellular or membrane-bound protein. Due to enzymatic mRNA degradation mechanisms in the cytoplasm, protein translation is controllable, as it is naturally self-limited.24Tavernier G. Andries O. Demeester J. Sanders N.N. De Smedt S.C. Rejman J. mRNA as gene therapeutic: how to control protein expression.J. Control. Release. 2011; 150: 238-247Crossref PubMed Scopus (177) Google Scholar, 25Yamamoto A. Kormann M. Rosenecker J. Rudolph C. Current prospects for mRNA gene delivery.Eur. J. Pharm. Biopharm. 2009; 71: 484-489Crossref PubMed Scopus (159) Google Scholar, 26Sahin U. Karikó K. Türeci Ö. mRNA-based therapeutics--developing a new class of drugs.Nat. Rev. Drug Discov. 2014; 13: 759-780Crossref PubMed Scopus (1061) Google Scholar Many of the initial obstacles of mRNA therapy, such as RNA instability and immunogenicity, have been solved, offering today a repertoire of techniques for designing chemically modified mRNA (cmRNA) with tailor-made pharmacodynamic properties.27Karikó K. Kuo A. Barnathan E. Overexpression of urokinase receptor in mammalian cells following administration of the in vitro transcribed encoding mRNA.Gene Ther. 1999; 6: 1092-1100Crossref PubMed Scopus (52) Google Scholar, 28Stepinski J. Waddell C. Stolarski R. Darzynkiewicz E. Rhoads R.E. Synthesis and properties of mRNAs containing the novel “anti-reverse” cap analogs 7-methyl(3′-O-methyl)GpppG and 7-methyl (3′-deoxy)GpppG.RNA. 2001; 7: 1486-1495PubMed Google Scholar, 29Gustafsson C. Govindarajan S. Minshull J. Codon bias and heterologous protein expression.Trends Biotechnol. 2004; 22: 346-353Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar, 30Karikó K. Buckstein M. Ni H. Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA.Immunity. 2005; 23: 165-175Abstract Full Text Full Text PDF PubMed Scopus (1180) Google Scholar, 31Karikó K. Muramatsu H. Welsh F.A. Ludwig J. Kato H. Akira S. Weissman D. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability.Mol. Ther. 2008; 16: 1833-1840Abstract Full Text Full Text PDF PubMed Scopus (765) Google Scholar However, clinical application of RTT is still at a pre-clinical stage, owing to challenges such as cell- or organ-specific delivery or the complexity of mRNA pharmacology.26Sahin U. Karikó K. Türeci Ö. mRNA-based therapeutics--developing a new class of drugs.Nat. Rev. Drug Discov. 2014; 13: 759-780Crossref PubMed Scopus (1061) Google Scholar As previously stated, promising results of ACE2 therapy in experimental liver and lung fibrosis have been reported.14Lubel J.S. Herath C.B. Tchongue J. Grace J. Jia Z. Spencer K. Casley D. Crowley P. Sievert W. Burrell L.M. Angus P.W. Angiotensin-(1-7), an alternative metabolite of the renin-angiotensin system, is up-regulated in human liver disease and has antifibrotic activity in the bile-duct-ligated rat.Clin. Sci. 2009; 117: 375-386Crossref PubMed Scopus (88) Google Scholar, 15Osterreicher C.H. Taura K. De Minicis S. Seki E. Penz-Osterreicher M. Kodama Y. Kluwe J. Schuster M. Oudit G.Y. Penninger J.M. Brenner D.A. Angiotensin-converting-enzyme 2 inhibits liver fibrosis in mice.Hepatology. 2009; 50: 929-938Crossref PubMed Scopus (105) Google Scholar, 16Mak K.Y. Chin R. Cunningham S.C. Habib M.R. Torresi J. Sharland A.F. Alexander I.E. Angus P.W. Herath C.B. ACE2 therapy using adeno-associated viral vector inhibits liver fibrosis in mice.Mol. Ther. 2015; 23: 1434-1443Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 17Warner F.J. Lubel J.S. McCaughan G.W. Angus P.W. Liver fibrosis: a balance of ACEs?.Clin. Sci. 2007; 113: 109-118Crossref PubMed Scopus (87) Google Scholar, 21Wang L. Wang Y. Yang T. Guo Y. Sun T. Angiotensin-converting enzyme 2 attenuates bleomycin-induced lung fibrosis in mice.Cell. Physiol. Biochem. 2015; 36: 697-711Crossref PubMed Scopus (41) Google Scholar, 22Rey-Parra G.J. Vadivel A. Coltan L. Hall A. Eaton F. Schuster M. Loibner H. Penninger J.M. Kassiri Z. Oudit G.Y. Thébaud B. Angiotensin converting enzyme 2 abrogates bleomycin-induced lung injury.J. Mol. Med. (Berl.). 2012; 90: 637-647Crossref PubMed Scopus (78) Google Scholar, 23Shenoy V. Ferreira A.J. Qi Y. Fraga-Silva R.A. Díez-Freire C. Dooies A. Jun J.Y. Sriramula S. Mariappan N. Pourang D. et al.The angiotensin-converting enzyme 2/angiogenesis-(1-7)/Mas axis confers cardiopulmonary protection against lung fibrosis and pulmonary hypertension.Am. J. Respir. Crit. Care Med. 2010; 182: 1065-1072Crossref PubMed Scopus (201) Google Scholar In human clinical trials, the safety and tolerability of systemically applied recombinant ACE2 was shown.32Apeiron Biologics. (2009). Safety and Tolerability Study of APN01 (Recombinant Human Angiotensin Converting Enzyme 2). ClinicalTrials.gov identifier NCT00886353. https://clinicaltrials.gov/ct2/show/study/NCT00886353?term=Apeiron&rank=2.Google Scholar, 33Haschke M. Schuster M. Poglitsch M. Loibner H. Salzberg M. Bruggisser M. Penninger J. Krähenbühl S. Pharmacokinetics and pharmacodynamics of recombinant human angiotensin-converting enzyme 2 in healthy human subjects.Clin. Pharmacokinet. 2013; 52: 783-792Crossref PubMed Scopus (260) Google Scholar However, localized translation of membrane-anchored ACE2 may be even more favorable. This may be achieved with recent advances in cmRNA technology.34Kaczmarek J.C. Patel A.K. Kauffman K.J. Fenton O.S. Webber M.J. Heartlein M.W. DeRosa F. Anderson D.G. Polymer-lipid nanoparticles for systemic delivery of mRNA to the lungs.Angew. Chem. Int. Ed. Engl. 2016; 55: 13808-13812Crossref PubMed Scopus (166) Google Scholar, 35Jarzębińska A. Pasewald T. Lambrecht J. Mykhaylyk O. Kümmerling L. Beck P. Hasenpusch G. Rudolph C. Plank C. Dohmen C. A single methylene group in oligoalkylamine-based cationic polymers and lipids promotes enhanced mRNA delivery.Angew. Chem. Int. Ed. Engl. 2016; 55: 9591-9595Crossref PubMed Scopus (67) Google Scholar, 36Fenton O.S. Kauffman K.J. McClellan R.L. Appel E.A. Dorkin J.R. Tibbitt M.W. Heartlein M.W. DeRosa F. Langer R. Anderson D.G. Bioinspired alkenyl amino alcohol ionizable lipid materials for highly potent in vivo mRNA delivery.Adv. Mater. 2016; 28: 2939-2943Crossref PubMed Scopus (134) Google Scholar Therefore, the objective of this study was to establish robust ACE2 translation from cmRNA in the liver or lung, respectively. First, we performed an in vitro validation of ACE2 cmRNA translation, protein activity, and integrity. In a second step, we designed eight different cmRNA sequences and screened them in cell culture to identify the optimal cmRNA composition for sustained protein translation and activity in liver and lung cells. Finally, this lead candidate was formulated for liver- or lung-specific delivery, which led to increased translation of ACE2 protein selectively in these organs. In our first set of experiments, we investigated RNA delivery of in vitro-transcribed chemically modified ACE2 RNA and its successful translation into ACE2 protein. As a generic test system, we chose HEK293 human embryonic kidney cells, which are frequently used for transient transfection experiments. With the aim of liver- and lung-targeted protein translation in subsequent in vivo studies, we selected A549 alveolar epithelial cells (AECs) and HepG2 hepatoma cells as representative human cell lines and hepatocytes and lung fibroblasts as representative primary murine cells. First, all cells were screened for their endogenous levels of ACE2 mRNA (Figure S1). Endogenous levels in pulmonary cells were either not detectable (as in the case of lung fibroblasts) or were at detection limit (in the case of A549 cells). All other cells showed moderate levels of ACE2 mRNA relative to three reference genes. Cellular uptake of cmRNA after transfection was analyzed by real-time PCR (Figure 1A). cmRNA uptake was quantified against a set of reference genes (which were not affected by the experimental conditions; data not shown). 24 hr after transfection, ACE2 cmRNA was successfully taken up in all ACE2 cmRNA-treated samples, while no ACE2 cmRNA could be detected in control cmRNA or untransfected samples. In a next step, we aimed at verifying whether ACE2 cmRNA is successfully translated into ACE2 protein. First, ACE2 protein abundance after transfection was analyzed by western blot. For this purpose, A549, HepG2, and HEK293 cells were transfected with two doses of ACE2 cmRNA, respectively. All three cell lines showed clear dose-dependent expression levels for ACE2 protein (Figure 1B). Likewise, transfection of primary liver and lung cells with ACE2 cmRNA also led to clearly detectable ACE2 protein levels. Second, enzymatic activity of ACE2 protein was analyzed by an ACE2 activity assay (Figure 1C). All ACE2 cmRNA-transfected samples showed a significant induction in ACE2 activity relative to untransfected samples. These findings demonstrate that ACE2 cmRNA transfection leads to translation of an enzymatically active protein in liver and lung cells. ACE2 is a typical type I integral membrane protein with the core domain located at the extracellular surface. The extracellular domain is flanked by a signal peptide followed by the catalytic domain, which has several glycosylation sites.37Tipnis S.R. Hooper N.M. Hyde R. Karran E. Christie G. Turner A.J. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase.J. Biol. Chem. 2000; 275: 33238-33243Crossref PubMed Scopus (1647) Google Scholar It was crucial for our in vivo studies to guarantee a locally expressed membrane-bound version of ACE2 protein; therefore, we specifically investigated post-translational modifications (e.g., glycosylation and intramolecular disulfide bonds) with regard to these biological properties. N-linked glycosylation is pivotal for proper folding, assembly, and trafficking of membrane proteins. Therefore, we investigated whether cmRNA-derived ACE2 protein is glycosylated and correctly integrated into the plasma membrane. Cells transfected with ACE2 cmRNA produced an ACE2 protein with a size of 120 kDa, corresponding to the mature, fully glycosylated form of the protein.38Lambert D.W. Yarski M. Warner F.J. Thornhill P. Parkin E.T. Smith A.I. Hooper N.M. Turner A.J. Tumor necrosis factor-alpha convertase (ADAM17) mediates regulated ectodomain shedding of the severe-acute respiratory syndrome-coronavirus (SARS-CoV) receptor, angiotensin-converting enzyme-2 (ACE2).J. Biol. Chem. 2005; 280: 30113-30119Crossref PubMed Scopus (500) Google Scholar The glycosylation process was successfully inhibited by treating cells with tunicamycin, an inhibitor of N-linked glycosylation.39Powell L.D. Inhibition of N-linked glycosylation.Curr. Protoc. Immunol. 2001; 8: 8.14.1-8.14.9Google Scholar Retrospective deglycosylation of mature ACE2 protein by enzymatic deglycosylation resulted in the same protein size as inhibition of glycosylation, confirming full protein glycosylation (Figure 2A). Additionally, the formation of disulfide bonds is shown in Figure S8. To verify correct protein integration and expression on the cell surface, ACE2 cmRNA-transfected cells were stained with anti-ACE2 antibody recognizing the ACE2 core domain located on the cell surface. Cells were then analyzed by flow cytometry (Figures 2B and 2C). In all three cell lines, ACE2 cmRNA-transfected samples showed a clear increase in ACE2 translation relative to untransfected samples. To localize cmRNA-derived protein, A549 and HepG2 cells were transfected with ACE2 cmRNA and ACE2 protein was visualized by fluorescence staining (HepG2 and A549 cells in Figures 2D and S3, respectively). In both cell lines, samples transfected with ACE2 cmRNA stained positive for ACE2 protein, while transfection with control cmRNA showed only a weak background signal in HepG2 cells. In light of potential future therapeutic applications for lung and liver fibrosis, endogenous levels of ACE2 protein are not sufficient to prevent disease onset and progress; hence, strong ACE2 translation is pivotal. The immunocytochemical images revealed ACE2 protein localization throughout the cytoplasm and on the plasma membrane. The accumulations found throughout the cytoplasm showed a dotted pattern, indicating protein enrichment in vesicular structures probably involved in protein maturation or trafficking to the plasma membrane. The presence of ACE2 protein at the plasma membrane was indicated by co-localization of ACE2 with wheat germ agglutinin, a plasma membrane marker (white staining patterns in overlay images). Taken together, we could prove that ACE2 cmRNA-derived protein undergoes biological post-translational modifications leading to correct integration into the plasma membrane. After having successfully verified that the sequence of ACE2 cmRNA leads to the translation of an active membrane-bound form of ACE2 protein, we wanted to further optimize the sequence for strong protein translation. Therefore, we designed eight different ACE2 cmRNA sequences, sharing the same open reading frame (ORF) encoding ACE2, a C1-m7G cap, and a poly(A) tail of ∼120 nucleotides, which was found to be the optimal length.40Holtkamp S. Kreiter S. Selmi A. Simon P. Koslowski M. Huber C. Türeci O. Sahin U. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells.Blood. 2006; 108: 4009-4017Crossref PubMed Scopus (357) Google Scholar In addition to the natural ACE2 mRNA sequence, we introduced three different modifications of the UTRs known for a high level of protein translation41Ferizi M. Leonhardt C. Meggle C. Aneja M.K. Rudolph C. Plank C. Rädler J.O. Stability analysis of chemically modified mRNA using micropattern-based single-cell arrays.Lab Chip. 2015; 15: 3561-3571Crossref PubMed Google Scholar, 42Babendure J.R. Babendure J.L. Ding J. Tsien R.Y. Control of mammalian translation by mRNA structure near caps.RNA. 2006; 12: 851-861Crossref PubMed Scopus (199) Google Scholar, 43Qadah, T.H. (2014). A study of molecular mechanisms regulating human alpha globin production: an in vitro comparative study between normal and α-thalassemia subtypes. PhD thesis (Perth: University of Western Australia).Google Scholar: namely, a minimal 5′ UTR, a human alpha globin (hαG) 5′ UTR, and a cytochrome b-245 alpha poly-peptide (CYBA) 5′ with 3′ UTR. For all four sequences, we designed one natural version and one codon-optimized version of the ORF. 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′ and 3′ 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 hαG 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 hαG 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 hαG 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 hαG cmRNA and codon-optimized minimal cmRNA relative to untransfected samples. Based on these results, codon-optimized minimal and codon-optimized hαG cmRNA were identified as the best performing sequences with regard to cmRNA stability, protein translation, and kinetics. As the codon-optimized hαG cmRNA sequence showed a slightly longer half-life than the minimal sequence, it was used in all subsequent in vivo studies.Table 1Half-Life of ACE2 cmRNA SequencesNaturalMinimalhαGCYBACodon OptimizedNaturalMinimalhαGCYBAA549 Half-life (hr)7.655.096.777.9910.2911.3513.3011.10 95% confidence interval6.01–10.544.69–5.556.52–7.035.71–13.359.27–11.578.23–18.279.44–22.538.48–16.08HepG2 Half-life (hr)7.225.665.669.296.957.819.1610.86 95% confidence interval5.35–11.115.24–6.165.11–6.357.83–11.425.66–9.006.86–9.076.95–13.427.71–18.37 Open table in a new tab Dysregulation of the local RAS contributes significantly to inflammation and fibrosis,44Bataller R. Sancho-Bru P. Ginès P. Lora J.M. Al-Garawi A. Solé M. Colmenero J. Nicolás J.M. Jiménez W. Weich N. et al.Activated human hepatic stellate cells express the renin-angiotensin system and synthesize angiotensin II.Gastroenterology. 2003; 125: 117-125Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 45Huang Q. Xie Q. Shi C.C. Xiang X.G. Lin L.Y. Gong B.D. Zhao G.D. Wang H. Jia N.N. Expression of angiotensin-converting enzyme 2 in CCL4-induced rat liver fibrosis.Int. J. Mol. Med. 2009; 23: 717-723PubMed Google Scholar a process that can best be counterbalanced by specific ACE2 translation in the affected organs. For the purpose of liver-targeted cmRNA delivery, we chose lipoplexes (referred to as the liver lipidoid formulation [LLF] in the following) as described by Jarzębińska et al.35Jarzębińska A. Pasewald T. Lambrecht J. Mykhaylyk O. Kümmerling L. Beck P. Hasenpusch G. Rudolph C. Plank C. Dohmen C. A single methylene group in oligoalkylamine-based cationic polymers and lipids promotes enhanced mRNA delivery.Angew. Chem. Int. Ed. Engl. 2016; 55: 9591-9595Crossref PubMed Scopus (67) Google Scholar The liver specificity of LLF was first confirmed in a formulation with firefly luciferase cmRNA. A 1 mg/kg dose of this formulation was administered intravenously to mice and luciferase protein activity was mea" @default.
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• W2607297673 title "Translation of Angiotensin-Converting Enzyme 2 upon Liver- and Lung-Targeted Delivery of Optimized Chemically Modified mRNA" @default.
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