Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2
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Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters through the airways and infects the lungs, causing lethal pulmonary damage in vulnerable patients. This virus contains spike proteins on its envelope that binds to human angiotensin-converting enzyme 2 (hACE2) expressed on the surface of airway cells, enabling entry of the virus for causing infection 1,2 . In severe cases, the virus enters the circulatory system, contributing to multiorgan failure. Soluble form of hACE2 binds to SARS-CoV-2 spike protein and prevents viral entry into target cells 3 . Moreover, soluble recombinant ACE2 ameliorates lung injury 4 but its short half-life limits its therapeutic utility 5 . Here, we engineered synthetic mRNA to encode a soluble form of hACE2 (hsACE2) to prevent viral infection. Novel lipid nanoparticles (LNPs) were used to package mRNA and transfect mammalian cells for enhanced production of secreted proteins. Intravenously administered LNP led to hepatic delivery of the mRNA. This elicited secretion of hsACE2 into the blood circulation within 2 h, and levels of circulating hsACE2 peaked at 6 h and gradually decreased over several days. Since the primary site of entry and pathogenesis for SARS-CoV-2 is the lungs, we instilled LNPs into the lungs and were able to detect hsACE2 in the bronchoalveolar lavage fluid within 24 h and lasted for 48 h. Through co-immunoprecipitation, we found that mRNA-generated hsACE2 was able to bind with the receptor binding domain of the SARS-CoV-2 spike protein. Furthermore, hsACE2 was able to strongly inhibit (over 90%) SARS-CoV-2 pseudovirus infection. Our proof of principle study shows that mRNA-based nanotherapeutics can be potentially deployed for pulmonary and extrapulmonary neutralization of SARS-CoV-2 and open new treatment opportunities for COVID-19.
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SciScore for 10.1101/2020.07.24.205583: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Animals: All animal studies were conducted at Oregon Health and Sciences University and approved by the Institutional Animal Care and Use Committee (IACUC, IP00001707). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable In vivo Fluc mRNA transfection via intravenous administration: Female BALB/c mice (8-12 weeks) were sedated using isoflurane, and LNPs encapsulating Fluc mRNA were intravenously administered via tail vein. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibodies used were: rabbit monoclonal anti-V5 tag at 1:1,000 (Cell Signaling Technology, … SciScore for 10.1101/2020.07.24.205583: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Animals: All animal studies were conducted at Oregon Health and Sciences University and approved by the Institutional Animal Care and Use Committee (IACUC, IP00001707). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable In vivo Fluc mRNA transfection via intravenous administration: Female BALB/c mice (8-12 weeks) were sedated using isoflurane, and LNPs encapsulating Fluc mRNA were intravenously administered via tail vein. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The primary antibodies used were: rabbit monoclonal anti-V5 tag at 1:1,000 (Cell Signaling Technology, 13202), rabbit monoclonal anti-6x-His tag at 1:1,000 (Thermo Fisher Scientific, MA5-33032), and mouse monoclonal anti-β-actin at 1:10,000 (R&D Systems, MAB8929). anti-6x-Hissuggested: (Thermo Fisher Scientific Cat# MA5-33032, RRID:AB_2810125)MA5-33032suggested: (Thermo Fisher Scientific Cat# MA5-33032, RRID:AB_2810125)anti-β-actinsuggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)The secondary antibodies used were goat polyclonal anti-rabbit HRP (Jackson ImmunoResearch, 111-035-003) and anti-mouse HRP (115-035-003). anti-rabbit HRPsuggested: (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567)anti-mouse HRPsuggested: (Jackson ImmunoResearch Labs Cat# 115-035-003, RRID:AB_10015289)The antibodies used for pull-down were mouse monoclonal anti-His tag (sc-8036) or anti-V5 tag (sc-81594) antibody (Santa Cruz Biotechnology). anti-His tagsuggested: Nonesc-81594suggested: (Santa Cruz Biotechnology Cat# sc-81594, RRID:AB_1131162)The collected BALF was incubated with Dynabeads having anti-V5 tag antibody for 20 min at room temperature with rotation. anti-V5suggested: NoneTo probe hACE2, anti-ACE2 antibody (Santa Cruz Biotechnology, sc-390851) and anti-mouse HRP were used as the primary and secondary antibodies at 1:200 and 1:2,000, respectively. anti-ACE2suggested: (Santa Cruz Biotechnology Cat# sc-390851, RRID:AB_2861379)sc-390851suggested: (Santa Cruz Biotechnology Cat# sc-390851, RRID:AB_2861379)Experimental Models: Cell Lines Sentences Resources 293T/17 cell line was purchased from ATCC (CRL-11268). 293T/17suggested: ATCC Cat# CRL-11268, RRID:CVCL_1926)Calu-3 cells were cultured in MEM supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, non-essential amino acids, and sodium pyruvate. Calu-3suggested: NoneIn vitro Fluc mRNA transfection assay: For in vitro Fluc mRNA transfection assays, cells were seeded on a white 96 well plate at 4×103 cells/well for 293T and Hep G2 cells or at 104 cells/well for Calu-3, followed by overnight incubation for cell attachment. 293Tsuggested: NoneHep G2suggested: NonePseudovirus neutralization assay: For neutralization assay, 293T-hACE2 cells were seeded into white 96-well plates at 2×104 cells/well and grown for 24 h. 293T-hACE2suggested: NoneExperimental Models: Organisms/Strains Sentences Resources In vivo hsACE2 mRNA transfection via intravenous administration: Female BALB/c mice (8-12 weeks) were sedated using isoflurane, and LNPs encapsulating hsACE2 mRNA were administered to animals via tail vein. BALB/csuggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on pages 33 and 34. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
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