Rapid generation of circulating and mucosal decoy ACE2 using mRNA nanotherapeutics for the potential treatment of SARS-CoV-2

  1. Jeonghwan Kim
  2. Anindit Mukherjee
  3. Dylan Nelson
  4. Antony Jozic
  5. Gaurav Sahay

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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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  1. ScreenIT

    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 StatementIACUC: Animals: All animal studies were conducted at Oregon Health and Sciences University and approved by the Institutional Animal Care and Use Committee (IACUC, IP00001707).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableIn 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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    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-His
    suggested: (Thermo Fisher Scientific Cat# MA5-33032, RRID:AB_2810125)
    MA5-33032
    suggested: (Thermo Fisher Scientific Cat# MA5-33032, RRID:AB_2810125)
    anti-β-actin
    suggested: (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 HRP
    suggested: (Jackson ImmunoResearch Labs Cat# 111-035-003, RRID:AB_2313567)
    anti-mouse HRP
    suggested: (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 tag
    suggested: None
    sc-81594
    suggested: (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-V5
    suggested: None
    To 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-ACE2
    suggested: (Santa Cruz Biotechnology Cat# sc-390851, RRID:AB_2861379)
    sc-390851
    suggested: (Santa Cruz Biotechnology Cat# sc-390851, RRID:AB_2861379)
    Experimental Models: Cell Lines
    SentencesResources
    293T/17 cell line was purchased from ATCC (CRL-11268).
    293T/17
    suggested: 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-3
    suggested: None
    In 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.
    293T
    suggested: None
    Hep G2
    suggested: None
    Pseudovirus 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-hACE2
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    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/c
    suggested: None

    Results 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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.1101/2020.07.24.205583v1 on bioRxiv