Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl

  1. Zhifen Cui
  2. Cong Zeng
  3. Furong Huang
  4. Fuwen Yuan
  5. Jingyue Yan
  6. Yue Zhao
  7. Jiaoti Huang
  8. Herman F. Staats
  9. Jeffrey I. Everitt
  10. Gregory D. Sempowski
  11. Hongyan Wang
  12. Yizhou Dong
  13. Shan-Lu Liu
  14. Qianben Wang

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Abstract

The COVID-19 pandemic persists as a global health crisis for which curative treatment has been elusive. Development of effective and safe anti-SARS-CoV-2 therapies remains an urgent need. SARS-CoV-2 entry into cells requires specific host proteases including TMPRSS2 and Cathepsin L (Ctsl) 1–3 , but there has been no reported success in inhibiting host proteases for treatment of SARS-CoV-2 pathogenesis in vivo . Here we have developed a lung Ctsl mRNA-targeted, CRISPR/Cas13d-based nanoparticle therapy to curb fatal SARS-CoV-2 infection in a mouse model. We show that this nanotherapy can decrease lung Ctsl expression in normal mice efficiently, specifically, and safely. Importantly, this lung-selective Ctsl -targeted nanotherapy significantly extended the survival of lethally SARS-CoV-2 infected mice by decreasing lung virus burden, reducing expression of pro-inflammatory cytokines/chemokines, and diminishing the severity of pulmonary interstitial inflammation. Additional in vitro analyses demonstrated that Cas13d-mediated Ctsl knockdown inhibited infection mediated by the spike protein of SARS-CoV-1, SARS-CoV-2, and more importantly, the authentic SARS-CoV-2 B.1.617.2 Delta variant, regardless of TMPRSS2 expression status. Our results demonstrate the efficacy and safety of a lung-selective, Ctsl -targeted nanotherapy against infection by SARS-CoV-2 and likely other emerging coronaviruses, forming a basis for investigation of this approach in clinical trials.

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    SciScore for 10.1101/2021.10.03.462915: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    EthicsIACUC: Mouse experiments and authentic SARS-CoV-2 virus infection of mice: Animal studies were approved by the Institutional Animal Care and Use Committee of Duke University.
    Sex as a biological variableC57BL/6 mice (Stock# B6-MPF, 5-8 weeks old, female or male) were purchased from Taconic (Rensselaer, NY).
    RandomizationThe percentage of pulmonary parenchyma affected was assessed on three randomly selected regions of interest from the digitized whole slide images using a grid overlay with Imagescope software.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibody against CTSL was purchased from R&D SYSTEMS (#AF1515)
    Antibody against CTSL
    suggested: (Abgent Cat# AP6788c, RRID:AB_1967364)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: HEK293T (ATCC #CRL-11268, RRID: CVCL_1926), Vero-ACE2 (Vero-E6 expressing high endogenous ACE2, BEI, NR-53726) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS, Thermo).
    HEK293T
    detected: (ATCC Cat# CRL-11268, RRID:CVCL_1926)
    Caco-2 (ATCC #HTB-37, RRID: CVCL_0025) cells were grown in DMEM supplemented with 1% penicillin/streptomycin and 20% FBS.
    Caco-2
    detected: (RCB Cat# RCB0988, RRID:CVCL_0025)
    Vero-ACE2 cells stably expressing TMPRSS2 were generated by transduction of Vero-ACE2 cells with a lentiviral vector expressing human TMPRSS2, followed by blasticidin S HCl selection (7.5 μg/mL) for 7 days.
    Vero-ACE2
    suggested: None
    Serial dilutions of lung homogenate were incubated with Vero E6 cells in a standard plaque assay.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Briefly, HEK293T cells were transfected with HIV-1 NL4.3-inGluc (a gift of Marc Johnson at the University of Missouri, Columbia, Missouri, USA) and pcDNA3.1-SARS-CoV2-S-C9 (obtained from Fang Li at the University of Minnesota, St. Paul, Minnesota, USA) constructs in a 2:1 ratio using polyelthylenimine (PEI).
    HEK293T
    suggested: None
    Pseudotyped virus infection: 3 × 105 of Vero-ACE2, Vero-ACE2-TMPRSS2 or Caco-2 were seeded into 6-well plate and transfected with CasRx mRNA and pre-gControl oligo (CasRx-pre-gControl) or pre-gCtsl oligo (CasRx-pre-gCtsl) by Lipofectamine MessengerMAX (Invitrogen, #LMRNA015).
    Caco-2
    suggested: None
    Authentic SARS-CoV-2 B.1.617.2 Delta variant and cell infection: The variant strain (USA/PHC658/2021) was obtained from BEI Resources NR-55611. 3 × 105 of Vero-ACE2-TMPRSS2 cells were seeded into 6-well plate and transfected with CasRx-pre-gControl or CasRx-pre-gCtsl by Lipofectamine MessengerMAX.
    Vero-ACE2-TMPRSS2
    suggested: None
    Briefly, 4 × 104 Vero-E6 cells were placed in each well of a 96-well tissue culture plate and allowed to adhere overnight.
    Vero-E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    C57BL/6 mice (Stock# B6-MPF, 5-8 weeks old, female or male) were purchased from Taconic (Rensselaer, NY).
    C57BL/6
    suggested: None
    Survival study: K18-hACE2 mice were lightly anesthetized with isoflurane and infected intranasally with 105 PFU of SARS-CoV-2 (USA-WA1/2020 strain) in a total volume of 50 μl DMEM on Day 0.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Recombinant DNA
    SentencesResources
    Briefly, HEK293T cells were transfected with HIV-1 NL4.3-inGluc (a gift of Marc Johnson at the University of Missouri, Columbia, Missouri, USA) and pcDNA3.1-SARS-CoV2-S-C9 (obtained from Fang Li at the University of Minnesota, St. Paul, Minnesota, USA) constructs in a 2:1 ratio using polyelthylenimine (PEI).
    pcDNA3.1-SARS-CoV2-S-C9
    suggested: None
    Software and Algorithms
    SentencesResources
    A pathology disease index of respiratory disease was created using semi-quantitative scoring on digitized lung sections (Aperio AT2, Leica Biosystems) with Imagescope (Leica Biosystems)
    Imagescope
    suggested: (ImageScope, RRID:SCR_014311)
    The protein expression was quantified by densitometry (ImageJ) and normalized to calnexin.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical analyses: All data were analyzed by GraphPad Prism 9.0 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 28. 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.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.10.03.462915v1 on bioRxiv