Intranasal gene therapy to prevent infection by SARS-CoV-2 variants

  1. Joshua J. Sims
  2. Jenny A. Greig
  3. Kristofer T. Michalson
  4. Sharon Lian
  5. R. Alexander Martino
  6. Rosemary Meggersee
  7. Kevin B. Turner
  8. Kalyani Nambiar
  9. Cecilia Dyer
  10. Christian Hinderer
  11. Makoto Horiuchi
  12. Hanying Yan
  13. Xin Huang
  14. Shu-Jen Chen
  15. James M. Wilson

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Abstract

SARS-CoV-2 variants have emerged with enhanced pathogenicity and transmissibility, and escape from pre-existing immunity, suggesting first-generation vaccines and monoclonal antibodies may now be less effective. Here we present an approach for preventing clinical sequelae and the spread of SARS-CoV-2 variants. First, we affinity matured an angiotensin-converting enzyme 2 (ACE2) decoy protein, achieving 1000-fold binding improvements that extend across a wide range of SARS-CoV-2 variants and distantly related, ACE2-dependent coronaviruses. Next, we demonstrated the expression of this decoy in proximal airway when delivered via intranasal administration of an AAV vector. This intervention significantly diminished clinical and pathologic consequences of SARS-CoV-2 challenge in a mouse model and achieved therapeutic levels of decoy expression at the surface of proximal airways when delivered intranasally to nonhuman primates. Importantly, this long-lasting, passive protection approach is applicable in vulnerable populations such as the elderly and immune-compromised that do not respond well to traditional vaccination. This approach could be useful in combating COVID-19 surges caused by SARS-CoV-2 variants and should be considered as a countermeasure to future pandemics caused by one of the many pre-emergent, ACE2-dependent CoVs that are poised for zoonosis.

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  1. Version published to 10.1371/journal.ppat.1009544
  2. ScreenIT

    SciScore for 10.1101/2021.04.09.439149: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Expression study in mice: All animal procedures were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.
    RandomizationNext-Generation Sequencing and Analysis: We performed 2×250 paired-end Illumina sequencing on randomly sheared and size-selected ACE2 amplicons from the yeast display rounds.
    BlindingSlides were blindly evaluated by a blinded pathologist using a severity score of 0 (no lesions observed), 1 (minimal), 2 (mild), 3 (moderate), 4 (marked) and 5 (severe) for each finding.
    Power Analysisnot detected.
    Sex as a biological variableFor the barcode study, 4×1012 GC of the pool AAV preps was delivered IN in a total volume of 0.28 ml to an adult male rhesus macaque using the MAD Nasal™ device.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    We followed up with Mouse anti His6 (Genscript), rabbit anti-HA-PE (Cell Signaling Technology), and goat anti mouse-488 secondary antibody (ThermoFisher) all in phosphate buffered saline (PBS) with 0.1% BSA.
    anti His6
    suggested: (G. Sengle; University of Cologne Cat# anti-rF11-C-term-His6, RRID:AB_2801629)
    anti-HA-PE (Cell Signaling Technology),
    suggested: None
    anti mouse-488
    suggested: None
    Wells were washed as described and biotin conjugated goat anti-human IgG (Jackson AffiniPure #109-065-098; 1:30,000 or Southern Biotech 2049-08; 1:1,000) in PBS/1.0% BSA detection antibody was added to the wells at 100ul/well and incubated for 2 hours at room temperature with shaking.
    anti-human IgG
    suggested: (Jackson ImmunoResearch Labs Cat# 109-065-098, RRID:AB_2337630)
    Experimental Models: Cell Lines
    SentencesResources
    We transfected RBD plasmids into HEK293 cells using PEI and collected supernatants 72 hrs later for clarification, concentration, and purification on Ni-NTA resin, followed by dialysis into PBS.
    HEK293
    suggested: None
    After 1 hour at 37°C, we added 20,000 cells/well in 50 ul of a HEK 293T cell line overexpressing ACE2 (Integral Molecular) and incubated the cells for 48 hours.
    HEK 293T
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    C57BL/6J mice were purchased from The Jackson Laboratory.
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Software and Algorithms
    SentencesResources
    After removing adapters and low-quality reads, we mapped clean reads no shorter than 200bp to the WT ACE2 nucleotide sequences using NovoAlign (v.4.03.01).
    NovoAlign
    suggested: (NovoAlign, RRID:SCR_014818)
    We exported and analyzed the data in GraphPad Prism Version 9.0.2.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    An additional two NHPs were administered with 5×1011 GC of AAVrh91.CDY14HL. All NHPs were negative for pre-existing neutralizing antibody titres to the administered AAV capsid prior to study initiation (Immunology Core at the Gene Therapy Program).
    Gene Therapy Program
    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: We did not find any issues relating to colormaps.

    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.

  3. Version published to 10.1101/2021.04.09.439149v2 on bioRxiv
  4. Version published to 10.1101/2021.04.09.439149v1 on bioRxiv