An intranasal ASO therapeutic targeting SARS-CoV-2

  1. Chi Zhu
  2. Justin Y. Lee
  3. Jia Z. Woo
  4. Lei Xu
  5. Xammy Nguyenla
  6. Livia H. Yamashiro
  7. Fei Ji
  8. Scott B. Biering
  9. Erik Van Dis
  10. Federico Gonzalez
  11. Douglas Fox
  12. Eddie Wehri
  13. Arjun Rustagi
  14. Benjamin A. Pinsky
  15. Julia Schaletzky
  16. Catherine A. Blish
  17. Charles Chiu
  18. Eva Harris
  19. Ruslan I. Sadreyev
  20. Sarah Stanley
  21. Sakari Kauppinen
  22. Silvi Rouskin
  23. Anders M. Näär

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Abstract

The COVID-19 pandemic is exacting an increasing toll worldwide, with new SARS-CoV-2 variants emerging that exhibit higher infectivity rates and that may partially evade vaccine and antibody immunity. Rapid deployment of non-invasive therapeutic avenues capable of preventing infection by all SARS-CoV-2 variants could complement current vaccination efforts and help turn the tide on the COVID-19 pandemic. Here, we describe a novel therapeutic strategy targeting the SARS-CoV-2 RNA using locked nucleic acid antisense oligonucleotides (LNA ASOs). We identify an LNA ASO binding to the 5′ leader sequence of SARS-CoV-2 that disrupts a highly conserved stem-loop structure with nanomolar efficacy in preventing viral replication in human cells. Daily intranasal administration of this LNA ASO in the COVID-19 mouse model potently suppresses viral replication (>80-fold) in the lungs of infected mice. We find that the LNA ASO is efficacious in countering all SARS-CoV-2 “variants of concern” tested both in vitro and in vivo. Hence, inhaled LNA ASOs targeting SARS-CoV-2 represents a promising therapeutic approach to reduce or prevent transmission and decrease severity of COVID-19 in infected individuals. LNA ASOs are chemically stable and can be flexibly modified to target different viral RNA sequences and could be stockpiled for future coronavirus pandemics.

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  1. Version published to 10.1038/s41467-022-32216-0
  2. ScreenIT

    SciScore for 10.1101/2021.05.17.444397: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All procedures involving the use of mice and hamsters were approved by the University of California, Berkeley Institutional Animal Care and Use Committee.
    Sex as a biological variableAnimals: C57BL/6J and K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice were purchased from the Jackson Laboratory and male golden Syrian hamsters at 4–5 weeks old were obtained from Charles River Labs (Strain Code:049).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies used were rabbit monoclonal CD3 primary antibody (Abcam, ab16669, 1:100), rabbit monoclonal B220 primary antibody (Novus, NB100-77420, 1:10000), rabbit monoclonal SARS-CoV-2 (COVID-19) nucleocapsid primary antibody (GeneTex, GTX635686, 1:8000) and rabbit anti-rat secondary (Vector, 1:100).
    CD3
    suggested: (Abcam Cat# ab16669, RRID:AB_443425)
    ab16669
    suggested: (Abcam Cat# ab16669, RRID:AB_443425)
    B220
    suggested: (Novus Cat# NB100-77420, RRID:AB_1083798)
    NB100-77420
    suggested: (Novus Cat# NB100-77420, RRID:AB_1083798)
    GTX635686
    suggested: (GeneTex Cat# GTX635686, RRID:AB_2888557)
    anti-rat secondary (Vector,
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture and viruses: Huh7 and Vero E6 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin/Streptomycin.
    Huh7
    suggested: None
    Infectious stocks were produced by inoculating Vero E6 cells and collecting the cell culture media upon observation of cytopathic effect; debris were removed by centrifugation and passage through a 0.22 μm filter.
    Vero E6
    suggested: RRID:CVCL_XD71)
    DMS modification of infected Huh-7 cells with ASO treatment: Huh-7 cells were transfected with LNA ASO (50 nM) 12 h before the infection.
    Huh-7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Experimental Models: Organisms/Strains
    SentencesResources
    Animals: C57BL/6J and K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice were purchased from the Jackson Laboratory and male golden Syrian hamsters at 4–5 weeks old were obtained from Charles River Labs (Strain Code:049).
    C57BL/6J
    suggested: None
    K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J]
    suggested: None
    K18-hACE2 mice or hamsters were anesthetized using isoflurane and inoculated with 1 × 104 TCID50 (for mice) or 10 TCID50 (for hamsters) of SARS-CoV-2 intranasally.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Animals: C57BL/6J and K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J] mice were purchased from the Jackson Laboratory and male golden Syrian hamsters at 4–5 weeks old were obtained from Charles River Labs (Strain Code:049).
    Charles River Labs
    suggested: None
    cDNA was prepared by iScript™ Reverse Transcription Supermix (BioRAD) and qPCR was performed with Fast SYBR™ Green Master Mix (Thermo Fisher) and the reaction was run on the QuantStudio6 System (Applied Biosystems).
    BioRAD
    suggested: None
    The data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO Series accession number GSE174382.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    STAR aligner35 was used to map sequencing reads to transcripts in the mouse mm10 reference genome.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Data analysis was performed using GraphPad Prism 8.
    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 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.

    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.

  3. Version published to 10.1101/2021.05.17.444397v1 on bioRxiv