Inhibition of SARS-CoV-2 in Vero cell cultures by peptide-conjugated morpholino oligomers

  1. Kyle Rosenke
  2. Shanna Leventhal
  3. Hong M Moulton
  4. Susan Hatlevig
  5. David Hawman
  6. Heinz Feldmann
  7. David A Stein

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Abstract

Background

As the causative agent of COVID-19, SARS-CoV-2 is a pathogen of immense importance to global public health. Development of innovative direct-acting antiviral agents is sorely needed to address this virus. Peptide-conjugated morpholino oligomers (PPMO) are antisense compounds composed of a phosphorodiamidate morpholino oligomer covalently conjugated to a cell-penetrating peptide. PPMO require no delivery assistance to enter cells and are able to reduce expression of targeted RNA through sequence-specific steric blocking.

Methods

Five PPMO designed against sequences of genomic RNA in the SARS-CoV-2 5′-untranslated region and a negative control PPMO of random sequence were synthesized. Each PPMO was evaluated for its effect on the viability of uninfected cells and its inhibitory effect on the replication of SARS-CoV-2 in Vero-E6 cell cultures. Cell viability was evaluated with an ATP-based method using a 48 h PPMO treatment time. Viral growth was measured with quantitative RT–PCR and TCID50 infectivity assays from experiments where cells received a 5 h PPMO treatment time.

Results

PPMO designed to base-pair with sequence in the 5′ terminal region or the leader transcription regulatory sequence region of SARS-CoV-2 genomic RNA were highly efficacious, reducing viral titres by up to 4–6 log10 in cell cultures at 48–72 h post-infection, in a non-toxic and dose-responsive manner.

Conclusions

The data indicate that PPMO have the ability to potently and specifically suppress SARS-CoV-2 growth and are promising candidates for further preclinical development.

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  1. Version published to 10.1093/jac/dkaa460
  2. ScreenIT

    SciScore for 10.1101/2020.09.29.319731: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: Biosafety: Work with infectious SARS-CoV-2 was approved by the Institutional Biosafety Committee (IBC) and performed in high biocontainment at Rocky Mountain Laboratories (RML), NIAID, NIH.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 cells grown in 96-well plates were incubated in the presence of PPMO in growth media for 48 hours, then assayed according to the manufacturer’s instructions.
    Vero E6
    suggested: None
    Subsequently, 100 ul of each virus dilution was transferred to Vero-E6 cells grown in 96-well plates containing 100ul DMEM.
    Vero-E6
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis was carried out using GraphPad Prizm (San Diego, CA)
    GraphPad
    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.

    About SciScore

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  3. Version published to 10.1101/2020.09.29.319731v1 on bioRxiv