Exosome-Mediated mRNA Delivery For SARS-CoV-2 Vaccination

  1. Shang Jui Tsai
  2. Chenxu Guo
  3. Alanna Sedgwick
  4. Saravana Kanagavelu
  5. Justin Nice
  6. Sanjana Shetty
  7. Connie Landaverde
  8. Nadia A. Atai
  9. Stephen J. Gould

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Abstract

Expression-dependent, Spike-only vaccines have been developed, deployed, and shown to be effective in the fight against SARS-CoV-2. However, additional approaches to vaccine development may be needed to meet existing and future challenges posed by emerging Spike variant strains, as well as a likely need for different antigen-delivery systems that are safe and effective for regular, periodic re-administration. We report here the development of mRNA-loaded exosomes, demonstrate that they can mediate the functional expression of heterologous proteins in vitro and in vivo , and have fewer adverse effects than comparable doses of lipid nanoparticles. Furthermore, we applied this approach to the development of an exosome-based, multiplexed mRNA vaccine that drives expression of immunogenic SARS-CoV-2 Nucleocapsid and Spike proteins. This vaccine elicited long-lasting cellular and humoral responses to Nucleocapsid and to Spike, demonstrating that exosome-based mRNA formulations represent a previously unexplored platform in the fight against COVID-19 and other infectious diseases.

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  1. Version published to 10.1101/2020.11.06.371419v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2020.11.06.371419: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal experimentation was performed following institutional guidelines for animal care and were approved by the Cedars-Sinai Medical Center IACUC (#8602).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableImmunizations were initiated on thirteen weeks- old, male C57BL/6J mice (Jackson Laboratory) housed under pathogen-free conditions at the Cedars-Sinai Medical Center animal facility.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were probed using antibodies directed against CD9, CD63 (System Biosciences EXOAB-CD9A-1 and EXOAB-CD63A-1, respectively), and actin (Sigma A2066), with HRP-conjugated goat anti-rabbit secondary antibody used for detection (Cell Signaling, #7074).
    CD9
    suggested: None
    CD63
    suggested: None
    EXOAB-CD63A-1 , respectively) , and actin
    suggested: None
    anti-rabbit
    suggested: None
    Histological analysis was performed by the service arm of the HIC/Comparative Pathology Program of the University of Washington. ELISA for SARS-CoV-2 antigen-specific antibody responses: Mouse IgG antibody production against SARS-CoV-2 antigens was measured by enzyme-linked immunosorbent assays (ELISA).
    antigen-specific
    suggested: None
    Mouse IgG
    suggested: None
    SARS-CoV-2
    suggested: None
    Mouse antibodies bound to the antigens coated on the ELISA plates were detected using HRP-conjugated goat anti-mouse secondary antibodies (Jackson Immuno Research Inc.) Plates were washed 4 times with washing buffer, and developed using TMB substrate (RayBiotech).
    anti-mouse
    suggested: None
    Spleen lymphocyte population characterization: Splenocytes (2 x 105 cells/mouse) were resuspended in 100 μL of 10% FBS in 1x PBS and incubated with fluorochrome-conjugated antibodies for surface staining of CD3 (Invitrogen, #17-0032-82
    CD3
    suggested: (Thermo Fisher Scientific Cat# 17-0032-82, RRID:AB_10597589)
    Cells were then stained with anti-CD3-APC (Invitrogen, #17-0032- 82), anti-CD4-PerCP-Cy5.5 (Biolegend, #100433), and anti-CD8-PE antibodies (Biolegend, #MCD0801) for 30 minutes at 4°C.
    anti-CD3-APC
    suggested: (Sigma-Aldrich Cat# SAB4700046, RRID:AB_10897585)
    anti-CD4-PerCP-Cy5.5
    suggested: None
    anti-CD8-PE
    suggested: (Sigma-Aldrich Cat# P5560, RRID:AB_477372)
    The cells were then washed with 10% FBS in 1x PBS and stained with anti-CD3-APC, anti- CD4-PerCP-Cy5.5, and anti-CD8-PE antibodies (Added above) for 30 minutes at 4°C in dark.
    anti-CD3-APC , anti- CD4-PerCP-Cy5.5
    suggested: None
    anti-
    suggested: (Sigma-Aldrich Cat# P5560, RRID:AB_477372)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: 293F cells (Gibco, Cat.# 51-0029) were tested for pathogens and found to be free of viral (cytomegalovirus, human immunodeficiency virus I and II, Epstein Barr virus, hepatitis B virus, and parvovirus B19), and bacterial (Mycoplasma) contaminants.
    293F
    suggested: None
    HEK293 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum.
    HEK293
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Immunizations were initiated on thirteen weeks- old, male C57BL/6J mice (Jackson Laboratory) housed under pathogen-free conditions at the Cedars-Sinai Medical Center animal facility.
    C57BL/6J
    suggested: RRID:IMSR_JAX:000664)
    Software and Algorithms
    SentencesResources
    The data analysis was performed using FlowJo 10 software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical Analysis: Statistical analysis was performed using GraphPad Prism 8 software for Windows/Mac (GraphPad Software, La Jolla California USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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 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: 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 10.1101/2020.11.06.371419v1 on bioRxiv