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Cited by 87RNA Interference and Gene DeliveryRNA and protein synthesis mechanismsSARS-CoV-2 and COVID-19 Research

Complete substitution with modified nucleotides in self-amplifying RNA suppresses the interferon response and increases potency

Open access
McGee, Joshua E., Kirsch, Jack R., Kenney, Devin, Cerbo, Faith, Chavez, Elizabeth C., Shih, Ting-Yu, Douam, Florian, Wong, Wilson W. +1 more·2025·Nature Biotechnology
DOI 10.1038/s41587-024-02306-zPMC PMC11707045

Published version of a preprint

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Abstract

The use of modified nucleotides to suppress the interferon response and maintain translation of self-amplifying RNA (saRNA), which has been achieved for mRNA, has not yet succeeded. We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression, and when formulated as a vaccine, protect against lethal SARS-CoV-2 challenge in mice. This discovery advances saRNA therapeutics by enabling prolonged protein expression at low doses.

AI snapshot

Written from the paper’s full text.

TL;DR

Complete substitution of self-amplifying RNA (saRNA) with selected modified nucleotides—especially 5-methylcytidine (m5C)—suppressed the interferon response, improved protein expression, and enabled low-dose protection against lethal SARS-CoV-2 challenge in mice. This challenges the prior view that modified nucleotides are incompatible with saRNA.

Key findings

  • In HEK293T cells, complete substitution with hm5C, m5C, or m5U gave transfection efficiencies 14-fold, 10-fold, and 8-fold higher than the N1mΨ-modified construct, respectively.
  • m5C saRNA loaded into lipid nanoparticles produced 4.9-fold higher protein expression than wild-type saRNA and 68-fold higher than N1mΨ mRNA in HEK293T cells at 10 ng; in C2C12 cells, m5C saRNA gave 3.5-fold higher expression than wild-type saRNA and 314-fold higher than N1mΨ mRNA.
  • hm5C or m5C incorporation reduced IFNα1 and IFNβ1 expression by 8.5-fold and 3-fold, respectively; 100% m5C substitution made IFNβ1 expression similar to untreated controls. In mice, serum IFNα1 at 6 h was 2.2-fold lower with m5C RNA than wild-type saRNA.
  • After lethal SARS-CoV-2 MA30 challenge, 10 ng m5C saRNA vaccination gave 75% survival, versus 20% for 10 ng N1mΨ mRNA and 45% for 10 ng wild-type saRNA; at 10 ng, m5C saRNA also gave 5-fold higher spike IgG titers than wild-type saRNA and 121-fold higher than N1mΨ mRNA.

Methods

  • saRNA constructs were synthesized by in vitro transcription using a Venezuela equine encephalitis virus replicon system, with complete substitution of modified nucleoside triphosphates, and formulated into lipid nanoparticles.
  • In vitro assays used HEK293T, C2C12, Jurkat, primary human CD3+ T cells, and human PBMCs; readouts included flow cytometry, luciferase expression, ELISA, and qPCR for interferon genes.
  • In vivo studies used C57BL/6 mice for bioluminescent imaging after intramuscular injection of 2.5 μg RNA, and vaccination with 10, 100, or 1,000 ng RNA, boosting at day 21, then intranasal challenge with 1 × 10^5 plaque-forming units of mouse-adapted SARS-CoV-2 MA30 at day 35; serum IgG and neutralization were measured.

Limitations

  • The paper states that future work will evaluate Fc effector functions and T cell responses, and that the next step is clinical-grade saRNA testing in non-human primates and humans.
  • The paper notes that serum neutralizing antibody titers were similar between wild-type and m5C saRNA-treated mice despite different survival, indicating protection correlates beyond neutralizing humoral responses.
  • Evident from the text: in vivo bioluminescent imaging showed no significant differences in expression levels or duration between wild-type and m5C saRNA groups, and the protective efficacy data are from mice only, with no non-human primate or human efficacy data reported here.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1038/s41587-024-02306-z

Cites

66 of 66 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

27 of 32 cited sources checked · 2 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    Previous efforts to decrease immunogenicity of saRNA focused on sequence evolution, co-expression of viral inhibitory proteins and optimization of the delivery vehicle.

    “Previous efforts to decrease the immunogenicity of saRNA focused on sequence evolution, co-expression of viral inhibitory proteins and optimization of the delivery vehicle16–18.”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [16] Li, Y. et al. In vitro evolution of enhanced RNA replicons for immunotherapy. Sci. Rep. 9, 6932 (2019). source

      The Introduction and Results report in vitro evolution of saRNA replicon sequences and identify mutations that reduced interferon responses in some variants. The paper also describes nanoparticle delivery, but does not present delivery-vehicle optimization as a way to decrease immunogenicity or describe co-expression of viral inhibitory proteins. No page numbers are available in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [17] Blakney, A. K. et al. Innate inhibiting proteins enhance expression and immunogenicity of self-amplifying RNA. Mol. Ther. 29, 1174–1185 (2021). source

      The Introduction describes viral innate-inhibiting proteins as a strategy to dampen the interferon response to RNA, supporting that part of the claim. It does not describe sequence evolution or delivery-vehicle optimization as efforts to decrease saRNA immunogenicity. The Abstract and Discussion instead report that the paper’s inhibitory-protein approach enhanced vaccine immunogenicity in rabbits. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [18] Kimura, T. et al. A localizing nanocarrier formulation enables multi-target immune responses to multivalent replicating RNA with limited systemic inflammation. Mol. Ther. 31, 2360–2375 (2023). source

      The Discussion (page unavailable) supports delivery-vehicle optimization as an alternative way to limit systemic innate responses to replicating RNA. The Results discuss prior efforts to inhibit type I interferon signaling, and the References name work on innate-inhibiting proteins, but our copy does not describe co-expression of viral inhibitory proteins. The Discussion says sequence optimization cannot be applied to replicating RNA to raise its dose ceiling, rather than identifying it as a prior saRNA strategy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    The early and intense type I IFN response induced by saRNA detection hinders replication and antigen expression.

    “The early and intense type I IFN response induced by saRNA detection hinders replication and antigen expression, similar to unmodified mRNA5,13–15.”
    Quote in the paperSubstantial claim4 of 4 sources checked
    • cites [5] Minnaert, A. K. et al. Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: getting the message across. Adv. Drug Deliv. Rev. 176, 113900 (2021). source

      In “Type I IFN-mediated effects: Beneficial or detrimental for mRNA vaccines?” (page unavailable), the review reports that an early, robust type I IFN response to a saRNA vaccine in mice negatively affected initial antigen expression. It does not establish in that passage that the response hindered saRNA replication; the “Innate immunity stimulation” section describes antiviral mechanisms generally, without making that specific link. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [13] Zhong, Z. et al. Corticosteroids and cellulose purification improve, respectively, the in vivo translation and vaccination efficacy of sa-mRNAs. Mol. Ther. 29, 1370–1381 (2021). source

      The Results report that the sa-mRNA vaccine induced a rapid IFN-β response peaking within five hours, and the Introduction says type I IFN can induce enzymes that inhibit mRNA translation. These passages support the early response and its potential to hinder antigen expression, but our copy does not establish that the response hinders sa-mRNA replication. The Discussion instead says the IFN-β peak occurs before replication, addressing what triggers the response rather than whether it inhibits replication. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [14] Pepini, T. et al. Induction of an IFN-mediated antiviral response by a self-amplifying RNA vaccine: implications for vaccine design. J. Immunol. 198, 4012–4024 (2017). source

      The Abstract (page unavailable) reports an early response to self-amplifying RNA and says type I IFN may inhibit replicon amplification and antigen expression. The antigen-expression effect is supported by the section “SAM-driven Ag expression is enhanced in IFNAR KO and MAR1-5A3-treated mice” (page unavailable), but the paper presents inhibition of replication as a possibility, not an established result. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [15] Huysmans, H. et al. Expression kinetics and innate immune response after electroporation and LNP-mediated delivery of a self-amplifying mRNA in the skin. Mol. Ther. Nucleic Acids 17, 867–878 (2019). source

      The Results report an early, stronger IFN-β response after LNP delivery of saRNA, and the Discussion links that response to reduced reporter-protein expression. But the paper presents the expression mechanism as a likely translation blockage, not a demonstrated inhibition of replication or antigen expression; it also says the weaker response after electroporation likely did not reduce expression. Page numbers are unavailable in our copy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Early saRNA vaccine trials showed lower efficacy and neutralizing antibody levels than mRNA vaccine trials.

    “However, early saRNA vaccine trials showed lower efficacy and neutralizing antibody levels than mRNA vaccine trials11.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Low, J. G. et al. A phase I/II randomized, double-blinded, placebo-controlled trial of a self-amplifying Covid-19 mRNA vaccine. NPJ Vaccines 7, 161 (2022). source

      The Discussion reports that neutralizing antibody titers for the saRNA vaccine ARCT-021 were low. It does not compare those titers with mRNA vaccine trials or report lower clinical efficacy for ARCT-021; the efficacy figure it mentions concerns a different saRNA vaccine, ARCT-154. No page number is available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Another study demonstrated that saRNA, when fully substituted with m5C, exhibits a notable decrease in the innate immune response compared to wild-type saRNA in vitro.

    “Another study48 demonstrated that saRNA, when fully substituted with m5C, exhibits a notable decrease in the innate immune response compared to wild-type saRNA in vitro.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [48] Komori, M. et al. Incorporation of 5 methylcytidine alleviates innate immune response to self-amplifying RNA vaccine. Preprint at bioRxiv https://doi.org/10.1101/2023.11.01.565056 (2023). source
  • Premisecitation

    Recently, a study reported that 5% m5C-modified saRNA affords a functional construct as a SARS-CoV-2 booster in a phase 1 human safety study.

    “Recently, a study reported that 5% m5C-modified saRNA affords a functional construct as a SARS-CoV-2 booster in a phase 1 human safety study47.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [47] Aboshi, M. et al. Safety and immunogenicity of VLPCOV-02, a SARS-CoV-2 self-amplifying RNA vaccine with a modified base, 5-methylcytosine. iScience 27, 108964 (2024). source

      The Introduction identifies VLPCOV-02 as a 5-methylcytosine–modified saRNA booster, and the Methods describe the reported phase 1 human safety study. The Conclusions report improved immune responses and a favorable safety profile, supporting the description of a functional construct. Our copy does not establish the claimed 5% modification level (searched: “5%” and “5% 5-methylcytosine modified saRNA”). (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • May combine two claims. 24
  • Classified as probably not a substantial claim. 4

  • May combine two claims.

    Premisecitation

    saRNA can address shortcomings of mRNA by decreasing the number of doses and administration frequency, mitigating side effects and bolstering manufacturing capacity.

    “saRNA can address the shortcomings of mRNA by decreasing the number of doses and the administration frequency, which will mitigate side effects8–10 and bolster manufacturing capacity”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [8] Trougakos, I. P. et al. Adverse effects of COVID-19 mRNA vaccines: the spike hypothesis. Trends Mol. Med. 28, 542–554 (2022). source

      The section “Anti-SARS-CoV-2 mRNA vaccines and their reported adverse effects” (page not provided) says frequent boosters may increase adverse-effect frequency or severity. That supports a possible benefit from fewer administrations, but it does not establish that saRNA reduces dosing or that fewer doses will mitigate side effects. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [9] Ndeupen, S. et al. The mRNA-LNP platform’s lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience 24, 103479 (2021). source

      The Discussion (page unavailable) says side effects after a second mRNA-LNP vaccination could be further exacerbated, which lends some support to the idea that fewer administrations might reduce side effects. It does not discuss saRNA or establish that saRNA requires fewer doses or mitigates side effects; the manufacturing-capacity claim is also unsupported by this source. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [10] Ju, Y. et al. Impact of anti-PEG antibodies induced by SARS-CoV-2 mRNA vaccines. Nat. Rev. Immunol. 23, 135–136 (2023). source

      The “Potential impact of anti-PEG antibodies” section reports a correlation between anti-PEG antibodies and systemic reactogenicity after two mRNA vaccine doses, and raises a concern about further booster doses. It does not show that saRNA reduces the number or frequency of doses or that doing so mitigates side effects; it also does not address manufacturing capacity. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    saRNAs use an RNA-dependent RNA polymerase from an alphavirus and conserved sequence elements at the 5′ and 3′ ends for replication, leading to robust and durable expression of encoded protein cargo.

    “saRNAs use an RNA-dependent RNA polymerase from an alphavirus and the corresponding conserved sequence elements at the 5′ and 3′ ends for replication, leading to robust and durable expression of encoded protein cargo5–7.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [5] Minnaert, A. K. et al. Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: getting the message across. Adv. Drug Deliv. Rev. 176, 113900 (2021). source

      The “Self-amplifying mRNA” section says most saRNAs are alphavirus-based, identifies nsP4 as an RNA-dependent RNA polymerase, and reports higher, longer-lasting protein expression from self-replication. It describes 5′ and 3′ UTRs and replication promoters, but does not identify conserved sequence elements at both ends as required for replication. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [6] Geall, A. J., Kis, Z. & Ulmer, J. B. Vaccines on demand, part II: future reality. Expert Opin. Drug Discov. 18, 119–127 (2023). source
    • cites [7] Bloom, K., van den Berg, F. & Arbuthnot, P. Self-amplifying RNA vaccines for infectious diseases. Gene Ther. 28, 117–129 (2021). source

      The Introduction’s Fig. 1 caption says alphavirus nsP1–4 proteins form an RNA-dependent RNA polymerase complex that recognizes 5′ and 3′ conserved sequence elements, amplifying vaccine-encoding transcripts and causing the encoded protein to accumulate. The Introduction also describes high-level antigen or therapeutic expression. These passages support robust expression, but they do not establish that protein expression is durable. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Native RNA activates pattern recognition receptors such as toll-like receptors and triggers type I interferon production, resulting in translational shutoff and systemic inflammation.

    “Native RNA activates pattern recognition receptors such as toll-like receptors and triggers type I interferon (IFN) production, resulting in translational shutoff and systemic inflammation3.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Anderson, B. R. et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38, 5884–5892 (2010). source

      The Introduction states that RNA can activate innate immune receptors, including Toll-like receptors, and that RNA-activated PKR represses translation. The Discussion likewise links PKR activation to translational inhibition. Our copy does not establish the claimed sequence of type I interferon production leading to translational shutoff or systemic inflammation. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Recent clinical trial results revealed comparable immunogenicity of a saRNA vaccine at one-tenth of the dose of an equivalent mRNA vaccine, but side effects were similar between treatment groups.

    “Recent clinical trial results revealed comparable immunogenicity of a saRNA vaccine at one-tenth of the dose of an equivalent mRNA vaccine. However, side effects were similar between the treatment groups12.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [12] Akahata, W. et al. Safety and immunogenicity of SARS-CoV-2 self-amplifying RNA vaccine expressing an anchored RBD: a randomized, observer-blind phase 1 study. Cell Rep. Med. 4, 101134 (2023). source

  • Classified as probably not a substantial claim.

    Premisecitation

    The current understanding of the field is that incorporating modified NTPs into saRNA abrogates downstream efficacy.

    “However, the current understanding of the field is that incorporating modified NTPs into saRNA abrogates downstream efficacy, as reported by multiple independent groups5,19–28.”
    Quote in the paperSubstantial claim7 of 10 sources checked
    • cites [5] Minnaert, A. K. et al. Strategies for controlling the innate immune activity of conventional and self-amplifying mRNA therapeutics: getting the message across. Adv. Drug Deliv. Rev. 176, 113900 (2021). source

      The ‘Modifications to the mRNA backbone itself’ section says modified nucleosides are expected to impair saRNA self-replication, and the Conclusions say nucleoside modification is not tolerated if saRNA functionality is to be retained (page numbers unavailable). The paper does not establish that modified NTPs abrogate downstream efficacy or report that this result was demonstrated by multiple independent groups. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [19] Kairuz, D., Samudh, N., Ely, A., Arbuthnot, P. & Bloom, K. Advancing mRNA technologies for therapies and vaccines: an African context. Front. Immunol. 13, 1018961 (2022). source

      Section 2.4 says modified nucleotides may be unsuitable for saRNA because its replication relies on stable RNA structures, and that their compatibility needs evaluation. It does not say incorporating modified NTPs abrogates saRNA efficacy; the citing sentence turns a qualified concern into a categorical conclusion. (LLM, conf=0.87)

      Experimental — accuracy not yet measured
    • cites [20] Voigt, E. A. et al. A self-amplifying RNA vaccine against COVID-19 with long-term room-temperature stability. NPJ Vaccines 7, 136 (2022). source

      The Results section (“Antigen optimization increases SARS-CoV-2 variant cross-neutralization”; page unavailable) reports that making this saRNA vaccine with modified nucleosides entirely abrogated its antibody and T-cell responses in mice. That supports the claim for this vaccine, but the paper’s brief mouse study does not establish that modified NTPs generally abrogate downstream efficacy across saRNA vaccines. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [21] Beissert, T. et al. A trans-amplifying RNA vaccine strategy for induction of potent protective immunity. Mol. Ther. 28, 119–128 (2020). source
    • cites [22] Geall, A., Hekele, A. & Mandl, C. RNA containing modified nucleotides and use thereof in vaccines. WIPO publication no. WO2011/005799A3 (2010); https://patentscope.wipo.int/search/en/detail.jsf?docId=WO2011005799

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

    • cites [23] Erasmus, J. H. et al. Intramuscular delivery of replicon RNA encoding ZIKV-117 human monoclonal antibody protects against Zika virus infection. Mol. Ther. Methods Clin. Dev. 18, 402–414 (2020). source

      The Results section (“RepRNA Mediates Higher Expression of Antibody Compared to Non-Replicating mRNA”; page not available in our copy) reports that pseudouridine-modified repRNA was non-functional, with no detectable ZIKV-117 antibody in mouse serum. This supports the claim for that modification and outcome, but not the broader assertion that incorporating modified NTPs into saRNA generally abrogates downstream efficacy. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [24] Pollock, K. M. et al. Safety and immunogenicity of a self-amplifying RNA vaccine against COVID-19: COVAC1, a phase I, dose-ranging trial. EClinicalMedicine 44, 101262 (2022). source

      The Discussion (page unavailable) says that the compatibility of modified bases with saRNA has not been established and that the bases would be rapidly lost during amplification. This supports a concern about their usefulness, but it does not establish that incorporating modified NTPs abrogates downstream efficacy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
    • cites [25] Blakney, A. K. et al. Effects of cationic adjuvant formulation particle type, fluidity and immunomodulators on delivery and immunogenicity of saRNA. J. Control. Release 304, 65–74 (2019). source
    • cites [26] Papukashvili, D. et al. Self-amplifying RNA approach for protein replacement therapy. Int. J. Mol. Sci. 23, 12884 (2022). source

      Section 4 (page unavailable) says modified nucleosides are not a reasonable way to avoid innate immune recognition in saRNA because the modifications would vanish after the first round of amplification. It does not say that incorporating modified NTPs abrogates downstream efficacy, nor does it report that multiple independent groups found that outcome. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [27] Kairuz, D., Samudh, N., Ely, A., Arbuthnot, P. & Bloom, K. Production, characterization, and assessment of permanently cationic and ionizable lipid nanoparticles for use in the delivery of self-amplifying RNA vaccines. Pharmaceutics 15, 1173 (2023). source

      The Discussion reports strong reporter-protein expression from saRNA made without modifications used in conventional mRNA, but does not say what happens when modified NTPs are incorporated into saRNA. The Results discuss NTP concentration and capping, not a comparison of modified and unmodified NTPs. Searches for modified NTPs, modified nucleotides, and effects on replication or efficacy found no passage addressing the claim. (LLM, conf=0.75)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Substitution of chemically modified nucleotides in mRNA enhances stability and transfection capability and decreases immunogenicity.

    “Substitution of chemically modified nucleotides in messenger ribonucleic acid (mRNA) enhances stability and transfection capability, and decreases immunogenicity1–3”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [1] Kariko, K. et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833–1840 (2008). source

      The Results (pages unavailable) show that pseudouridine-modified mRNA persists longer, produces more protein in transfected cells, and induces less IFN-α in mice than unmodified mRNA. But the claim is too broad for chemically modified nucleotides generally: the Results also report that mRNAs containing s2U or m6A were poorly translated or not translated at all. The paper demonstrates improved expression after transfection, rather than improved delivery into cells. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [2] Kormann, M. S. et al. Expression of therapeutic proteins after delivery of chemically modified mRNA in mice. Nat. Biotechnol. 29, 154–157 (2011). source
    • cites [3] Anderson, B. R. et al. Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation. Nucleic Acids Res. 38, 5884–5892 (2010). source

      The Discussion says modified nucleosides reduce host-defense responses, supporting decreased immunogenicity. The Introduction describes mRNA transfection as efficient, and the Results show increased translation from pseudouridine-containing mRNA, but neither establishes that substitution improves transfection capability. The paper’s References name a study of increased biological stability; the cited paper’s own discussion and results do not establish that stability claim. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    Mice vaccinated with 1,000 ng of wild-type saRNA, m5C saRNA or N1mΨ mRNA show 100% survival and minimal weight loss after lethal SARS-CoV-2 challenge.

    “The mice vaccinated with 1,000 ng of wild-type saRNA, m5C saRNA or N1mΨ mRNA show 100% survival and minimal weight loss”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Wild-type saRNA contain dsRNA impurities approximately fivefold and threefold higher than hm5C and m5C saRNAs, respectively.

    “By contrast, wild-type saRNA contain dsRNA impurities, approximately fivefold and threefold higher than hm5C and m5C saRNAs, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For mice vaccinated with 100 ng of wild-type saRNA or m5C saRNA, titers vary between 10^5 and 10^6 ng ml−1, showing no significant difference between the two groups.

    “For mice vaccinated with 100 ng of wild-type saRNA or m5C saRNA, titers vary between 105 and 106 ng ml−1, showing no significant difference between the two groups”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Groups administered wild-type or m5C saRNA both display significantly prolonged protein expression throughout 28-day study duration.

    “Notably, the groups that were administered wild-type or m5C saRNA both display significantly prolonged protein expression throughout the 28-day study duration”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Mice vaccinated with 100 ng of wild-type or m5C saRNA survive, with average weight loss of 5%.

    “Similarly, mice vaccinated with 100 ng of wild-type or m5C saRNA survive, with an average weight loss of 5%”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 10-ng dose, animals vaccinated with m5C saRNA display significantly elevated titers, with a 5-fold higher titer than wild-type saRNA vaccinated animals and a 121-fold higher titer than N1mΨ mRNA-vaccinated animals.

    “In contrast, at the 10-ng dose, animals vaccinated with m5C saRNA display significantly elevated titers, with a 5-fold higher titer than wild-type saRNA vaccinated animals and a 121-fold higher titer N1mΨ mRNA-vaccinated animals”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In mice, N1mΨ mRNA attains peak protein expression at 24 h and then signal rapidly decreases.

    “The group that received N1mΨ mRNA attains peak protein expression at 24 h, and then the signal rapidly decreases”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    hm5C and m5C saRNA transfection efficiencies are significantly greater than wild-type saRNA.

    “The transfection efficiencies of the hm5C and m5C constructs are also significantly greater than the wild-type (WT) saRNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    SARS-CoV-2 spike protein-reactive IgG titers in serum on day 35 ranged from 10^6 to 10^7 ng ml−1, with no significant differences between 1,000-ng wild-type saRNA, m5C saRNA and N1mΨ mRNA treatment groups.

    “Titers ranged from 106 to 107 ng ml−1, with no significant differences between the 1,000-ng wild-type saRNA, m5C saRNA and N1mΨ mRNA treatment groups”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In mice, wild-type or m5C saRNA show consistent increase in signal and reach peak protein expression 7 days after injection that is four times greater than N1mΨ mRNA group.

    “By contrast, the groups treated with wild-type or m5C saRNA show a consistent increase in the signal and, 7 days after the initial injection, reached peak protein expression that is four times greater than the N1mΨ mRNA group”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    No significant differences in expression levels or duration of expression between groups that received wild-type or m5C saRNA in mice.

    “We observed no significant differences in expression levels or duration of expression between the groups that received wild-type or m5C saRNA.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Expression profile of influenza hemagglutinin antigen is consistent with previous experiments.

    “We performed similar in vitro experiments to evaluate the expression of the influenza hemagglutinin antigen, and the expression profile is consistent with our previous experiments”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In primary human foreskin fibroblasts, N1mΨ mRNA exhibits a linear dose response, whereas saRNA shows a nonlinear dose response.

    “In primary human foreskin fibroblasts, N1mΨ mRNA exhibits a linear dose response, whereas saRNA shows a nonlinear dose response”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Survival rates are 20% and 45% for mice receiving 10-ng dose of N1mΨ mRNA or wild-type saRNA, respectively.

    “However, the survival rates are 20% and 45% for mice receiving the 10-ng dose of N1mΨ mRNA or wild-type saRNA, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 10 ng, purified hm5C and m5C saRNAs afford 36-fold and 18-fold greater protein production than N1mΨ mRNA, respectively.

    “and 36-fold and 18-fold greater protein production than N1mΨ mRNA, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 10 ng, purified hm5C and m5C saRNAs afford 146-fold and 75-fold greater protein production than wild-type saRNA, respectively.

    “At 10 ng, purified hm5C and m5C saRNAs affords 146-fold and 75-fold greater protein production than wild-type saRNA”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Across all samples, IFNβ is not detectable at 24 h or 48 h.

    “Across all samples, IFNβ is not detectable at 24 h or 48 h”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Complete substitution of saRNA with modified nucleotides can confer innate immune evasion and robust long-term protein expression.

    “We identify modified nucleotides that, when substituted at 100% in saRNA, confer innate immune evasion and robust long-term protein expression”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    This discovery enhances the potency of RNA vaccines and broadens the utility of saRNA.

    “This discovery enhances the potency of RNA vaccines and broadens the utility of saRNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Denaturing gel electrophoresis reveals comparable transcript length and quality for modified saRNAs encoding mCherry.

    “Denaturing gel electrophoresis reveals comparable transcript length and quality for modified saRNAs encoding mCherry (Supplementary Fig. 3a).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    All RNAs show minimal degradation.

    “All RNAs show minimal degradation (Supplementary Fig. 5a).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    Encapsulation of RNA is similar between modified constructs (>90% encapsulation efficiency).

    “Furthermore, encapsulation of the RNA is similar between modified constructs (>90% encapsulation efficiency; Supplementary Fig. 3b).”
    Quote in the paperNot a substantial claim
28 of 28

Cites

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3 of the 69 references couldn’t be matched to a known paper.

Cited by

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Related

Authors
McGee, J. E., Kirsch, J. R., Kenney, D., Cerbo, F., Chavez, E. C., Shih, T.-Y., Douam, F., Wong, W. W., & Grinstaff, M. W.
Affiliations
Boston University (US)
Corresponding author
  • Douam, Florian
  • Wong, Wilson W.
  • Grinstaff, Mark W.
ORCID
Venue
Nature Biotechnology
Publisher
Springer Science and Business Media LLC
Published
2024-07-08
Volume
43
Issue
5
Pages
720-726
ISSN
  • 0733-222X (ISSN-L)
  • 1087-0156
  • 1546-1696
DOI
10.1038/s41587-024-02306-z
PMC
PMC11707045
OpenAlex
W4400422913
Cited by
87
References
69
Citations by year
2026: 35 · 2025: 37 · 2024: 13 · 2023: 2
FWCI
10.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Biotechnology (publishedVersion)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion)
  • Europe PMC (PubMed Central) (submittedVersion)
Funders
  • U.S. Department of Health & Human Services | NIH | National Cancer Institute (U01CA265713)
  • U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (R01EB029483, R01EB031904, T32EB006359)
  • U.S. Department of Health & Human Services | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (R01DK132576)
  • NSF | BIO | Division of Molecular and Cellular Biosciences (2234657)
  • U.S. Department of Health & Human Services | NIH | National Institute of Allergy and Infectious Diseases (T32AI007309, K22AI144050)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
Keywords
self-amplifying RNA, modified nucleotides, innate immune evasion, RNA vaccines, SARS-CoV-2, RNA therapeutics, viral challenge protection

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-11.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-11.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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