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Cited by 62RNA Interference and Gene DeliveryRNA and protein synthesis mechanismsRNA Research and Splicing

Combining an optimized mRNA template with a double purification process allows strong expression of in vitro transcribed mRNA

Open access
Linares-Fernández, Sergio, Moreno, Julien, Lambert, Elise, Mercier-Gouy, Perrine, Vachez, Laetitia, Verrier, Bernard, Exposito, Jean-Yves·2021·Molecular Therapy - Nucleic Acids
DOI 10.1016/j.omtn.2021.10.007PMC PMC8523304
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Abstract

mRNA is a blooming technology for vaccination and has gained global attention during the SARS-CoV-2 pandemic. However, the recent clinical trials have highlighted increased reactogenicity when using high mRNA doses. Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process. We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5′ untranslated region (UTR) and two 3′ UTRs of the human β-globin as a reference mRNA and identified the most promising mRNA sequence using in vitro and in vivo models. First, we assessed the impact of different poly(A) sizes on translation and selected the most optimal sequence. Then, we selected the best 5′ UTR among synthetic sequences displaying a high ribosome loading. Finally, we evaluated the transfection efficiency of our standard mRNA template after two capping strategies and purification using either double-stranded RNA (dsRNA) depletion or dephosphorylation of 5′PPP RNA or both combined. Double purification was shown to give the best results. Altogether, the use of a newly defined 5′ UTR coupled to post-transcriptional treatments will be of great interest in the mRNA vaccine field, by limiting the amount of the antigen-coding transcript and subsequently the formulation components needed for an efficient vaccination.

AI snapshot

Written from the paper’s full text.

TL;DR

The paper optimizes an in vitro transcribed mRNA backbone and purification process to increase expression. It identifies a 148A poly(A) tail, synthetic UTR4, vaccinia capping, and combined dsRNA depletion plus 5′PPP dephosphorylation as improvements, with up to a 3-fold increase in dendritic cells.

Key findings

  • A 148A poly(A) tail gave the highest eGFP expression among 76A, 112A, and 148A mRNAs; in DC2.4 cells, 148A-mRNA increased eGFP expression by 65% versus 76A-mRNA and 25% versus 112A-mRNA by AUC.
  • Synthetic 5′ UTR4 produced the highest translation among UTR1–UTR6; compared with standard β-globin 148A-mRNA, UTR4 increased eGFP expression by 33% in HeLa cells, 37% in DC2.4 cells, and 80% in human moDCs.
  • Vaccinia virus capping was more efficient than ARCA capping, giving +67% eGFP expression in HeLa cells and +45% in DC2.4 cells.
  • Double purification—cellulose dsRNA depletion plus Antarctic phosphatase dephosphorylation—gave the best eGFP expression in DC2.4 and moDCs; a 3-fold increase was observed between dephosphorylated/dsRNA-depleted vaccinia mRNA and untreated ARCA mRNA in DC2.4 cells, and a 3-fold increase between dephosphorylated/dsRNA-depleted and untreated ARCA mRNA in moDCs. dsRNA depletion also lowered IFN-α and TNF-α in moDCs, while phosphatase treatment did not decrease those cytokines versus untreated mRNA.

Methods

  • The eGFP reporter was flanked by a 5′ UTR and two tandem copies of the human β-globin 3′ UTR; plasmids carried poly(A) tails of 76, 112, or 148 units, and synthetic 5′ UTRs UTR1–UTR6 were selected from a high-ribosome-loading library.
  • IVT mRNA was produced with ARCA or vaccinia virus capping and then treated by cellulose chromatography for dsRNA depletion and/or Antarctic phosphatase for 5′PPP dephosphorylation.
  • Expression was tested in HeLa, DC2.4, and human moDCs by fluorescence microscopy and flow cytometry, with AUC analysis and ELISA for IFN-α/TNF-α; in vivo work used zebrafish embryo microinjection and adult zebrafish intramuscular injection of naked mRNA or mRNA-LNP complexes.

Limitations

  • The paper states that it will be important to compare UTR4’s added value relative to the 5′ β-globin UTR in mouse and non-human primate models.
  • The paper notes that vaccinia capping is more complex, requires extra production/purification steps, must be validated for every batch, and is more time-consuming and laborious than ARCA capping.
  • The paper states that high ribosome loading is not always correlated with high protein expression, so selected 5′ and 3′ UTRs should be validated for the gene of interest and target cell type; it also notes that the commercial L7601 mRNA’s untranslated and codon-optimized sequences are unknown, so the cell-line discrepancy cannot be discussed. Evident from the text: adult zebrafish in vivo results are described as qualitative, and moDC transfection efficiency and viability were lower than in HeLa and DC2.4 cells.

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

Integrity signals

Registry

Crossref · 10.1016/j.omtn.2021.10.007

Cites

35 of 35 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

14 of 32 cited sources checked · 6 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    Double-stranded RNA generated during in vitro transcription synthesis is the major contributor to immunogenicity.

    “Double-stranded RNA (dsRNA) generated during in vitro transcription (IVT) synthesis is the major contributor in the immunogenicity.12”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [12] Mu, 2018, An origin of the immunogenicity of in vitro transcribed RNA, Nucleic Acids Res. source
  • Premisecitation

    dsRNA removal through HPLC or cellulose methods drastically enhances the translational efficiency of IVT mRNAs.

    “It has been shown that dsRNA removal through HPLC or cellulose methods drastically enhances the translational efficiency of IVT mRNAs.13,14”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [13] Karikó, 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucleic Acids Res. source
    • cites [14] Baiersdörfer, 2019, A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA, Mol. Ther. Nucleic Acids source

      The Results section (“Cellulose-Purified IVT mRNA Translates Better In Vivo”; page unavailable) reports substantially higher protein levels after purification of U-containing mRNA and says cellulose and HPLC similarly increase its translational capacity. But for m1Ψ-containing mRNA, the increase after either method was minor and not statistically significant. The unqualified claim that purification drastically enhances translation of IVT mRNAs overstates the finding. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Premisecitation

    High doses of mRNA formulated with lipid nanoparticles caused side effects in the clinical trial candidates mRNA-1273 and BNT162b1.

    “Indeed, the use of high doses of mRNA formulated with lipid nanoparticles (LNPs) resulted in some side effects in the candidates mRNA-1273 and BNT162b1.3,4”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [3] Jackson, 2020, An mRNA Vaccine against SARS-CoV-2 — Preliminary Report, N. Engl. J. Med source
    • cites [4] Mulligan, 2020, Phase I/II study of COVID-19 RNA vaccine BNT162b1 in adults, Nature source
    • Rests on an assumption: Increasing mRNA expression or potency will allow decreasing the therapeutic dose and reactogenicity.
  • Findingcitation

    Using a new set of 5′ UTR synthetic sequences based on selection of synthetic 5′ UTRs with high ribosome loading improved mRNA translation.

    “Here, we improved the mRNA translation by using a new set of 5′ UTR synthetic sequences based on the selection of synthetic 5′ UTRs with high ribosome loading.22”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [22] Sample, 2019, Human 5′ UTR design and variant effect prediction from a massively parallel translation assay, Nat. Biotechnol. source
  • Findingown data

    In human monocyte-derived dendritic cells, a 3-fold increase of eGFP expression was observed between dephosphorylated/dsRNA-depleted mRNA and untreated mRNA.

    “The results obtained were comparable with those generated with DC2.4 cells but revealed a higher increase of eGFP expression between the dephosphorylated/dsRNA depleted mRNA and untreated mRNA (3-fold).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 18
  • May combine two claims. 21
  • The quote only partly states this claim. 1

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

    Premisecitation

    A poly(A) tail of 120 units is considered the standard size for mRNA vaccines.

    “A poly(A) tail of 120 units is considered the standard size for mRNA vaccines.9,20,21”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [9] Holtkamp, 2006, Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells, Blood source

      The Discussion says antigen-encoding RNAs then used in preclinical studies had 30–70-nucleotide poly(A) tails, while those in clinical trials had 64-nucleotide tails. It proposes 120 nucleotides as an improvement, not an established standard; the section on combining modifications calls a 67-base-tail RNA the standard comparator. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [20] Oliveira, 1995, The relationship between eukaryotic translation and mRNA stability. A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNA degradation in the yeast Saccharomyces cerevisiae, J. Biol. Chem. source

      The Materials and Methods and Results passages discuss yeast cat mRNA constructs and translation, not mRNA vaccines. Searches for “poly(A),” “120 units,” and vaccine-related tail length found no passage in our copy addressing a standard poly(A)-tail size for mRNA vaccines. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [21] Oh, 2018, Design, Assembly, Production, and Transfection of Synthetic Modified mRNA, Methods source

      The Introduction says this protocol attaches a consistent, standardized 120-base poly(A) tail to each mRNA vector. It does not say that 120 bases is the standard size for mRNA vaccines generally; it also describes a 100–250-base range for actively translated eukaryotic mRNAs. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    An alternative strategy to avoid poly(A) instability consists of including an internal 10-nucleotide linker into the poly(A) stretch.

    “An alternative strategy has been developed to avoid this instability, consisting of including an internal 10-nucleotide linker into the poly(A) stretch.25,26”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [25] Stadler, 2017, Elimination of large tumors in mice by mRNA-encoded bispecific antibodies, Nat. Med. source
    • cites [26] Trepotec, 2019, Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life, RNA source

      The Results and Discussion section (“Design of modified/segmented poly(A) tails”; page unavailable) describes inserting internal spacers to reduce instability of plasmid-encoded poly(A) tails. It reports spacers of 1, 6, 12, and 24 nucleotides, but not the claimed 10-nucleotide linker. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    dsRNA depletion leads to a decrease of type I IFN and a translational enhancement of the mRNA into transfected DC cells.

    “This dsRNA depletion leads to a decrease of type I IFN and a translational enhancement of the mRNA into transfected DC cells.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [13] Karikó, 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucleic Acids Res. source

      The Results report that HPLC purification removed dsRNA contaminants, eliminated type I interferon induction by nucleoside-modified mRNA in transfected dendritic cells, and enhanced translation in those cells. But dsRNA depletion alone is not sufficient to explain the interferon claim: HPLC-purified mRNA without nucleoside modification remained a potent IFN-α inducer. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [14] Baiersdörfer, 2019, A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA, Mol. Ther. Nucleic Acids source

      The Results section reports that removing dsRNA by HPLC had been shown to eliminate IFN-α secretion by transfected dendritic cells. It also reports enhanced translation after purification, but that experiment measured protein production in mice, not in transfected dendritic cells. The paper further limits the IFN finding: cellulose-purified m1Ψ-containing mRNA no longer induced IFN-α, whereas U-containing mRNA still did. Page numbers are not provided in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    dsRNA contaminants can be purified by high-performance liquid chromatography or cellulose chromatography.

    “dsRNA contaminants can be purified by high-performance liquid chromatography (HPLC) or cellulose chromatography13,14”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [13] Karikó, 2011, Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA, Nucleic Acids Res. source

      The Results report that HPLC removed dsRNA contaminants from in vitro-transcribed RNA. Our copy does not report cellulose chromatography as a method for removing them, so it supports only the HPLC part of the cited sentence. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [14] Baiersdörfer, 2019, A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA, Mol. Ther. Nucleic Acids source

  • May combine two claims.

    Findingown data

    The study's results showed a difference in mRNA expression patterns, and this difference was consistent in all models.

    “Altogether, these results showed a difference in mRNA expression patterns, and this difference was consistent in all models.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: eGFP reporter mRNA expression results generalize to antigen-coding mRNA and mRNA vaccines.
    • Rests on an assumption: In vitro cell lines and zebrafish models predict human mRNA vaccine performance.

  • May combine two claims.

    Findingown data

    At 24 h post-transfection, 148A-mRNA provided the highest eGFP expression in HeLa cells by qualitative fluorescence microscopy, with the same transfection efficiency and cell viability for all assays.

    “At 24 h post-transfection, qualitative fluorescence microscopy analysis showed that the 148A-mRNA provided the highest eGFP expression (Figure 2A) for the same transfection efficiency and cell viability for all assays (Figures S2A and S2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The eGFP expression level of the mRNAs tested is comparable between HeLa and DC2.4 cell lines, except for the L7601 commercial mRNA.

    “The eGFP expression level of the mRNAs tested is comparable between HeLa and DC2.4 cell lines, except for the L7601 commercial mRNA.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    In human monocyte-derived dendritic cells, eGFP expression was significantly more important in primary cells transfected with UTR4 mRNA than with 148A and UTR2 mRNAs.

    “As for HeLa and DC2.4 cell lines, the eGFP expression was significantly more important in primary cells transfected with UTR4 mRNA.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A low level of eGFP production is obtained with UTR2 and UTR5 mRNAs in both HeLa and DC2.4 cell lines, whereas UTR2 presented a higher eGFP expression in moDCs.

    “Hence, a low level of eGFP production is obtained with UTR2 and UTR5 mRNAs in both cell lines (Figures 4 and 5), whereas UTR2 presented a higher eGFP expression in moDCs (Figures 6A and 6B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    UTR4 allows a high level of eGFP expression in human and mouse cells as well as in vivo after intramuscular injection of adult zebrafish.

    “Here, we have shown that UTR4 allows a high level of eGFP expression in human and mouse cells as well as in vivo after intramuscular injection of adult zebrafish.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In HeLa cells, all poly(A) mRNAs showed similar eGFP expression kinetics, with maximal eGFP expression at 24 h post-transfection followed by a gradual decrease over time.

    “A similar eGFP expression kinetic was observed with all the poly(A) mRNA, with a maximal eGFP expression at 24 h post-transfection followed by a gradual decrease of eGFP over time (Figure 2B).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Combining dsRNA depletion and 5′PPP dephosphorylation produces a great synergic improvement in mRNA potency in DC2.4 cells and even more in human moDCs.

    “From a more general view, our results demonstrated a great synergic improvement in mRNA potency by combining dsRNA depletion and 5′PPP dephosphorylation in our APC model, the DC2.4 cells, and even more in human moDCs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Selection of high-ribosome loading 5′ UTR sequence is a promising tool to improve mRNA potency, although it is not always correlated with high protein expression.

    “From this study, we showed that the selection of high-ribosome loading 5′ UTR sequence is a promising tool to improve mRNA potency, although it is not always correlated with high protein expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    The poly(A) region contributes to translation efficiency, mRNA half-life, and therefore mRNA potency.

    “The poly(A) is a critical region that contributes to translation efficiency, mRNA half-life, and therefore mRNA potency.19”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Sachs, 1990, The role of poly(A) in the translation and stability of mRNA, Curr. Opin. Cell Biol. source

  • May combine two claims.

    Interpretationreasoning

    dsRNA depletion and 5′PPP dephosphorylation of mRNA are important for vaccine purposes, since immune cells are more sensitive to PAMPs.

    “This highlights the importance of dsRNA depletion and 5′PPP dephosphorylation of mRNA used for vaccine purposes, since immune cells are more sensitive to pathogen-associated molecular patterns (PAMPs).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Immune cells are more sensitive to pathogen-associated molecular patterns (PAMPs) than non-immune cells.

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

    Premisecitation

    Strategies to impair pattern recognition receptor recognition of IVT mRNA include modified nucleosides, uridine depletion in the coding sequence, or dephosphorylation of 5′PPP mRNAs.

    “Others strategies aim to impair the PRR recognition of IVT mRNA with the use of modified nucleosides,16 uridine depletion in the coding sequence,17 or dephosphorylation of 5′PPP mRNAs.18”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [16] Karikó, 2005, Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA, Immunity source
    • cites [17] Vaidyanathan, 2018, Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification, Mol. Ther. Nucleic Acids source

      Queued — the background check reaches it, usually within a day.

    • cites [18] Schmidt, 2009, 5′-triphosphate RNA requires base-paired structures to activate antiviral signaling via RIG-I, Proc. Natl. Acad. Sci. USA source

      Queued — the background check reaches it, usually within a day.

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

    Study design and conductnone given

    Data were determined as mean of three independent experiments, each including four technical replicates.

    “Data were determined as mean of three independent experiments, each of them including four technical replicates.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    After cellulose chromatography and phosphatase treatment, dsRNA contaminants are depleted; thus the decrease of 5′PPP mRNA is beneficial for eGFP expression.

    “After cellulose chromatography and phosphatase treatment, dsRNA contaminants are depleted; thus the decrease of 5′PPP mRNA is beneficial for eGFP expression.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Translational efficiency of mRNA improves with increasing poly(A) size up to a length of 300 A.

    “who showed that the translational efficiency of mRNA was improved in conjunction with increasing poly(A) size until a length of 300 A”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Grier et al.,10

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

    • cites [24] Nicholson, 2019, Tales of Detailed Poly(A) Tails, Trends Cell Biol. source

      Checked before — its earlier verdict is held back until a new check runs.

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

    Study design and conductnone given

    The enzymatic capping efficiency was validated by fluorescence microscopy after mRNA transfection into HeLa cells.

    “The enzymatic capping efficiency was validated by fluorescence microscopy after mRNA transfection into HeLa cells.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    The more pronounced effect of double purification in DCs may be due to higher expression of PRRs in DCs and their sensing capacity to contaminants.

    “possibly because of the higher expression of PRRs into DCs and therefore their sensing capacity to contaminants.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All plasmids were propagated with NEB stable competent E. coli cells, and sequences were checked by Sanger sequencing.

    “All plasmids were propagated with NEB stable competent E. coli cells (NEB, France), and sequences were checked by Sanger sequencing.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Use of modified nucleosides such as methyl-pseudo uridine is a successful approach to avoid overactivation of innate immunity and increase translation of mRNA.

    “For instance, a successful approach to avoid overactivation of the innate immunity and increase translation of mRNA is the use of modified nucleosides such as methyl-pseudo uridine.29”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [29] Karikó, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol. Ther. source

      Not checked in the free run.

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

    Study design and conductnone given

    MFIs are represented as mean ± SD, and experimental values were compared by one-way ANOVA with Tukey's post hoc test.

    “MFIs are represented as the mean ± standard deviation of the mean (SD), and experimental values were compared by one-way analysis of variance (ANOVA) with Tukey’s post hoc test.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The procedures on adult zebrafish were conducted in accordance with European Union and French laws and approved by the local animal ethic committee.

    “The procedures on adult zebrafish were conducted in accordance with the guidelines of the European Union and French laws and approved by the local animal ethic committee under regulatory governmental authority (CECCAPP, Comité d’Evaluation Commun au Centre Léon Bérard, à l’Animalerie de transit de l’ENS, au PBES et au laboratoire P4 [no. C2EA15], APAFIS #16888-2018082321405041).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The threshold between eGFP-positive and eGFP-negative cells was defined so that >99.5% of non-transfected cells are considered not expressing eGFP.

    “The threshold between eGFP-positive and eGFP-negative cells was defined so that >99.5% of non-transfected cells are considered not expressing eGFP.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Specific UTRs, such as one or two copies of the 3′ UTR of globin or a short 14-nucleotide 5′ UTR, increase the translational efficiency of mRNAs.

    “numerous works have developed specific UTRs to increase the translational efficiency of mRNAs, as the use of one or two copies of the 3′ UTR of the globin or other known stable mRNA8,9 or short 14-nucleotide 5′ UTR.27”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [8] Orlandini von Niessen, 2019, Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3′ UTRs Identified by Cellular Library Screening, Mol. Ther. source
    • cites [9] Holtkamp, 2006, Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells, Blood source

      Not checked in the free run.

    • cites [27] Trepotec, 2019, Maximizing the Translational Yield of mRNA Therapeutics by Minimizing 5′-UTRs, Tissue Eng. Part A source

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

    Study design and conductcitation

    Data were tested for normality with the Shapiro-Wilk test.

    “Data were tested for normality with the Shapiro-Wilk test.33”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [33] Shapiro, 1965, An analysis of variance test for normality (complete samples), Biometrika source

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

    Premisecitation

    A comparable result had been previously observed after intramuscular injection of plasmid DNA into adult zebrafish, showing nucleic acid entering myocytes and antigen-presenting cells at the site of administration.

    “A comparable result had been previously observed after intramuscular injection of plasmid DNA into adult zebrafish and showed the nucleic acid entering the cells (myocytes and antigen-presenting cells [APCs]) at the site of administration.28”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [28] Myllymäki, 2018, Immunization of Adult Zebrafish for the Preclinical Screening of DNA-based Vaccines, J. Vis. Exp source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The dsRNA elimination was checked by dot-blotting.

    “The dsRNA elimination was checked by dot-blotting.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    All analyzed mRNAs showed sharp bands by agarose gel electrophoresis, confirming their overall integrity.

    “All these mRNAs were analyzed by agarose gel electrophoresis, and the presence of sharp bands confirmed their overall integrity (Figure S8A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Activation by dsRNA leads to detrimental strong innate immune activation, impairing the cellular translational machinery and therefore eGFP expression.

    “Activation by dsRNA leads to a detrimental strong innate immune activation, impairing the cellular translational machinery, and therefore the eGFP expression.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The presence of uncapped mRNA and dsRNA are sources of innate immunity activation.

  • May combine two claims.

    Premisecitation

    Contaminants such as dsRNA or 5′PPP uncapped mRNA activate pattern recognition receptors and block cellular protein synthesis.

    “Indeed, those contaminants, such as dsRNA or to a lesser extent 5′PPP uncapped mRNA, activate PRRs and block the cellular protein synthesis.12”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [12] Mu, 2018, An origin of the immunogenicity of in vitro transcribed RNA, Nucleic Acids Res. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Premisecitation

    Poly(A) tail plays an important function in the stability and regulation of translation efficiency of mRNA.

    “Poly(A) tail plays an important function in the stability and regulation of translation efficiency of the mRNA.24”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [24] Nicholson, 2019, Tales of Detailed Poly(A) Tails, Trends Cell Biol. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Premisecitation

    A high capping efficiency in the adequate Cap-1 conformation evades host responses by IFIT family members and favors mRNA translation.

    “Indeed, a high capping efficiency in the adequate Cap-1 conformation evades host responses by interferon (IFN)-induced protein with tetratricopeptide repeats (IFIT) family members and favors mRNA translation.23”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [23] Kumar, 2014, Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5’ppp- mRNAs, Nucleic Acids Res. source

      Checked before — its earlier verdict is held back until a new check runs.

35 of 35

  • The paper relies on this without support.

    Increasing mRNA expression or potency will allow decreasing the therapeutic dose and reactogenicity.

    “Intending to increase the potency of mRNA therapeutics and to decrease the therapeutic dose, we designed a mRNA backbone and optimized the mRNA purification process.”

  • The paper relies on this without support.

    eGFP reporter mRNA expression results generalize to antigen-coding mRNA and mRNA vaccines.

    “We used the enhanced green fluorescent protein (eGFP) reporter gene flanked by one 5′ untranslated region (UTR) and two 3′ UTRs of the human β-globin as a reference mRNA”

  • The paper relies on this without support.

    In vitro cell lines and zebrafish models predict human mRNA vaccine performance.

    “Those results were then confirmed in vivo with the embryonic zebrafish model.”

  • The paper relies on this without support.

    The poly(A) tail has been limited to 120 units because of its instability during bacterial plasmid production.

    “However, because of its instability during bacterial plasmid production, the poly(A) tail has been limited to 120 units.”

  • The paper relies on this without support.

    The presence of uncapped mRNA and dsRNA are sources of innate immunity activation.

    “However, other factors like the presence of uncapped mRNA and dsRNA are still a source of innate immunity activation.”

  • The paper relies on this without support.

    Immune cells are more sensitive to pathogen-associated molecular patterns (PAMPs) than non-immune cells.

    “since immune cells are more sensitive to pathogen-associated molecular patterns (PAMPs).”
6 of 6

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Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • moDC measurement time differs between results and Figure 6 caption

    Minor

    The results say moDC eGFP expression was evaluated 72 hours after transfection, but the Figure 6 caption lists measurements at 24, 48, and 96 hours, with no 72-hour measurement.

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Authors
Linares-Fernández, S., Moreno, J., Lambert, E., Mercier-Gouy, P., Vachez, L., Verrier, B., & Exposito, J.-Y.
Affiliations
Lyon 1 Université (FR)
Corresponding author
  • Verrier, Bernard
  • Exposito, Jean-Yves
ORCID
Venue
Molecular Therapy - Nucleic Acids
Publisher
Elsevier BV
Published
2021-12
Volume
26
Pages
945-956
ISSN
2162-2531 (ISSN-L)
DOI
10.1016/j.omtn.2021.10.007
PMC
PMC8523304
OpenAlex
W3205624390
Cited by
62
References
34
Citations by year
2026: 10 · 2025: 19 · 2024: 12 · 2023: 12 · 2022: 8 · 2021: 1
FWCI
2.59
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Nucleic Acids (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, other-oa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • European Commission
  • Agence nationale de la recherche (ANR-16-CE20-0002-01 554)
  • Agence Nationale de Recherches sur le Sida et les Hépatites Virales (ECTZ 119388)
  • Université Claude Bernard Lyon 1
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
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
mRNA vaccines, mRNA purification, poly(A) tail length, 5' untranslated region, 3' untranslated region, mRNA capping, in vitro transcribed mRNA, EGFP reporter, ribosome loading, transfection efficiency, reactogenicity, SARS-CoV-2

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 2026-11-05.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-05.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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