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Cited by 514RNA Interference and Gene DeliveryRNA and protein synthesis mechanismsRNA Research and Splicing

Sequence-engineered mRNA without chemical nucleoside modifications enables an effective protein therapy in large animals

Open access
Thess, Andreas, Grund, Stefanie, Mui, Barbara L, Hope, Michael J, Baumhof, Patrick, Fotin-Mleczek, Mariola, Schlake, Thomas·2015·Molecular Therapy
DOI 10.1038/mt.2015.103PMC PMC4817881
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Abstract

Being a transient carrier of genetic information, mRNA could be a versatile, flexible, and safe means for protein therapies. While recent findings highlight the enormous therapeutic potential of mRNA, evidence that mRNA-based protein therapies are feasible beyond small animals such as mice is still lacking. Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system. Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules. Using erythropoietin (EPO) driven production of red blood cells as the biological model, engineered Epo mRNA elicited meaningful physiological responses from mice to nonhuman primates. Even in pigs of about 20 kg in weight, a single adequate dose of engineered mRNA encapsulated in lipid nanoparticles (LNPs) induced high systemic Epo levels and strong physiological effects. Our results demonstrate that sequence-engineered mRNA has the potential to revolutionize human protein therapies.

AI snapshot

Written from the paper’s full text.

TL;DR

Sequence-engineered, chemically unmodified mRNA encoding erythropoietin produced high protein expression and physiological responses in mice, pigs (~20 kg), and cynomolgus monkeys without requiring nucleoside modifications. The work provides proof of feasibility for unmodified mRNA protein therapy in large animals, though clinical proof remains pending.

Key findings

  • In vitro, sequence-engineered unmodified luciferase and EPO mRNAs gave higher protein expression than their nucleoside-modified counterparts; with a less advanced luciferase mRNA, pseudouridine incorporation showed the previously described superiority (P < 0.0001 for all doses, Student’s t-test).
  • In BALB/c mice, intraperitoneal TransIT-complexed sequence-engineered mEpo mRNA produced higher EPO levels than its pseudouridine-modified counterpart (P < 0.0005 at all times), and doses down to 10 ng unmodified mRNA significantly increased reticulocytes (P < 0.05 versus untreated).
  • Repeated treatment with unmodified mRNA did not elicit substantial TNF-α, IL-6, or IFN-γ release; all measurements lay within the range observed for formulation reagent only, and no anti-EPO antibodies were detected (assay detection limit < 0.1 ng/ml).
  • In large animals, a single intravenous dose of LNP-encapsulated unmodified engineered mRNA produced high serum EPO and strong physiological effects: pigs (~20 kg) received 0.065 mg/kg and showed sustained hematocrit elevation (P < 0.05 for day 12 versus pretreatment); cynomolgus monkeys received 0.037 mg/kg and showed increased EPO, reticulocytes, and hematocrit (P < 0.05 for 96 hours versus pretreatment) with no detectable cytokine release.

Methods

  • Sequence engineering adapted codons to the most GC-rich codons for each amino acid and used optimized regulatory elements: a 5′-UTR from HSD17B4, a 3′-UTR from ALB, a polyA, and a histone stem loop; mRNAs were T7-transcribed, capped, and purified by reversed-phase chromatography.
  • Formulations: TransIT-mRNA for intraperitoneal administration in mice and pigs; LNPs from Acuitas for intravenous administration in pigs and macaques (ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid at 50:10:38.5:1.5 mol/mol, RNA-to-total lipid ratio ~0.05 wt/wt, diameter ~80 nm).
  • Animal experiments: female BALB/c mice 7–9 weeks old; cynomolgus monkeys 2.5–2.8 kg given a single 100 μg LNP-formulated EPO mRNA dose; Hungarian large white domestic pigs (~20 kg) given either 360 μg TransIT-formulated porcine EPO mRNA intraperitoneally on two consecutive days or 1.3 mg LNP-formulated porcine Epo mRNA intravenously. Analyses included EPO ELISA, reticulocyte counts, hematocrit, cytokine bead array, anti-EPO ELISA, and spleen histology.

Limitations

  • The paper does not include a formal limitations section; it states that the approach “currently awaits its first clinical proof of principle” and that formulations differ and “have probably to be selected on a case-by-case basis.”
  • Evident from the text: no human data are reported, and large-animal immunogenicity/safety assessment is limited—cytokine release was examined in monkeys, but anti-EPO antibody testing was performed only in mice.
  • Evident from the text: the therapeutic model is restricted to erythropoietin, and large-animal experiments used single or short dosing regimens rather than long-term repeated administration.

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

Integrity signals

Registry

Crossref · 10.1038/mt.2015.103

Cites

45 of 45 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

21 of 48 cited sources checked · 11 supported · 2 flagged · accuracy not yet measured

  • Interpretationown data

    Sequence-engineered, unmodified mRNA is competitive with nucleoside-modified mRNA with respect to protein yield in vivo.

    “In summary, these data demonstrate that sequence-engineered, unmodified mRNA is competitive with nucleoside-modified mRNA with respect to protein yield in vivo.”
    • Also stated in: § Discussion · hedged
      “Using a common model protein, erythropoietin, as well as a formulation reagent and an administration route that were previously applied to state-of-the-art pseudouridine mRNA, our data suggest that sequence-engineered mRNA is competitive with modified mRNA with respect to protein yield and physiological efficacy.27”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] Karikó, 2012, Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin, Mol Ther source

      The Results (page not provided) compare codon-optimized EPO mRNAs differing in whether they contain uridine or pseudouridine. In mice, the unmodified mRNA produced much lower EPO levels; 0.001 µg of pseudouridine-containing mRNA was as potent as 1 µg of unmodified mRNA in raising reticulocyte counts. This is contrary to the claimed comparable in-vivo yield. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
  • Premisecitation

    Two independent studies found superiority of nucleoside-modified compared to unmodified Epo mRNA.

    “Two independent studies on erythropoietin-encoding mRNA found a superiority of nucleoside-modified compared to unmodified mRNA.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [26] Kormann, 2011, Expression of therapeutic proteins after delivery of chemically modified mRNA in mice, Nat Biotechnol source
    • cites [27] Karikó, 2012, Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin, Mol Ther source

      The Results (page unavailable) show that pseudouridine-modified EPO mRNA produced higher, longer-lasting EPO levels than uridine-containing EPO mRNA in mice. The Introduction mentions another study of modified mRNA for therapeutic proteins, but does not establish that it independently compared modified and unmodified EPO mRNA. This source therefore supports the superiority finding for one study, not the claim about two independent EPO studies. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    The physiological effect of administering engineered Epo mRNA is as strong as that observed with pseudouridine-modified mRNA.

    “Taking into account the lower reticulocyte numbers obtained with 100 U of recombinant EPO protein as well as the lower background levels compared to a recent publication,27 the physiological effect of administering our engineered Epo mRNA is as strong as that observed with pseudouridine-modified mRNA.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [27] Karikó, 2012, Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin, Mol Ther source
    • Rests on an assumption: Administration of erythropoietin causes a burst release of reticulocytes into the blood stream.
  • Interpretationreasoning

    The lack of biological efficacy of HPLC-purified basic Epo mRNA highlights the importance and potency of sequence-engineering.

    “Although high performance liquid chromatography (HPLC) purification depletes contaminants and, thus, contributes to reduced immunostimulation as well as improves mRNA expression to some extent,36,37,38 the lack of biological efficacy of HPLC-purified basic Epo mRNA highlights the importance and potency of sequence-engineering.”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites [36] Pascolo, 2004, Messenger RNA-based vaccines, Expert Opin Biol Ther source
    • cites [37] Probst, 2007, Spontaneous cellular uptake of exogenous messenger RNA in vivo is nucleic acid-specific, saturable and ion dependent, Gene Ther source
    • cites [38] 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
  • Premisecitation

    Nucleoside modifications are well known to limit or even inhibit RNA sensor engagement.

    “In addition, nucleoside modifications are well known to limit or even inhibit RNA sensor engagement.20,21,22”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [20] Karikó, 2005, Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA, Immunity source
    • cites [21] Anderson, 2011, Nucleoside modifications in RNA limit activation of 2’-5’-oligoadenylate synthetase and increase resistance to cleavage by RNase L, Nucleic Acids Res source
    • cites [22] Anderson, 2010, Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Nucleic Acids Res source
  • Classified as probably not a substantial claim. 14
  • May combine two claims. 19
  • The quote only partly states this claim. 2

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

    Premisecitation

    LNPs used in this study are similar in composition to those described previously, which contain an ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid (50:10:38.5:1.5 mol/mol), encapsulated RNA-to-total lipid ratio of ~0.05 (wt/wt) and a diameter of ~80 nm.

    “LNPs used in this study are similar in composition to those described previously, which contain an ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid (50:10:38.5:1.5 mol/mol), encapsulated RNA-to-total lipid ratio of ~0.05 (wt/wt) and a diameter of ~80 nm.28,43”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [28] Jayaraman, 2012, Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo, Angew Chem Int Ed Engl source

      The paper describes an optimized LNP formulation with the stated 50/10/38.5/1.5 molar composition. In its formulation description (page not available), it gives an approximately 0.05 siRNA-to-lipid ratio and a 70–90 nm particle-size range, but those measurements are stated for the earlier 40/10/40/10 formulation, not explicitly for the optimized one. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [43] Maier, 2013, Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics, Mol Ther source

      The Materials and Methods describe the same four lipid components but give a molar ratio of 55:10:32.5:2.5 and a total-lipid-to-siRNA weight ratio of ~10:1 (siRNA-to-lipid ~0.1), rather than the claimed 50:10:38.5:1.5 and ~0.05. The in vivo efficacy section reports a mean particle diameter of ~60 nm, not ~80 nm. No page numbers are available in our copy. (LLM, conf=0.88)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

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

    Premisecitation

    Pseudouridine-containing mRNAs reduced activation of known RNA sensors substantially.

    “Transferring this concept to in vitro transcribed mRNA, for instance, pseudouridine-containing mRNAs reduced activation of known RNA sensors substantially.”
    Quote in the paperNot a substantial claim2 of 3 sources checked
    • cites [20] Karikó, 2005, Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA, Immunity source
    • cites [21] Anderson, 2011, Nucleoside modifications in RNA limit activation of 2’-5’-oligoadenylate synthetase and increase resistance to cleavage by RNase L, Nucleic Acids Res source
    • cites [22] Anderson, 2010, Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Nucleic Acids Res source

      The Results (‘Conventional in vitro transcribed mRNA activates PKR’) show that pseudouridine-containing mRNA activates the RNA sensor PKR less than unmodified mRNA. The Introduction says pseudouridine-modified RNA can avoid activation of other receptors, including TLRs and RIG-I, but this paper does not establish a substantial reduction across multiple sensors specifically for pseudouridine-containing mRNAs. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Pseudouridine-containing mRNAs were still translated and produced even more protein compared to unmodified mRNA.

    “Although pseudouridine is primarily found in tRNA, rRNA, and small nuclear RNAs, pseudouridine-containing mRNAs were still translated and produced even more protein compared to unmodified mRNA.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [22] Anderson, 2010, Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation, Nucleic Acids Res source
    • cites [23] Karikó, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther source

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Nucleoside modifications can strongly enhance protein expression and suppress cytokine secretion.

    “Using enhanced green fluorescent protein mRNA, Rossi and colleagues confirmed that nucleoside modifications can strongly enhance protein expression and suppress cytokine secretion.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] Warren, 2010, Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA, Cell Stem Cell source

      In Results, “Development of modified-RNAs for directing cell fate” (page unavailable), the paper reports that nucleoside modifications in GFP-encoding mRNA markedly increased protein expression and dramatically attenuated interferon signaling. It assessed interferon-response gene expression by qRT-PCR, not cytokine secretion, so the secretion claim is not established by our copy. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data

    Conventional unmodified mRNAs are inferior to molecules harboring specific nucleoside modifications, but the picture changes dramatically with sophisticated sequence-engineering.

    “Our work principally confirms that conventional unmodified mRNAs are inferior to molecules harboring specific nucleoside modifications as suggested by various recent reports,23,24,25,26,27 however, the picture changes dramatically with a sophisticated sequence-engineering.”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [23] Karikó, 2008, Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability, Mol Ther source
    • cites [24] Warren, 2010, Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA, Cell Stem Cell source
    • cites [25] Zangi, 2013, Modified mRNA directs the fate of heart progenitor cells and induces vascular regeneration after myocardial infarction, Nat Biotechnol source
    • cites [26] Kormann, 2011, Expression of therapeutic proteins after delivery of chemically modified mRNA in mice, Nat Biotechnol source
    • cites [27] Karikó, 2012, Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin, Mol Ther source

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

    Premisecitation

    The Acuitas mRNA delivery platform has been developed from technology shown previously to enable siRNA-dependent hepatic gene silencing across species in rodents, nonhuman primates and humans.

    “The Acuitas mRNA delivery platform has been developed from technology shown previously to enable siRNA-dependent hepatic gene silencing across species in rodents, nonhuman primates and humans.28,43,44”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [28] Jayaraman, 2012, Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo, Angew Chem Int Ed Engl source

      The study reports siRNA-mediated hepatic gene silencing in mice and cynomolgus monkeys (Figure 7; page not available). Its conclusion mentions a related formulation in a human clinical trial and preliminary LDL-C decreases, but does not demonstrate hepatic gene silencing in humans or identify the Acuitas mRNA platform as derived from this technology. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
    • cites [43] Maier, 2013, Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics, Mol Ther source

      The 'In vivo efficacy evaluation in mouse FVII model' section reports siRNA-dependent gene silencing in mice and rats, and 'Efficacy and rapid elimination properties translate to higher species' reports about 70% silencing of a hepatic gene in nonhuman primates. The paper does not demonstrate hepatic gene silencing in humans or establish that the Acuitas mRNA platform was developed from this technology; its Introduction mentions human clinical trials without making those links. Our copy provides no page numbers for these passages. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [44] Coelho, 2013, Safety and efficacy of RNAi therapy for transthyretin amyloidosis, N Engl J Med source

      The Discussion (page not supplied) reports that lipid-nanoparticle siRNA delivery silenced liver-expressed genes in animals, including nonhuman primates, and that this study demonstrated RNAi-mediated gene modulation in human liver. The paper also discusses results across multiple species, but our copy does not establish that the Acuitas mRNA platform was developed from this technology. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    At blood pH, LNPs exhibit a net neutral surface charge but become positively charged in acidified endosomes following ApoE-mediated endocytosis by hepatocytes in vivo, resulting in endosome disruption and release of mRNA into the cytoplasm.

    “At blood pH, LNPs exhibit a net neutral surface charge but become positively charged in acidified endosomes following ApoE-mediated endocytosis by hepatocytes in vivo, resulting in endosome disruption and release of mRNA into the cytoplasm.28,41,45”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites [28] Jayaraman, 2012, Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo, Angew Chem Int Ed Engl source

      The main text around Figure 5 (page not provided) describes minimal LNP charge at blood pH, ApoE-mediated hepatocyte uptake, and positive charge in acidified endosomes promoting membrane disruption. It says siRNA, not mRNA, is released into the cytoplasm; the cited study concerns siRNA delivery. (LLM, conf=0.91)

      Experimental — accuracy not yet measured
    • cites [41] Hope, 2014, Enhancing siRNA delivery by employing lipid nanoparticles, Therapeutic Delivery source
    • cites [45] Akinc, 2010, Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms, Mol Ther source

  • May combine two claims.

    Findingown data

    With a less advanced luciferase mRNA, pseudouridine incorporation showed superiority over unmodified mRNA, albeit at a low expression level.

    “Here, incorporation of pseudouridine showed the previously described superiority regarding protein expression compared to the corresponding unmodified mRNA, albeit at a low expression level (Figure 1c) (P < 0.0001 for all doses, student's t-test).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    mRNA-injected animals had strongly elevated hematocrits and erythropoiesis-induced splenomegaly without other spleen disturbances.

    “As opposed to mice receiving murine recombinant EPO protein, mRNA-injected animals were characterized by strongly elevated hematocrits (P < 0.01 for 1 μg versus untreated on all days, Student's t-test) as well as erythropoiesis induced splenomegaly in the absence of any other disturbances of the spleen (Figure 5c and Supplementary Figure S4).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Sequence-engineered, chemically unmodified mRNA can achieve sufficient protein expression and avoid innate immune activation.

    “Here we show that chemically unmodified mRNA can achieve those goals as well by applying sequence-engineered molecules.”
    • Also stated in: § Introduction · boosted
      “Here, using sequence-engineered mRNA, we demonstrate that unmodified mRNA is fully suited for use in protein therapies.”
    • Also stated in: § Discussion · boosted
      “Further, strong evidence is provided that such an mRNA does not cause inappropriate immunostimulation.”
    Quote in the paperSubstantial claim

  • The quote only partly states this claim.

    Findingown data

    Sequence-engineered mRNA gave rise to longer lasting protein expression than pseudouridine-modified mRNA in mice.

    “However, our sequence-engineered mRNA gave rise to longer lasting protein expression.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    None of the mice showed substantial cytokine release upon mRNA administration; all measurements lay within the range observed for the formulation reagent only.

    “None of the mice showed any substantial cytokine release upon mRNA administration; all measurements lay within the range observed for the formulation reagent only (which by itself could have some immunostimulatory potential) (Figure 4a).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Cytokine release from unmodified mRNA may cause unwanted or detrimental side effects, including a possibly fatal immune response against the encoded protein.

  • The quote only partly states this claim.

    Findingown data

    A similar result was obtained with a sequence-engineered mRNA coding for erythropoietin: unmodified mRNA gave higher expression than modified mRNA.

    “This effect was not specific to luciferase as a similar result was obtained with a sequence-engineered mRNA coding for erythropoietin (Figure 1b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In pigs, reticulocyte numbers were strongly increased and animals showed a substantial and sustained elevation of the hematocrit after LNP-encapsulated mRNA.

    “As a consequence, reticulocyte numbers were strongly increased and animals showed a substantial and sustained elevation of the hematocrit (P < 0.05 for day 12 versus pretreatment, Student's t-test) (Figure 6a).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Administration of erythropoietin causes a burst release of reticulocytes into the blood stream.

  • May combine two claims.

    Findingown data

    In vitro, sequence-engineered unmodified luciferase mRNA gave very high luciferase activity, while its nucleoside-modified counterparts gave substantially lower protein levels.

    “Notably, in vitro protein expression revealed very high luciferase activity with the unmodified mRNA, while its nucleoside-modified counterparts gave rise to substantially lower protein levels (Figure 1a).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Sequence-engineering effectiveness requires both adaptation of the open reading frame sequence and potent regulatory untranslated regions.

    “Finally, comparison to a modified luciferase mRNA from a commercial supplier confirmed the effectiveness of sequence-engineering for which both adaptation of the open reading frame sequence as well as potent regulatory untranslated regions are essential parameters (Supplementary Figure S1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In cynomolgus monkeys, a single intravenous dose (0.037 mg/kg) of LNP-encapsulated unmodified Epo mRNA increased serum EPO levels, reticulocyte counts, and hematocrit, with no detectable cytokine release.

    “As observed in pigs, nonhuman primates revealed a considerable increase of serum EPO levels and reticulocyte counts as well as a meaningful raise of the hematocrit (increase for each individual from pretreatment to 96 hours after treatment; P < 0.05 for 96 hours versus pretreatment, Student's t-test) while there was no detectable cytokine release upon treatment (Figure 6b and Supplementary Figure”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Administration of erythropoietin causes a burst release of reticulocytes into the blood stream.

  • May combine two claims.

    Findingown data

    Engineered mRNA neither induced substantial cytokine release nor elicited a protein-specific antibody response.

    “Interestingly, as being attributed to nucleoside-modified mRNA, engineered mRNA neither induced substantial cytokine release nor elicited a protein-specific antibody response.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Cytokine release from unmodified mRNA may cause unwanted or detrimental side effects, including a possibly fatal immune response against the encoded protein.
    • Rests on an assumption: A potential antibody response should be fully developed 4 weeks after the last treatment.

  • May combine two claims.

    Findingown data

    TransIT-complexed Epo-mRNA induces massive erythropoiesis in the spleen without causing histological or immunological disturbances.

    “Figure S4. TransIT-complexed Epo-mRNA induces massive erythropoiesis in the spleen without causing histological or immunological disturbances.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Unmodified Epo mRNA was immunostimulatory and failed to induce sufficient protein expression in mice, whereas nucleoside-modified mRNA achieved detectable protein expression and physiological effects.

    “Whereas unmodified Epo mRNA was immunostimulatory and failed to induce sufficient protein expression in mice, a few micrograms of nucleoside-modified mRNA were enough to achieve detectable protein expression and physiological effects upon direct administration to murine muscle.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [26] Kormann, 2011, Expression of therapeutic proteins after delivery of chemically modified mRNA in mice, Nat Biotechnol source

  • May combine two claims.

    Interpretationown data

    Choosing less effective sequences appears to dampen the negative effect of chemical modifications, suggesting an interference between chemical modifications and regulatory RNA elements.

    “Interestingly, choosing less effective sequences appears to dampen the negative effect of chemical modifications (Supplementary Figure S7a), suggesting an interference between chemical modifications and regulatory RNA elements.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system.

    “Previous studies imply that mRNA therapeutics require chemical nucleoside modifications to obtain sufficient protein expression and avoid activation of the innate immune system.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    A dose of recombinant erythropoietin of about 600 IU/kg appears to be among the highest usually applied in clinical settings.

    “(which appears to be among the highest usually applied in clinical settings)”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [39] Cheung, 1998, Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin after single and multiple subcutaneous doses to healthy subjects, Clin Pharmacol Ther source
    • cites [40] McGowan, 2008, Pharmacokinetic and pharmacodynamic profiles of extended dosing of epoetin alfa in anemic patients who have chronic kidney disease and are not on dialysis, Clin J Am Soc Nephrol source

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

    Interpretationreasoning

    The approach currently awaits its first clinical proof of principle.

    “Thus, the approach currently awaits its first clinical proof of principle.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The present study provides the first proof of mRNA-based therapies being feasible in large animals, even for systemically acting proteins.

    “as well as the first proof of mRNA-based therapies being feasible in large animals, even for systemically acting proteins.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Porcine EPO levels in the supernatant were measured using a mouse EPO ELISA kit with cross reactivity to pig EPO.

    “Porcine EPO levels in the supernatant were measured using a mouse EPO ELISA kit (R&D Systems) with cross reactivity to pig EPO and recombinant pig EPO protein (Cusabio, Wuhan, China) as standard.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All experiments were approved by the responsible authorities (Regional Council Tuebingen) and carried out in accordance with national laws.

    “All experiments were approved by the responsible authorities (Regional Council Tuebingen) and carried out in accordance with national laws.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S2 (checkable), S3 (load-bearing).

    Interpretationnone given

    Formulations differ with respect to their characteristics and have probably to be selected on a case-by-case basis.

    “However, formulations differ with respect to their characteristics and have probably to be selected on a case-by-case basis.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Serum of cynomolgus monkeys was analyzed with a human EPO ELISA kit that cross reacts with macaque EPO.

    “Serum of cynomolgus monkeys was analyzed with a human EPO ELISA kit (R&D Systems) that cross reacts with macaque EPO.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    A protective formulation will be mandatory for systemic delivery due to the ubiquity and abundance of RNases.

    “a protective formulation will be mandatory for systemic delivery due to the ubiquity and abundance of RNases.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: RNases are ubiquitous and abundant.

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

    Study design and conductnone given

    The ELISA allowed detection of a concentration of anti-EPO antibody as low as about 100 pg/ml.

    “The ELISA allowed detection of a concentration of anti-EPO antibody as low as about 100 pg/ml.”
    Quote in the paperNot a substantial claim
31 of 31

  • The paper relies on this without support.

    Administration of erythropoietin causes a burst release of reticulocytes into the blood stream.

    “Administration of erythropoietin causes a burst release of reticulocytes representing precursors of mature erythrocytes into the blood stream.”

  • The paper relies on this without support.

    Cytokine release from unmodified mRNA may cause unwanted or detrimental side effects, including a possibly fatal immune response against the encoded protein.

    “This cytokine release may cause unwanted or even detrimental side effects such as a possibly fatal immune response against the encoded protein and, thus, should be minimized.”

  • The paper relies on this without support.

    A potential antibody response should be fully developed 4 weeks after the last treatment.

    “At that time, a potential antibody response should be fully developed.”

  • The paper relies on this without support.

    The effective dose of systemically acting proteins is a function of animal size.

    “Particularly for systemically acting proteins, the effective dose will be a function of animal size.”

  • The paper relies on this without support.

    It has yet to be shown that meaningful expression levels can be obtained in animals close to humans in weight with a feasible mRNA dose.

    “It has yet to be shown that meaningful expression levels can be obtained in animals close to humans in weight with a feasible mRNA dose.”

  • The paper relies on this without support.

    In vitro expression data may not faithfully predict in vivo features.

    “However, in vitro expression data may not faithfully predict in vivo features.”

  • The paper relies on this without support.

    EPO protein has a very short half-life in serum and mRNA is transient, so measurements reflect EPO expression in response to the latest treatment only.

    “Due to the very short half-life of EPO protein in serum and the transient nature of mRNA, all measurements reflect EPO expression in response to the latest treatment only.”

  • The paper relies on this without support.

    RNases are ubiquitous and abundant.

    “a protective formulation will be mandatory for systemic delivery due to the ubiquity and abundance of RNases.”
8 of 8

Claims other papers make about this one

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

  • Our proprietary mRNA technology employs sequence optimization and selected untranslated regions (UTRs) to achieve high antigen expression.

    “In recent publications chemically modified nucleosides were used to reach sufficient antigen expression,12–15 which is in contrast to our proprietary mRNA technology, which employs sequence optimization and selected untranslated regions (UTRs) to achieve high antigen expression.16”

    From Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines (2017) · cites this paper as [16]

    The Materials and Methods describe codon optimization and selection of 5′ and 3′ UTRs, and the Results report high expression from sequence-engineered mRNA. However, the expression experiments described in the Results use luciferase and erythropoietin, not an antigen; the source does not establish high antigen expression specifically. Page numbers were not provided in our copy. (LLM, conf=0.85)

    Experimental — accuracy not yet measured

  • Physiological responses elicited in primates and domestic pigs treated with an mRNA-encoded hormone have suggested the feasibility of mRNA for large animal therapies.

    “Physiological responses elicited in primates and domestic pigs treated with an mRNA‐encoded hormone have suggested the feasibility of mRNA for large animal therapies (Thess et al, 2015).”

    From mRNA mediates passive vaccination against infectious agents, toxins, and tumors (2017) · cites this paper as Thess et al, 2015

  • It is likely that target-specific mRNA optimizations and further improvements to the formulation can substantially increase efficacy (Thess et al, 2015).

    “Thirdly, in light of previous findings, it is likely that target‐specific mRNA optimizations and further improvements to the formulation can substantially increase efficacy (Thess et al, 2015).”

    From mRNA mediates passive vaccination against infectious agents, toxins, and tumors (2017) · cites this paper as Thess et al, 2015

  • Short-lived proteins can significantly profit by being expressed from mRNA (Kariko et al, 2012; Thess et al, 2015).

    “By contrast, short‐lived proteins can significantly profit by being expressed from mRNA (Kariko et al, 2012; Thess et al, 2015).”

    From mRNA mediates passive vaccination against infectious agents, toxins, and tumors (2017) · cites this paper as Thess et al, 2015

  • An earlier study on erythropoietin revealed that findings in a murine model translated to larger animals such as domestic pigs as well as to primates (Thess et al, 2015).

    “An earlier study on a different secreted protein, erythropoietin, revealed that findings in a murine model translated to larger animals such as domestic pigs as well as to primates (Thess et al, 2015).”

    From mRNA mediates passive vaccination against infectious agents, toxins, and tumors (2017) · cites this paper as Thess et al, 2015

Cites

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2 of the 47 references couldn’t be matched to a known paper.

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Related

Authors
Thess, A., Grund, S., Mui, B. L., Hope, M. J., Baumhof, P., Fotin-Mleczek, M., & Schlake, T.
Affiliations
  • CureVac (Germany) (DE)
  • Acuitas Therapeutics (Canada) (CA)
Corresponding author
Schlake, Thomas
ORCID
Venue
Molecular Therapy
Publisher
Elsevier BV
Published
2015-09
Volume
23
Issue
9
Pages
1456-1464
ISSN
1525-0016 (ISSN-L)
DOI
10.1038/mt.2015.103
PMC
PMC4817881
OpenAlex
W1538766821
Semantic Scholar
ba6194e7bb15ea1a7e676b261ee348f90c99f8b7
Cited by
513
References
47
Citations by year
2026: 33 · 2025: 53 · 2024: 70 · 2023: 51 · 2022: 80 · 2021: 82 · 2020: 37 · 2019: 30 · 2018: 31 · 2017: 21 · 2016: 19 · 2015: 5
FWCI
9.81
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by-nc-nd)
  • PubMed Central (submittedVersion, cc-by-nc-nd)
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
erythropoietin, lipid nanoparticles, non-human primates, pigs, innate immune activation, erythropoiesis, large animal model, mRNA therapeutics
MeSH terms
Silent Mutation, Animals, Cell Line, Erythrocyte Indices, Erythropoietin, Humans, Lipids, Macaca fascicularis, RNA, Messenger, Genetic Therapy, Gene Expression, Genes, Reporter, Models, Animal, Sus scrofa, Mice, Nanoparticles

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.

What counts as a claim ›How we measure confidence ›How we check this ›

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