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Cited by 100Extracellular vesicles in diseaseRNA Interference and Gene DeliveryNanoparticle-Based Drug Delivery

GAPDH controls extracellular vesicle biogenesis and enhances the therapeutic potential of EV mediated siRNA delivery to the brain

Open access
Dar, Ghulam Hassan, Mendes, Cláudia C., Kuan, Wei-Li, Speciale, Alfina A., Conceição, Mariana, Görgens, André, Uliyakina, Inna, Lobo, Miguel J. +8 more·2021·Nature Communications
DOI 10.1038/s41467-021-27056-3PMC PMC8602309

Published version of a preprint

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Abstract

Extracellular vesicles (EVs) are biological nanoparticles with important roles in intercellular communication, and potential as drug delivery vehicles. Here we demonstrate a role for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in EV assembly and secretion. We observe high levels of GAPDH binding to the outer surface of EVs via a phosphatidylserine binding motif (G58), which promotes extensive EV clustering. Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering. Fusion of the GAPDH-derived G58 peptide to dsRNA-binding motifs enables highly efficient loading of small interfering RNA (siRNA) onto the EV surface. Such vesicles efficiently deliver siRNA to multiple anatomical regions of the brain in a Huntington’s disease mouse model after systemic injection, resulting in silencing of the huntingtin gene in different regions of the brain.

AI snapshot

Written from the paper’s full text.

TL;DR

GAPDH binds the outer surface of extracellular vesicles via a phosphatidylserine-binding G58 motif, promotes EV clustering, and is required for normal exosome/ILV biogenesis in a Drosophila model. G58-based chimeric proteins load siRNA onto EVs and, with brain-targeting RVG, achieve ~40% huntingtin silencing in the cortex of Q140 Huntington’s disease mice after systemic injection.

Key findings

  • GAPDH is present on the outer surface of EVs from HEK293T, HeLa, MSCs, SKOV-3 and B16 lymphoma cells, and exogenous GAPDH induces EV clustering; the GAPDH tether width was 13.54 ± 5.5 nm (mean ± s.d., n = 30). G58 peptide binding was quantified at ~1200 sites per MSC-derived EV and ~1400 sites per HEK293T-derived EV.
  • In Drosophila secondary cells, GAPDH2 knockdown severely disrupted dense-core granule formation and ILV clustering and significantly reduced exosome secretion; GAPDH1 knockdown reduced exosome secretion without significantly affecting ILV/DCG biogenesis. Human GAPDH overexpression increased ILV and luminal exosome clustering.
  • G58T bound siRNA at ~500–700 siRNA molecules per EV and protected siRNA from RNase A degradation. G58TF improved silencing to ~60% in cells, and chloroquine increased silencing to ~80%; in Q140 mice, systemic G58TF/siRNA-RVG EVs gave almost 40% Htt silencing in cortex and a significant decrease in p62 inclusion bodies.
  • After systemic administration, ~1% of injected EVs were found in the brain at 4 h; binding of G58TF or G58TF/siRNA did not change RVG-EV biodistribution.

Methods

  • EVs were isolated from cultured cells by differential centrifugation, ultrafiltration/TFF and size-exclusion chromatography, then characterized by NTA, electron microscopy, western blotting, protease protection, GAPDH activity assays, gel-shift assays and high-resolution single-EV imaging flow cytometry.
  • GAPDH, G58T, G58T(TAT)2 and G58TF proteins were expressed in E. coli, purified, and incubated with EVs; siRNA loading and silencing were tested in N2a cells and HeLa cells, with RT-qPCR and western blotting.
  • In vivo work used C57BL/6 mice for EV biodistribution and one-year-old Q140 Huntington’s disease mice for four weekly intravenous doses of G58TF/siRNA-RVG EVs, followed by brain Htt mRNA analysis, immunohistochemistry and p62 inclusion-body quantification; Drosophila secondary-cell genetics and live/confocal imaging assessed exosome biogenesis and secretion.

Limitations

  • The paper states that low Htt silencing was observed in brain regions other than cortex for reasons not understood, and that the slow progression of Q140 mice makes it difficult to determine morphological and phenotypic changes of Huntington’s disease; further studies in R6/1 or R6/2 models are said to be necessary.
  • The paper states that human GAPDH knockdown in cultured cells was too variable and toxic to assess its role in human EV production, that the G∆(71–90) GAPDH mutant could not be expressed, and that HA2 chimeric proteins could not be expressed due to E. coli toxicity.
  • The paper notes that the proportion of EVs bearing both GAPDH and CD63 may be affected by low GAPDH antibody affinity, GAPDH binding to CD63-negative EVs, or CD63-GFP overexpression; it also states that the biochemical nature of the ~12 nm ILV tether remains to be determined. Evident from the text: in vitro silencing with G58T alone was low (~15%) and required endosomolytic peptides or chloroquine for stronger effects, and only ~1% of injected EVs reached the brain.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-021-27056-3

Cites

95 of 95 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

30 of 48 cited sources checked · 16 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    p62 inclusion bodies are an important phenotypic HD marker of disease progression.

    “Analysis of brain tissues revealed almost 40% silencing of the Htt gene in mouse brain cortex and a highly significant decrease of p62 inclusion bodies in the cortical neurons of the treated animals, an important phenotypic HD marker of disease progression (Fig. 4e, f).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [56] Kurosawa, M. et al. Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington’s model mice. Hum. Mol. Genet. 24, 1092–1105 (2015). source

      The Abstract calls neuronal inclusions of mutant huntingtin a neuropathological hallmark of HD. The section on nuclear inclusion formation reports changes in inclusions with age in HD model mice, but it does not establish that p62 inclusion bodies specifically are a marker of disease progression. Page numbers were not supplied for these passages. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    Due to the limited capacity of bacterial cells to introduce PTM, post-translational modifications are not required for GAPDH binding to EVs.

    “However, due to the limited capacity of bacterial cells to introduce PTM60, it appears that these modifications are not required for EV binding.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [60] Tokmakov, A. A. et al. Multiple post-translational modifications affect heterologous protein synthesis. J. Biol. Chem. 287, 27106–27116 (2012). source
  • Interpretationcitation

    The observations are unlikely to be Drosophila-specific.

    “so our observations are unlikely to be Drosophila-specific44.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [44] Fan, S. J. et al. Glutamine deprivation regulates the origin and function of cancer cell exosomes. EMBO J 39, 0261–4189 (2020). source
  • Premisecitation

    R6/1 or R6/2 models demonstrate a much more aggressive phenotype than Q140 mice.

    “R6/1or R6/2 models, which demonstrate a much more aggressive phenotype than Q140 mice70”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [70] Lerner, R. P., Trejo Martinez Ldel, C., Zhu, C., Chesselet, M. F. & Hickey, M. A. Striatal atrophy and dendritic alterations in a knock-in mouse model of Huntington’s disease. Brain Res. Bull. 87, 571–578 (2012). source

      The Introduction (page unavailable) describes the progressive course of deficits in CAG140 mice, and Results §3.2 mentions similar dendritic changes in R6/2 mice. Neither passage compares phenotype severity or progression between these models, and the searches for R6/1, R6/2 progression, and an aggressive phenotype found no such comparison in our copy. (LLM, conf=0.73)

      Experimental — accuracy not yet measured
  • Findingown data

    GAPDH promotes vesicle clustering in a Drosophila EV biogenesis model.

    “Further studies in a Drosophila EV biogenesis model reveal that GAPDH is required for the normal generation of intraluminal vesicles in endosomal compartments, and promotes vesicle clustering.”
    • Also stated in: § GAPDH regulates exosome clustering, biogenesis and secretion in vivo · plain
      “Overexpression of human GAPDH specifically in adult SCs produced larger clusters of Btl-GFP-positive ILVs (Fig. 3b), and increased clustering of Btl-GFP puncta, representing secreted exosomes, in the AG lumen (Fig. 3c).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 45
  • May combine two claims. 30
  • The quote only partly states this claim. 3

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

    Premisecitation

    Expanded polyglutamine repeats of mutant huntingtin protein associate with GAPDH, potentially disrupting GAPDH-mediated trafficking of damaged mitochondria into the lysosomal system.

    “The expanded polyglutamine repeats of mutant huntingtin protein have been shown to associate with GAPDH, potentially disrupting GAPDH-mediated trafficking of damaged mitochondria into the lysosomal system31.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [31] Hwang, S., Disatnik, M. H. & Mochly-Rosen, D. Impaired GAPDH-induced mitophagy contributes to the pathology of Huntington’s disease. EMBO Mol. Med. 7, 1307–1326 (2015). source

  • May combine two claims.

    Premisecitation

    Current methods of loading therapeutic molecules into EVs are limited by low efficiency, toxicity and lack of scalability.

    “Current methods of loading therapeutic molecules into EVs such as electroporation, genetic engineering of host cells and chemical conjugation, are limited by low efficiency, toxicity and lack of scalability20,21.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [20] Kooijmans, S. A. A. et al. Electroporation-induced siRNA precipitation obscures the efficiency of siRNA loading into extracellular vesicles. J. Control Release 172, 229–238 (2013). source
    • cites [21] Smyth, T. et al. Surface functionalization of exosomes using click chemistry. Bioconjug Chem. 25, 1777–1784 (2014). source

      The Introduction (page unavailable) says a surface targeting moiety may be needed for efficient exosome delivery, but does not assess the efficiency of loading therapeutic molecules. The Discussion (page unavailable) describes click-chemistry surface functionalization; our copy does not address whether electroporation, host-cell engineering, or chemical conjugation are limited by loading efficiency, toxicity, or scalability. Searches for those methods and limitations found no supporting passage. (LLM, conf=0.72)

      Experimental — accuracy not yet measured

  • Classified as probably not a substantial claim.

    Premisecitation

    EVs have potential as cell-free therapeutics.

    “Given their unique biological and pharmacological characteristics, EVs have attracted interest as mediators of physiological and pathophysiological processes, and have potential as cell-free therapeutics4.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [4] Iraci, N., Leonardi, T., Gessler, F., Vega, B. & Pluchino, S. Focus on extracellular vesicles: physiological role and signalling properties of extracellular membrane vesicles. Int. J. Mol. Sci. 17, 171 (2016). source

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

    Premisecitation

    Newly formed ILVs from the limiting membrane of some MVBs are physically attached via electron dense tethers with an approximate width of ~12 nm.

    “it has recently been observed that newly formed intraluminal vesicles (ILVs) from the limiting membrane of some multi-vesicular bodies (MVBs), the compartments that produce exosomes, are physically attached to each other via electron dense tethers with an approximate width of ~12 nm36.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [36] Frankel, E. B. et al. Ist1 regulates ESCRT-III assembly and function during multivesicular endosome biogenesis in Caenorhabditis elegans embryos. Nat. Commun. 8, 1439 (2017). source

      The Results section, “ILVs remain physically connected following internalization” (page not provided), reports newly formed ILVs clustered near the limiting membrane and connected to one another by electron-dense tethers. The cited ~12 nm measurement is tether length (12 ± 5 nm), not width; the reported width is 18 ± 7 nm in zygotes. (LLM, conf=0.93)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Structural analysis of GAPDH identified NAD+-binding and catalytic domains of the protein.

    “Structural analysis of GAPDH identified NAD+-binding and catalytic domains of the protein58.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [58] White, M. R. et al. A dimer interface mutation in glyceraldehyde-3-phosphate dehydrogenase regulates its binding to AU-rich RNA. J. Biol. Chem. 290, 1770–1785 (2015). source

  • May combine two claims.

    Findingown data

    Formation and clustering of ILVs and DCG biogenesis in Rab11 compartments of secondary cells are regulated by GAPDH2 and appear functionally linked.

    “Overall, our data indicate that the formation and clustering of ILVs and DCG biogenesis in Rab11 compartments of SCs are regulated by GAPDH2 and appear to be functionally linked processes, a result supported by a recent analysis of ESCRT function in SCs47.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [47] Marie, P. P. et. al. Accessory ESCRT-III proteins selectively regulate Rab11-exosome biogenesis in Drosophila secondary cells. bioRxiv 2020.06.18.158725; https://doi.org/10.1101/2020.06.18.158725. source

  • May combine two claims.

    Findingown data

    Treatment of cells with chloroquine enhanced gene silencing efficiency to around 80%, consistent with release of entrapped siRNA from late-endosomes.

    “Further treatment of cells with chloroquine, an endosomolytic molecule50, enhanced gene silencing efficiency to around 80%, consistent with the release of entrapped siRNA from late-endosomes (Fig. 4c; Supplementary Fig. 7g and 7h).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [50] Hou, K. K., Pan, H., Ratner, L., Schlesinger, P. H. & Wickline, S. A. Mechanisms of nanoparticle-mediated siRNA transfection by melittin-derived peptides. ACS Nano 7, 8605–8615 (2013). source

  • May combine two claims.

    Findingown data

    GAPDH-GFP is predominantly associated with non-EV protein fractions, suggesting non-vesicular routes of GAPDH secretion.

    “Analysis of GAPDH-GFP fluorescence from EVs and non-EV protein fractions reflected predominant association of GAPDH-GFP in the non-EV protein fractions, suggesting the existence of non-vesicular routes of GAPDH secretion, consistent with reports from others (Fig. 2g)34,43.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [34] Chauhan, A. S. et al. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways. FASEB J. 33, 5626–5640 (2019). source
    • cites [43] Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445 e418 (2019). source

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

    Premisecitation

    p62 is an important regulatory protein of selective autophagy.

    “p62 is an important regulatory protein of selective autophagy, and a reduction in p62 aggregates in HD mouse models has previously been shown to restore HD-associated phenotypes56.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [56] Kurosawa, M. et al. Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington’s model mice. Hum. Mol. Genet. 24, 1092–1105 (2015). source

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

    Premisecitation

    A reduction in p62 aggregates in HD mouse models has previously been shown to restore HD-associated phenotypes.

    “p62 is an important regulatory protein of selective autophagy, and a reduction in p62 aggregates in HD mouse models has previously been shown to restore HD-associated phenotypes56.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [56] Kurosawa, M. et al. Depletion of p62 reduces nuclear inclusions and paradoxically ameliorates disease phenotypes in Huntington’s model mice. Hum. Mol. Genet. 24, 1092–1105 (2015). source

      The Discussion reports that genetic ablation of p62 ameliorated disease phenotypes in three HD mouse models and reduced nuclear inclusions. It does not report that reducing p62 aggregates restored the phenotypes: the reduced nuclear inclusions were of mutant huntingtin, and the reported phenotypes improved rather than fully recovered. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Huntington's disease is a neurodegenerative disease caused by mutation in the huntingtin gene that results in polyglutamine expansion in the protein, causing death of neurons.

    “HD is a neurodegenerative disease caused by mutation in the huntingtin gene that results in polyglutamine (poly Q) expansion in the protein, causing death of neurons55.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [55] Glorioso, J. C., Cohen, J. B., Carlisle, D. L., Munoz-Sanjuan, I. & Friedlander, R. M. Moving toward a gene therapy for Huntington’s disease. Gene Ther. 22, 931–933 (2015). source

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

    Premisecitation

    Endocytosed EVs in large endosomal compartments traffic towards the endoplasmic reticulum and finally sort into late-endosomes.

    “Recently a detailed study has demonstrated that endocytosed EVs in large endosomal compartments traffic towards the endoplasmic reticulum and finally sort into late-endosomes67.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [67] Heusermann, W. et al. Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER. J. Cell Biol. 213, 173–184 (2016). source

      The Results and discussion section reports that internalized exosomes travel within larger endocytic vesicles and move along the ER. It describes their subsequent sorting more specifically as movement to lysosomes, with increasing lysosomal colocalization over time but minimal colocalization with the multivesicular-body marker. Thus the claim's final destination, stated simply as late endosomes, is imprecise. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Interpretationcitation

    The MVBs involved in these studies may not be classical late-endosomes.

    “One common feature in these studies is that the MVBs involved may not be classical late-endosomes.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [36] Frankel, E. B. et al. Ist1 regulates ESCRT-III assembly and function during multivesicular endosome biogenesis in Caenorhabditis elegans embryos. Nat. Commun. 8, 1439 (2017). source
    • cites [64] Muratori, C. et al. Massive secretion by T cells is caused by HIV Nef in infected cells and by Nef transfer to bystander cells. Cell Host Microbe 6, 218–230 (2009). source

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

    Premisecitation

    Some studies have reported GAPDH within the EV lumen, while others have identified it on the EV outer surface.

    “Some studies have reported the presence of GAPDH within the EV lumen, but others have identified it on the EV’s outer surface33,62.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [33] Malhotra, H. et al. Exosomes: tunable nano vehicles for macromolecular delivery of transferrin and lactoferrin to specific intracellular compartment. J. Biomed. Nanotechnol. 12, 1101–1114 (2016). source
    • cites [62] Jella, K. K. et al. Exosomal GAPDH from proximal tubule cells regulate ENaC activity. PLoS ONE 11, e0165763 (2016). source

      The Results section ('Exosomes from LLC-PK1 cells contain active GAPDH'; page unavailable) reports GAPDH activity after exosomes were lysed and suggests that GAPDH may remain active inside them. It does not identify GAPDH on the EV outer surface, and the wording about its internal location is tentative. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

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

    Premisecitation

    Exosome clustering can be induced when the endolysosomal V-ATPase is inhibited.

    “Indeed, exosome clustering can be induced when the endolysosomal V-ATPase is inhibited”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [65] Edgar, J. R., Manna, P. T., Nishimura, S., Banting, G. & Robinson, M. S. Tetherin is an exosomal tether. Elife 5, 17180 (2016). source

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

    Premisecitation

    Exosome and DCG biogenesis are inter-dependent.

    “consistent with recent ESCRT knockdown experiments in SCs47, which suggest that exosome and DCG biogenesis are inter-dependent.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [47] Marie, P. P. et. al. Accessory ESCRT-III proteins selectively regulate Rab11-exosome biogenesis in Drosophila secondary cells. bioRxiv 2020.06.18.158725; https://doi.org/10.1101/2020.06.18.158725. source

      In 'Core ESCRTs control Rab11- and late endosomal exosome biogenesis' (page unavailable), the paper says dense-core granule formation and Rab11-exosome biogenesis appear functionally associated, and several ESCRT knockdowns affect both. It also says the link is not understood and that some knockdowns strongly suppress intraluminal vesicle formation without changing DCG number. The experiments therefore suggest an association, but do not establish that the two processes are inter-dependent. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

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

    Premisecitation

    In one recent study, GAPDH was identified in non-vesicular aggregates from small EV preparations and was not pulled-down using immunocapture with CD63 beads.

    “In one recent study, GAPDH was identified in non-vesicular aggregates from small EV preparations and was not pulled-down using immunocapture with magnetic beads coupled to CD63, employed in this analysis as a putative exosome marker43.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [43] Jeppesen, D. K. et al. Reassessment of exosome composition. Cell 177, 428–445 e418 (2019). source

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

    Premisecitation

    Clustered ILVs have been observed in other cell types in mammals and nematodes in the absence of DCGs and in specific pathological scenarios.

    “Indeed, clustered ILVs have been observed in other cell types in mammals and nematodes in the absence of DCGs and in specific pathological scenarios36,64.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [36] Frankel, E. B. et al. Ist1 regulates ESCRT-III assembly and function during multivesicular endosome biogenesis in Caenorhabditis elegans embryos. Nat. Commun. 8, 1439 (2017). source

      The Results section, “ILVs remain physically connected following internalization” (page unavailable), reports contacting, tethered ILVs in C. elegans hypodermal cells, supporting the nematode-cell observation. Our copy does not establish that DCGs were absent in those cells, and it does not report the claimed mammalian or pathological observations. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [64] Muratori, C. et al. Massive secretion by T cells is caused by HIV Nef in infected cells and by Nef transfer to bystander cells. Cell Host Microbe 6, 218–230 (2009). source

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

    Premisecitation

    GAPDH has proposed roles in membrane fusion that regulate secretion of other DCG compartments.

    “the proposed roles of GAPDH in membrane fusion, which have been shown to regulate secretion of other DCG compartments63.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [63] Han, X., Ramanadham, S., Turk, J. & Gross, R. W. Reconstitution of membrane fusion between pancreatic islet secretory granules and plasma membranes: catalysis by a protein constituent recognized by monoclonal antibodies directed against glyceraldehyde-3-phosphate dehydrogenase. Biochim. Biophys. Acta 1414, 95–107 (1998). source

      The Discussion (page not available in our copy) reports that a GAPDH-related protein catalyzes fusion of isolated pancreatic islet secretory granules with plasma membranes in vitro. It proposes a role in regulating insulin secretion, but does not show that GAPDH regulates secretion in cells or in vivo. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Premisecitation

    GAPDH is one of the top five most common proteins associated with EVs.

    “GAPDH is one of the top five most common proteins associated with EVs61.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [61] Kalra, H. et al. Vesiclepedia: A Compendium for Extracellular Vesicles with Continuous Community Annotation. PLOS Biology 10, e1001450 (2012). source

      The Vesiclepedia section describes a compendium of proteins identified in EVs, but does not name GAPDH or rank proteins by frequency. Searches for GAPDH, glyceraldehyde-3-phosphate dehydrogenase, and top-five EV proteins found no passage establishing the claimed ranking; page numbers were not available in our copy. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    For electron microscopy analysis of EVs, samples were prepared as per the protocol described previously.

    “For electron microscopy (EM) analysis of EVs, samples were prepared as per the protocol described previously72.”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [72] Thery, C., Amigorena, S., Raposo, G. & Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr. Protoc. Cell Biol. Unit 3, 22 (2006). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Currently, there are no successful drugs to cure Huntington's disease.

    “Currently, there are no successful drugs to cure the disease68.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [68] Ross, C. A. et al. Huntington disease: natural history, biomarkers and prospects for therapeutics. Nat. Rev. Neurol. 10, 204–216 (2014). source

  • May combine two claims.

    Premisecitation

    siRNAs located on either side of the EV membrane can induce gene knockdown, indicating both routes are possible.

    “the ability of siRNAs located on either side of the EV membrane to induce gene knockdown indicates both routes are possible15,16.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [15] Kamerkar, S. et al. Exosomes facilitate therapeutic targeting of oncogenic KRAS in pancreatic cancer. Nature 546, 498–503 (2017). source

      The Results describe electroporating siRNA into exosomes, and the Extended Data (Figure 4) report targeting of KRASG12D expression by siRNA-loaded exosomes. The Methods also describe mixing siRNA with exosomes as a control, but the passages do not establish that siRNA on the outside of the EV membrane induced knockdown. The claim that both locations are effective is therefore not supported in full. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [16] Gao, X. et al. Anchor peptide captures, targets, and loads exosomes of diverse origins for diagnostics and therapy. Sci. Transl. Med. 10, eaat0195 (2018). source

  • Classified as probably not a substantial claim.

    Premisecitation

    Reducing expression of mutant HTT gene using siRNA has emerged as a promising treatment.

    “Recently, reducing the expression of mutant HTT gene using siRNA has emerged as a promising treatment69.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [69] Harper, S. Q. et al. RNA interference improves motor and neuropathological abnormalities in a Huntington’s disease mouse model. Proc. Natl Acad. Sci. USA 102, 5820–5825 (2005). source

      The Abstract and Discussion (page numbers unavailable) support RNA interference as a promising way to reduce mutant htt expression: the authors report improved abnormalities in an HD mouse model. The tested treatment used AAV-delivered short hairpin RNA (shRNA), however, rather than siRNA as the citing sentence specifies. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • Rests on an assumption: Huntington's disease is caused by toxic gain of gene function.

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

    Study design and conductcitation

    Six-day-old adult virgin males were used throughout this study to ensure that age- and mating-dependent effects on SC biology were mitigated.

    “Six-day-old adult virgin males were used throughout this study to ensure that age- and mating-dependent effects on SC biology were mitigated44,45.”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [44] Fan, S. J. et al. Glutamine deprivation regulates the origin and function of cancer cell exosomes. EMBO J 39, 0261–4189 (2020). source
    • cites [45] Corrigan, L. et al. BMP-regulated exosomes from Drosophila male reproductive glands reprogram female behavior. J. Cell Biol. 206, 671–688 (2014). source

      Figure 5 describes analysis of SCs from 6-day-old virgin males. The Results section on exosome transfer also reports that older, multiply mated males can transfer SCs and says this paper used 4-day-old males for its mating experiments to avoid that complication. These passages support controlling age and mating history, but do not establish that using 6-day-old virgins throughout the citing study mitigates age- and mating-dependent effects on SC biology generally. (LLM, conf=0.83)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The lactoferrin N domain is physically associated with GAPDH in cells and on the surface of EVs.

    “Co-immunoprecipitation experiments of HEK293T cell lysates and EVs demonstrated that the lactoferrin N domain is physically associated with GAPDH in cells and on the surface of EVs (Supplementary Fig. 1d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Binding of GAPDH to EVs occurs for all cell sources tested and is not affected by serum proteins.

    “Binding of GAPDH to EVs occurred for all cell sources tested, and the presence of serum proteins in the tissue culture media did not affect binding of GAPDH to EVs (Fig. 1e & Supplementary Fig. 2b).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    An electron dense corona of GAPDH tethers EVs, with tether width around 13.54 ± 5.5 nm.

    “An electron dense corona of GAPDH was observed, tethering the EVs (Supplementary Fig. 2d). The width of this tether was found to be around 13.54 ± 5.5 nm (mean ± s.d, n = 30).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Four hours after systemic administration, ~1% of injected RVG-EVs were found in the brain of C57BL/6 mice, corresponding to ~4.0 × 1011 EVs for a 20 g mouse.

    “Four hours after administration, ~1% of injected EVs were found in the brain, which is ~4.0 × 1011 EVs for a 20 g of mice.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Substitution of R80 and K84 to alanine does not affect expression levels or glycolytic activity of GAPDH.

    “Substitution of R80 and K84 to alanine did not affect expression levels of the mtGAPDH protein produced, and it had similar glycolytic activity to wtGAPDH (Supplementary Fig. 3e–g).”
    Quote in the paperSubstantial claim

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

    Findingown data

    HA2 chimeric proteins could not be expressed due to toxicity of the peptide in E. coli producer cells.

    “HA2 chimeric proteins could not be expressed due to toxicity of the peptide in the E. coli producer cells.”
    Quote in the paperNot a substantial claim

  • The quote only partly states this claim.

    Findingown data

    Chloroquine enhances silencing of GAPDH protein by G58T and G58TF EVs in N2a cells.

    “Western blot showing effect of chloroquine (CQ) on silencing of GAPDH protein by G58T and G58TF EVs in N2a cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    Addition of chloroquine and/or G58TF peptide increased siRNA efficacy.

    “The addition of chloroquine and/or G58TF peptide increased siRNA efficacy”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Knockdown of the single GAPDH gene in human cells produced variable levels and excessive toxicity.

    “we found that the levels of knockdown of the single GAPDH gene in human cells were too variable and led to excessive toxicity (data not shown)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    When overexpressed, human GAPDH trafficked into endosomal compartments of SCs and induced vesicle clustering both inside and outside of these cells.

    “When overexpressed, human GAPDH trafficked into endosomal compartments of SCs and induced vesicle clustering both inside and outside of these cells”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingnone given

    GAPDH chimeric proteins retain the G58 EV-binding domain but do not induce EV clustering.

    “by engineering GAPDH chimeric proteins that have retained the G58 EV-binding domain, but do not induce EV clustering”
    Quote in the paperSubstantial claim

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

    Premisecitation

    GAPDH binds to the nuclear membrane via a conserved PS-binding domain located between amino acids 70-94.

    “GAPDH protein has been reported to bind to the nuclear membrane via a conserved PS-binding domain, located between amino acids 70–94 (Supplementary Fig. 2c)39.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Kaneda, M., Takeuchi, K., Inoue, K. & Umeda, M. Localization of the phosphatidylserine-binding site of glyceraldehyde-3-phosphate dehydrogenase responsible for membrane fusion. J. Biochem. 122, 1233–1240 (1997). source

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

    Premisecitation

    GAPDH becomes associated with exosomes during formation of intraluminal vesicles and exocytosis of exosomes is one pathway for secretion of GAPDH from cells.

    “Recently, it has been observed that GAPDH becomes associated with exosomes during formation of intraluminal vesicles and that exocytosis of exosomes represents one pathway for secretion of GAPDH from cells, a process that is enhanced when cells are depleted of iron34.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Chauhan, A. S. et al. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways. FASEB J. 33, 5626–5640 (2019). source

  • May combine two claims.

    Premisecitation

    GAPDH is one of the most abundant proteins associated with EVs and binds to the EV outer membrane.

    “GAPDH is one of the most abundant proteins associated with EVs, and has been reported to bind to the EV outer membrane, where it interacts with the iron-binding proteins transferrin and lactoferrin and traffics them into cells from tissue culture medium33.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [33] Malhotra, H. et al. Exosomes: tunable nano vehicles for macromolecular delivery of transferrin and lactoferrin to specific intracellular compartment. J. Biomed. Nanotechnol. 12, 1101–1114 (2016). source

  • May combine two claims.

    Interpretationreasoning

    One function of GAPDH-EV interaction is to promote clustering of exosomes, which appears to play an important role in Rab11-exosome biogenesis and organizing the large secretory compartments produced by Drosophila secondary cells.

    “One function of this interaction is to promote clustering of exosomes, which appears to play an important role in Rab11-exosome biogenesis and organizing the large secretory compartments produced by Drosophila secondary cells.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    TAT, HA2 and FHV peptides have previously been used to enhance the release of drugs from late-endosomes.

    “These peptides have previously been successfully used to enhance the release of drugs from late-endosomes48,49.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [48] Wadia, J. S., Stan, R. V. & Dowdy, S. F. Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nat. Med. 10, 310–315 (2004). source
    • cites [49] Nakase, I. et al. Cell-surface accumulation of flock house virus-derived peptide leads to efficient internalization via macropinocytosis. Mol. Ther. 17, 1868–1876 (2009). source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    The Amnis Cellstream facilitates detection of single fluorescent EVs.

    “By staining CD63-neon GFP-tagged EVs with APC-labelled anti-CD63 antibodies, we confirmed that the Amnis Cellstream facilitates detection of single fluorescent EVs (Fig. 2d).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The PS-binding domain is not required for binding to NADH or glyceraldehyde-3-phosphate, or for the catalytic activity of GAPDH.

    “the ability of specific amino acid changes to alter binding of GAPDH to the EV surface without affecting glycolytic activity of the enzyme suggests that the PS-binding domain is not required to mediate binding to NADH or glyceraldehyde-3-phosphate, or for the catalytic activity of the enzyme.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    GAPDH isolated from rabbit brain was found to induce fusion of synthetic liposomes but lacked glycolytic activity.

    “An enzymatic assay confirmed that GAPDH bound to the EV surface is catalytically active, which is in contrast to earlier reports where GAPDH isolated from rabbit brain was found to induce fusion of synthetic liposomes but lacked glycolytic activity37.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [37] Glaser, P. E. & Gross, R. W. Rapid plasmenylethanolamine-selective fusion of membrane bilayers catalyzed by an isoform of glyceraldehyde-3-phosphate dehydrogenase: discrimination between glycolytic and fusogenic roles of individual isoforms. Biochemistry 34, 12193–12203 (1995). source

  • May combine two claims.

    Interpretationown data

    G58T-functionalized EVs highly efficiently load and deliver siRNA into cells, resulting in potent gene silencing when combined with fusion of endosomolytic peptides to G58T protein.

    “Taken together, these results show highly efficient loading and delivery of siRNA into cells by G58T-functionalized EVs, resulting in potent gene silencing when combined with fusion of endosomolytic peptides to G58T protein.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    GAPDH2 knockdown specifically affects ILV clustering and biogenesis.

    “confirming that GAPDH2 knockdown specifically affects ILV clustering and biogenesis.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    The slow progression of disease phenotypes in Q140 mice makes it difficult to determine morphological and phenotypic changes of HD symptoms in treated mice.

    “Moreover, the slow progression of disease phenotypes in Q140 mice makes it difficult to determine morphological and phenotypic changes of HD symptoms in treated mice.”
    Quote in the paperNot a substantial claim

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

    Interpretationcitation

    V-ATPase inhibition may suggest a requirement for more neutral pH.

    “which may suggest a requirement for more neutral pH”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The dsRBD of TARBP2 protects siRNA from degradation by nucleases and significantly reduces activation of the innate immune response to siRNA compared to naked siRNA.

    “a previous siRNA binding study using the dsRBD of TARBP2 protein has shown that this domain protects siRNA from degradation by nucleases and significantly reduces activation of the innate immune response to siRNA when compared to naked siRNA”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [66] Dar, G. H., Gopal, V. & Rao, M. Conformation-dependent binding and tumor-targeted delivery of siRNA by a designed TRBP2: affibody fusion protein. Nanomedicine 11, 1455–1466 (2015). source

  • Classified as probably not a substantial claim.

    Interpretationown data

    The presence of two Drosophila GAPDH isoforms may have been critical in allowing definition of this enzyme's specific roles in exosome clustering and biogenesis.

    “In our analysis, the presence of two Drosophila GAPDH isoforms may have been critical in allowing us to define this enzyme’s specific roles in exosome clustering and biogenesis.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    GAPDH is secreted on exosomes and, through interaction with transferrin and lactoferrin, can act as a sponge permitting uptake of free iron.

    “This GAPDH is secreted on exosomes and through its interaction with transferrin and lactoferrin can act as a sponge, which permits the uptake of free iron.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [34] Chauhan, A. S. et al. Trafficking of a multifunctional protein by endosomal microautophagy: linking two independent unconventional secretory pathways. FASEB J. 33, 5626–5640 (2019). source

  • The quote only partly states this claim.

    Interpretationown data

    The reduction of p62 aggregates highlights the therapeutic potential of this approach in HD treatment.

    “highlights the therapeutic potential of our approach in the treatment of HD.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Huntington's disease is caused by toxic gain of gene function.

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

    Study design and conductnone given

    18 S rRNA and HPRT mRNA were used as reference genes for normalization.

    “18 S rRNA and Hypoxanthine-guanine phosphoribosyl transferase (HPRT) mRNA was used as reference genes for normalization.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The accessory glands were maintained in a small drop of PBS surrounded by oxygen-diffusible hydrocarbon oil and secured with a cover glass to avoid dehydration and hypoxia during imaging.

    “To avoid dehydration and hypoxia, the glands were carefully maintained in a small drop of PBS, surrounded by 10 S Voltalef (VWR Chemicals), an oxygen-diffusible hydrocarbon oil, and kept stably in place by the application of a small cover glass (VWR).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Six-week-old female C57BL/6 mice were randomly assigned into four groups (n = 3).

    “Six-week-old female C57BL/6 mice were randomly assigned into four groups (n = 3).”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The immunoblot for HTT protein in brain tissues could not be analyzed due to high background.

    “However, we could not analyze the immunoblot due to high background.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    PCR efficiencies were determined using LinRegPCR.

    “PCR efficiencies were determined using LinRegPCR.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). The quote only partly states this claim.

    Study design and conductnone given

    Identical microscope settings and equipment were used throughout imaging of living SCs.

    “Identical microscope settings and equipment were used throughout.”
    • Also stated in: § Animal experiments · plain
      “Living SCs from 6-day-old males of each genotype were imaged using identical settings by wide-field microscopy.”
    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 conductown data

    The purified GAPDH protein was analysed by SDS-PAGE and its size was confirmed by MALDI-TOF-MS.

    “The protein was analysed by SDS-PAGE, and the size of the protein was confirmed by mass spectrometry, using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    All data are representative from at least two to three independent experiments.

    “All data are representative from at least two to three independent experiments.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    For B16-F10, SKOV-3 and MSCs cells, alix protein was used as a positive control for proteins residing in the lumen of the vesicles.

    “For B16-F10, SKOV-3 and MSCs cells, alix protein was used as a positive control for proteins residing in the lumen of the vesicles.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The modular design can be employed for delivery of other types of cargoes, including guide RNAs, proteins and peptides, and for targeting EVs to other specific cells or tissues.

    “the modular design employed for the delivery of other types of cargoes, ranging from different types of nucleic acids including guide RNAs, to proteins and peptides, and for targeting EVs to other specific cells or tissues”
    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 distribution of residuals was tested for normality using Q-Q plots and the appropriate statistical test was applied.

    “The distribution of residuals was tested for normality using Q–Q plots and the appropriate statistical test was applied.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All animal experiments were performed in compliance with the Animals (Scientific Procedures) Act 1986, revised 2012 (ASPA, UK).

    “All animal experiments were performed in compliance with the Animals (Scientific Procedures) Act 1986, revised 2012 (ASPA, UK).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Small EVs were defined as SSC (low) by using neon GFP-tagged EVs as biological reference material, and regions to quantify fluorescence-positive populations were set according to unstained samples and single-stained controls.

    “Small EVs were defined as SSC (low) by using neon GFP-tagged EVs as biological reference material and regions to quantify fluorescence-positive populations were set according to unstained samples and single-stained controls.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All NTA measurements were carried out in triplicate.

    “All NTA measurements were carried out in triplicate.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    Purified G58 chimeric proteins were analysed by SDS-PAGE and MALDI-TOF-MS.

    “Purified proteins were analysed by SDS-PAGE and MALDI-TOF-MS, as described above.”
    Quote in the paperNot a substantial claim
67 of 67

  • The paper relies on this without support.

    The observed ~40% silencing of Htt in the cortex and reduction of p62 inclusion bodies in Q140 mice are sufficient to indicate therapeutic promise for Huntington's disease.

    “Taken together, our study demonstrated a promising application of G58-coupled EVs in delivery of siRNA to brain with the potential for pre-clinical and clinical development.”

  • The paper relies on this without support.

    G58-coupled EVs are safe for pre-clinical and clinical development.

    “Taken together, our study demonstrated a promising application of G58-coupled EVs in delivery of siRNA to brain with the potential for pre-clinical and clinical development.”

  • The paper relies on this without support.

    The reduction in p62 inclusion bodies observed in treated Q140 mice is caused by the siRNA-mediated silencing of Htt.

    “Analysis of brain tissues revealed almost 40% silencing of the Htt gene in mouse brain cortex and a highly significant decrease of p62 inclusion bodies in the cortical neurons of the treated animals, an important phenotypic HD marker of disease progression (Fig. 4e, f).”

  • The paper relies on this without support.

    The siRNA mixture targeting different regions of the Htt exon silences mutant Htt without affecting wild-type Htt.

    “In this study, using a mixture of siRNA targeting different regions of the Htt exon of Q140 mice, we systemically administered a total of four doses, once per week.”

  • The paper relies on this without support.

    Huntington's disease is caused by toxic gain of gene function.

    “Huntington’s (HD) disease is a progressive neurological disorder that is caused due to toxic gain of gene function.”

  • The authors state this as an assumption.

    Clustered ILVs play a role in stabilizing a large DCG in SCs.

    “Our current model is that clustered ILVs play a role in stabilizing a large DCG in SCs46”

  • The paper relies on this without support.

    Slow progression of disease symptoms (>12 months) in Q140 mice makes it difficult to rapidly assess behavioural and phenotypic changes of disease pathology in this model.

    “However, slow progression of disease symptoms (>12 months) in Q140 mice makes it difficult to rapidly assess the behavioural and phenotypic changes of disease pathology in this model.”

  • The paper relies on this without support.

    The GAPDH gene encodes a PS-binding domain (designated G58).

    “The DNA sequence of GAPDH gene, which encodes the PS-binding domain (designated as G58), was fused with the second domain of the human TAR RNA-binding protein”

  • The paper relies on this without support.

    The PS-binding domain of GAPDH lies between amino acids 70 and 90.

    “(PS-binding domain lies between 70–90 amino acids)”

  • The paper relies on this without support.

    In the Q140 mouse model, exon 1 of mouse Htt is humanized and contains 140 CAG repeats, and the mice have a slow progression of disease phenotype starting at 6 months.

    “In the Q140 mouse model, exon 1 of mouse Htt is humanized and contains 140 CAG repeats. The mice have a slow progression of disease phenotype, which starts to appear at the age of 6 months.”
10 of 10

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.

  • Fig. 4d identifies different siRNA-loaded EV constructs

    Minor

    The Fig. 4d caption identifies the siRNA-loaded condition as G58T/siRNA-RVG-EVs, whereas the results describe the condition shown in Fig. 4d as G58TF/siRNA bound to RVG-EVs.

  • Conflicting incubation times for the Fig. 4a uptake image

    Minor

    The Fig. 4a caption says the N2a cells were incubated for 4 hours, while the confocal microscopy method says the labelled EV-siRNA complexes were incubated with the cells for 6 hours.

2 of 2

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Authors
Dar, G. H., Mendes, C. C., Kuan, W.-L., Speciale, A. A., Conceição, M., Görgens, A., Uliyakina, I., Lobo, M. J., Lim, W. F., EL Andaloussi, S., Mäger, I., Roberts, T. C., Barker, R. A., Goberdhan, D. C. I., Wilson, C., & Wood, M. J. A.
Affiliations
  • University of Oxford (GB)
  • University of Cambridge (GB)
  • Karolinska Institutet (SE)
Corresponding author
  • Wilson, Clive
  • Wood, Matthew J. A.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2021-11-18
Volume
12
Issue
1
Pages
6666-6666
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-021-27056-3
PMC
PMC8602309
OpenAlex
W3214573095
Semantic Scholar
e641c9032457a49fd4bb061ccbec61c483b39680
Cited by
100
References
95
Citations by year
2026: 11 · 2025: 25 · 2024: 23 · 2023: 21 · 2022: 16 · 2021: 2 · 2020: 2
FWCI
4.16
Type
journal article
Language
en
Update notices
correction (2021-12-16)
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Apollo (University of Cambridge) (submittedVersion, cc-by)
  • Apollo
  • Apollo (cc-by)
Funders
RCUK | Medical Research Council (MR/M007715/1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
glyceraldehyde-3-phosphate dehydrogenase, extracellular vesicles, vesicle biogenesis, siRNA delivery, brain drug delivery, vesicle clustering, intraluminal vesicles, endosomes, Huntington's disease, Drosophila model, systemic injection, drug carriers
MeSH terms
Extracellular Vesicles, Huntingtin Protein, Animals, Brain, Disease Models, Animal, Glyceraldehyde-3-Phosphate Dehydrogenases, HeLa Cells, Humans, Huntington Disease, Mice, Inbred C57BL, Phosphatidylserines, Protein Binding, Drug Delivery Systems, RNA, Small Interfering, Cell Line, Tumor, Mice, HEK293 Cells, Mesenchymal Stem Cells

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 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

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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