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Cited by 60Virus-based gene therapy researchRNA Interference and Gene DeliveryBarrier Structure and Function Studies

Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes

Open access
von Jonquieres, Georg, Fröhlich, Dominik, Klugmann, Claudia B., Wen, Xin, Harasta, Anne E., Ramkumar, Roshini, Spencer, Ziggy H. T., Housley, Gary D. +1 more·2016·Frontiers in Molecular Neuroscience
DOI 10.3389/fnmol.2016.00013PMC PMC4763065
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Abstract

Leukodystrophies are hereditary central white matter disorders caused by oligodendrocyte dysfunction. Recent clinical trials for some of these devastating neurological conditions have employed an ex vivo gene therapy approach that showed improved endpoints because cross-correction of affected myelin-forming cells occurred following secretion of therapeutic proteins by transduced autologous grafts. However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins. Recombinant adeno-associated viral (AAV) vectors are versatile tools for gene transfer to the central nervous system (CNS) and proof-of-concept studies in rodents have shown that the use of cellular promoters is sufficient to target AAV-mediated transgene expression to glia. The potential of this strategy has not been exploited. The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters. Here, we characterize the human myelin associated glycoprotein (MAG) promoter for reliable targeting of AAV-mediated transgene expression to oligodendrocytes in vivo. A homology screen revealed highly conserved genomic regions among mammalian species upstream of the transcription start site. Recombinant AAV expression cassettes carrying the cDNA encoding enhanced green fluorescent protein (GFP) driven by truncated versions of the recombinant MAG promoter (2.2, 1.5 and 0.3 kb in size) were packaged as cy5 vectors and delivered into the dorsal striatum of mice. At 3 weeks post-injection, oligodendrocytes, neurons and astrocytes expressing the reporter were quantified by immunohistochemical staining. Our results revealed that both 2.2 and 1.5 kb MAG promoters targeted more than 95% of transgene expression to oligodendrocytes. Even the short 0.3 kb fragment conveyed high oligodendroglial specific transgene expression (>90%) in vivo. Moreover, cy5-MAG2.2-GFP delivery to the neonate CNS resulted in selective GFP expression in oligodendrocytes for at least 8 months. Broadly, the characterization of the extremely short yet oligodendrocyte-specific human MAG promoter may facilitate modeling neurological diseases caused by oligodendrocyte pathology and has translational relevance for leukodystrophy gene therapy.

AI snapshot

Written from the paper’s full text.

TL;DR

The human myelin-associated glycoprotein (MAG) promoter was characterized for AAV-mediated transgene expression and selectively targeted oligodendrocytes in the mouse CNS. Even a truncated 0.3 kb MAG promoter drove high oligodendroglial specificity in adult striatum, and neonatal cy5-MAG2.2-GFP delivery sustained oligodendroglial expression for at least 8 months.

Key findings

  • In adult mouse striatum at 3 weeks after cy5-AAV delivery, MAG2.2, MAG1.5, and MAG0.3 promoters restricted GFP expression to oligodendrocytes at 98.4 ± 0.8%, 96.0 ± 0.9%, and 90.7 ± 2.1%, respectively; neuronal and astrocytic GFP expression was low.
  • Neonatal cy5-MAG2.2-GFP delivery resulted in oligodendrocyte-enriched GFP expression at 8 months: 82.2 ± 0.4% of GFP+ cells were oligodendrocytes, versus 10.3 ± 1.4% neurons and 7.6 ± 1.4% astrocytes; 32.8 ± 2.8% of all oligodendrocytes expressed GFP.
  • In an Mbp promoter comparison, adult striatal oligodendroglial GFP expression was 91.0 ± 1.5% for rh39, 87.0 ± 5.1% for cy5, 78.2 ± 4.4% for AAV1/2, and 78.0 ± 1.7% for rh20; rh39 and cy5 did not differ significantly.
  • In vitro, MAG promoter constructs produced low GFP expression in HEK 293 cells and GFP was below immunoblot detection in Oli-neu cells, but single-cell GFP immunoreactivity was seen in Oli-neu cells with oligodendrocyte morphology.

Methods

  • Bioinformatics identified conserved human MAG promoter regions and transcription factor binding sites; AAV plasmids expressed GFP under 2.2, 1.5, or 0.3 kb human MAG promoter fragments, packaged as cy5 vectors, with AAV1/2, rh20, and rh39 used for Mbp promoter comparison.
  • Adult C57BL/6J mice (2–4 months, both sexes) and neonatal mice (P0) received intrastriatal injections of 1 µl AAV-GFP at 2 × 10^12 vg/ml (2 × 10^9 vg); brains were analyzed at 3 weeks or 8 months by immunohistochemistry for GFP with ASPA (oligodendrocytes), NeuN (neurons), and GFAP (astrocytes).
  • GFP+ cell identity was quantified by counting at least 50 cells from each of three non-adjacent sections for at least 150 GFP+ cells; statistics used one-way or two-way ANOVA with Tukey’s post hoc test, with values as mean ± SEM.

Limitations

  • The paper states that a direct comparison between MAG and Mbp promoter transduction profiles was not feasible, that neonatal delivery yielded only a moderate total number of GFP-expressing oligodendrocytes, and that GFAP heterogeneity precluded some astroglial analysis.
  • Evident from the text: adult experiments used n = 3 and neonatal long-term experiments used n = 4, analyses focused on the dorsal striatum, and no disease model, therapeutic outcome, self-complementary AAV testing, or Schwann cell activity was evaluated.

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

Integrity signals

Registry

Crossref · 10.3389/fnmol.2016.00013

Cites

45 of 45 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

20 of 41 cited sources checked · 8 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    The proximal 300 bp human MAG promoter fragment contains evolutionarily conserved binding sites for MRF and RNF10.

    “Finally, we also selected the proximal 300 bp fragment for further characterization as it contains evolutionary conserved binding sites for myelin gene regulatory factor (MRF) and Ring Finger Protein 10 (RNF10), critical activators of myelination in oligodendrocytes or Schwann cells, respectively (Hoshikawa et al., 2008; Emery et al., 2009; Bujalka et al., 2013).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites Hoshikawa et al., 2008 source
    • cites Emery et al., 2009 source

      The Discussion (page unavailable) identifies MRF as a regulator required for oligodendrocyte maturation and CNS myelination. It does not establish that the proximal 300 bp human MAG promoter contains conserved MRF or RNF10 binding sites; the binding-site claim is unsupported by this source. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites Bujalka et al., 2013 source

      The Results section, “MYRF Binds DNA Via a Defined Consensus Sequence” (page not provided), identifies a MYRF-binding motif near Mag and reports that mutating it abolished MYRF-dependent reporter activity. The cited passages do not establish that this site lies within the proximal 300 bp of the human MAG promoter, that it is evolutionarily conserved, or that the fragment contains an RNF10-binding site. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
  • Findingcitation

    The distal segment of the 2.2 kb human MAG promoter fragment contains binding sites for YY1 and Id4.

    “The distal segment of this fragment contained binding sites for both positive and negative transcriptional regulators such as Yin Yang 1 (YY1) and Inhibitor of DNA Binding 4 (Id4; Marin-Husstege et al., 2006; He et al., 2007; Zolova and Wight, 2011).”
    Quote in the paperNot a substantial claim1 of 3 sources checked
    • cites Marin-Husstege et al., 2006 source
    • cites He et al., 2007

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

    • cites Zolova and Wight, 2011 source

      The Results section identifies a YY1-binding motif in the mouse Plp1 promoter, not in a human MAG promoter fragment. Searches for MAG, Id4, and a 2.2 kb promoter fragment found no passage addressing the claimed binding sites; the paper’s findings about Plp1 do not establish them for MAG. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
  • Premisecitation

    Preclinical proof-of-concept studies in rodents have shown that using cellular promoters can target AAV-mediated transgene expression to glia.

    “Preclinical proof-of-concept studies showing successful modification of the AAV system towards selective transgene expression in oligodendrocytes used “neurotropic” serotypes but employed the promoter of the mouse myelin basic protein (Mbp) gene (Chen et al., 1998; Lawlor et al., 2009; von Jonquieres et al., 2013).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Chen et al., 1998 source
    • cites Lawlor et al., 2009 source
    • cites von Jonquieres et al., 2013 source
  • Premisecitation

    To date, the only cellular promoter used for AAV-mediated transgene expression in oligodendrocytes is the murine 1.9 kb Mbp cis-regulating element and a truncated version spanning the proximal 1.3 kb upstream region including parts of Mbp exon 1.

    “To date, the only cellular promoter used for AAV-mediated transgene expression in oligodendrocytes is the murine 1.9 kb Mbp cis-regulating element and a truncated version spanning the proximal 1.3 kb upstream region including parts of Mbp exon 1 (Chen et al., 1998; Lawlor et al., 2009; von Jonquieres et al., 2013).”
    Quote in the paperSubstantial claim1 of 3 sources checked
    • cites Chen et al., 1998 source
    • cites Lawlor et al., 2009 source
    • cites von Jonquieres et al., 2013 source

      The Discussion (page unavailable) says there was no appropriately sized, oligodendrocyte-specific alternative to the 1.3 kb and 1.9 kb MBP promoter variants used in AAV studies. Materials and Methods identifies the longer promoter as mouse MBP. Our copy does not establish that the 1.3 kb variant spans the proximal upstream region or includes part of exon 1. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Premisecitation

    Somatic transgenesis in the mouse, achieved by vector delivery at P0, is instrumental for disease modeling.

    “However, somatic transgenesis in the mouse, achieved by vector delivery at P0, is instrumental for disease modeling (Dayton et al., 2012).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Dayton et al., 2012 source

      Section 9 describes somatic vector-based gene transfer as a way to model neurodegenerative disease, and Section 3 describes neonatal mouse AAV9 delivery as useful for mammalian modeling. Neither passage specifies delivery at P0 or establishes that P0 delivery in mice is the disease-modeling approach described; the paper’s specific disease-model examples in Section 9 are largely in rats. Page numbers were not available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • May combine two claims. 24
  • Classified as probably not a substantial claim. 13
  • The quote only partly states this claim. 4

  • May combine two claims.

    Findingcitation

    The proximal 1.5 kb human MAG promoter fragment lacks YY1 and Id4 motifs but contains binding sites for Olig1 and Sox10 and an evolutionarily highly conserved sequence.

    “We therefore decided to also investigate the proximal 1.5 kb of the long fragment since this fragment was devoid of these motifs but contained binding sites for myelin cell lineage factors Oligodendrocyte Transcription Factor 1 (Olig1) and SRY-Box 10 (Sox10) and an evolutionary highly conserved sequence (Wang et al., 2014).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Wang et al., 2014 source

      The Results discuss a conserved SOX10 enhancer, not the human MAG promoter. Searches for the 1.5 kb MAG promoter fragment, YY1 and Id4 motifs, and Olig1/Sox10 binding sites found no passage addressing the claimed promoter features. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The mouse Mbp promoter, despite promise, has caveats of murine origin, relatively large size, and poor specificity after neonatal vector delivery.

    “While the Mbp promoter holds promise for potential clinical applications, its murine origin, relative big size and poor specificity following neonatal vector delivery, are potential caveats (von Jonquieres et al., 2013).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites von Jonquieres et al., 2013 source

      The Materials and Methods identify the promoter as a 1.94 kb sequence from the mouse mbp gene, and the Discussion reports substantial MBP-promoter leakiness after neonatal delivery. The paper discusses AAV size limits, but does not characterize the MBP promoter’s size or murine origin as clinical caveats; it calls the MBP variants appropriately sized. (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

    GFAP is generally regarded as pan-astrocytic but is heterogeneously expressed with a bias towards white matter and reactive astrocytes.

    “GFAP is a marker, generally regarded as pan-astrocytic. However, recent evidence has shown that GFAP is heterogeneously expressed in the astroglial compartment with a bias towards white matter and reactive astrocytes (Cahoy et al., 2008).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Cahoy et al., 2008 source

      The sections on cell-type-specific markers and Aldh1L1 (page not provided) say GFAP labels only some astrocytes and is more prominent in white matter than gray matter, supporting heterogeneous expression and the white-matter bias. Our copy does not establish the claimed bias toward reactive astrocytes. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

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

    Study design and conductcitation

    Transduced astrocytes were identified by GFAP presence and morphological criteria because GFAP is heterogeneously expressed in the astroglial compartment.

    “GFAP is a marker, generally regarded as pan-astrocytic. However, recent evidence has shown that GFAP is heterogeneously expressed in the astroglial compartment with a bias towards white matter and reactive astrocytes (Cahoy et al., 2008). Therefore, transduced astrocytes were identified based on the presence of GFAP and morphological criteria.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Cahoy et al., 2008 source

      The ‘Identification of new cell-type-specific markers’ section says GFAP is not uniformly expressed in astrocytes and is preferentially expressed in white matter; the Aldh1L1 marker section likewise reports that GFAP labels only some astrocytes. The passages do not establish a bias toward reactive astrocytes. The citing authors’ use of GFAP and morphology to identify transduced cells is their own method, not a finding attributed to Cahoy et al. (LLM, conf=0.84)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Vector spread depends on the developmental stage of the target tissue at the time of infusion but not on the cellular promoter used in the expression cassette.

    “We have shown previously that the vector spread depends on the developmental stage of the target tissue at the time of infusion but not on the cellular promoter used in the expression cassette (von Jonquieres et al., 2013).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites von Jonquieres et al., 2013 source

  • May combine two claims.

    Premisecitation

    cy5 is a variant of AAV7 that, based on extremely weak immunogenicity, has been proposed to have strong potential as a clinical gene therapy vector.

    “For all other experiments we focused on cy5 as it is a variant of AAV7 that, based on its extremely weak immunogenicity, has been proposed to have a strong potential to be developed as a clinical gene therapy vector (Gao et al., 2002).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites Gao et al., 2002 source

      The Discussion (page not available) proposes AAV7 for further development as a human gene-therapy vector, citing its favorable muscle tropism and relative lack of preexisting immunity. The paper does not identify cy5 as an AAV7 variant, and its finding of low preexisting antibody reactivity does not establish that cy5 has extremely weak immunogenicity. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    ASPA is a marker of mature oligodendrocytes, and ASPA+ cells overlap 100% with GSTπ and 93% with CC1.

    “ASPA is a marker of mature oligodendrocytes. In this population, ASPA positive cells have been reported to overlap 100 and 93% with the widely used oligodendrocyte soma markers glutathione S-transferase π isoform and CC1, respectively (Kawai et al., 2009).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites Kawai et al., 2009 source

      The Results section reports that ASPA marks mature oligodendrocytes and that ASPA-positive cells overlapped with almost 100% of GSTπ-positive cells and 93 ± 4% of CC1-positive cells in three adult mice. The citing sentence makes the first figure an exact 100% and omits the uncertainty around the second. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    In HEK 293 cells, MAG promoter-driven constructs produced low-level GFP expression, whereas CAG- and Mbp-driven constructs produced robust GFP expression.

    “Low level GFP expression was detectable following transfection with the MAG promoter-driven constructs. In contrast, the control plasmids in which reporter gene transcription was driven by the CAG or Mbp promoter produced robust GFP expression.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Another conserved DNA stretch is located between −1315 and −1176 in the human MAG promoter.

    “Another conserved DNA stretch was located between −1315 to −1176.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    rh39-Mbp-GFP yielded a higher percentage of GFP+ oligodendrocytes (91.0 ± 1.5%) than AAV1/2-Mbp-GFP (78.2 ± 4.4%) and rh20-Mbp-GFP (78.0 ± 1.7%), p<0.05.

    “Two-way analysis of variance (ANOVA) with Tukey’s post hoc test revealed differences in oligodendroglial GFP expression between AAV1/2 (78.2 ± 4.4%) and rh39 (91.0 ± 1.5%; p < 0.05) as well as rh20 (78.0 ± 1.7%) and rh39 (p < 0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Following cy5-MAG1.5-GFP injection into the dorsal striatum of adult mice, GFP expression in neurons was 2.8 ± 0.5% and was significantly less than in oligodendrocytes.

    “Expression in neurons (2.8 ± 0.5%) and astrocytes (1.3 ± 0.6%) was significantly less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Following cy5-MAG1.5-GFP injection into the dorsal striatum of adult mice, GFP expression in astrocytes was 1.3 ± 0.6% and was significantly less than in oligodendrocytes.

    “Expression in neurons (2.8 ± 0.5%) and astrocytes (1.3 ± 0.6%) was significantly less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In adult mouse striatum, AAV1/2-Mbp-GFP and rh20-Mbp-GFP produced highly preferential oligodendroglial GFP expression, with rh39 and cy5 exceeding them.

    “We found that AAV1/2 and rh20 resulted in highly preferential oligodendroglial GFP expression, exceeded by rh39 and cy5.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Compared with the CAG promoter, the 1.9 kb Mbp promoter showed weaker yet robust GFP expression in HEK 293 cells.

    “Compared with the CAG promoter, the 1.9 kb Mbp promoter showed weaker yet robust GFP expression in HEK 293 cells.”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Following neonatal cy5-MAG2.2-GFP delivery, GFP expression in astrocytes was 7.6 ± 1.4% and was significantly less than in oligodendrocytes.

    “Expression in neurons (10.3 ± 1.4%) and astrocytes (7.6 ± 1.4%) was significantly less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Following cy5-MAG0.3-GFP injection into the dorsal striatum of adult mice, GFP expression in astrocytes was 6.8 ± 2.4% and was significantly less than in oligodendrocytes.

    “Expression in neurons (2.5 ± 1.7%) and astrocytes (6.8 ± 2.4%) was significantly less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All three recombinant MAG promoters showed very little activity in HEK 293 cells despite good transfection efficacy confirmed to be greater than 80% in CAG-GFP samples.

    “All three recombinant MAG promoters showed very little activity in HEK 293 cells despite good transfection efficacy confirmed to be greater than 80% in CAG-GFP samples.”
    Quote in the paperSubstantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Following neonatal cy5-MAG2.2-GFP delivery, GFP expression in neurons was 10.3 ± 1.4% and was significantly less than in oligodendrocytes.

    “Expression in neurons (10.3 ± 1.4%) and astrocytes (7.6 ± 1.4%) was significantly less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    GFP in the striatum of neonatally injected animals was predominantly found in oligodendrocytes, but also in some neurons and astrocytes.

    “GFP in the striatum of animals injected neonatally was predominantly found in oligodendrocytes, but also in some neurons and astrocytes (Figure 7B).”
    Quote in the paperSubstantial claim

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

    Findingnone given

    A similar trend was observed in neonatally infused animals that were analyzed after 3 weeks.

    “A similar trend was observed in neonatally infused animals that were analyzed after 3 weeks (not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims. The quote only partly states this claim.

    Findingown data

    Following cy5-MAG0.3-GFP injection into the dorsal striatum of adult mice, GFP expression in neurons was 2.5 ± 1.7% and was significantly less than in oligodendrocytes.

    “Expression in neurons (2.5 ± 1.7%) and astrocytes (6.8 ± 2.4%) was significantly less.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    MAG is a pre-myelinating marker responsible for oligodendroglial recognition of axons and myelin maintenance.

    “In the CNS, myelin-associated glycoprotein (MAG) is a pre-myelinating marker responsible for oligodendroglial recognition of axons and myelin maintenance (Martini and Schachner, 1997).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Martini and Schachner, 1997 source

  • May combine two claims.

    Premisecitation

    The AAV vector system has a small DNA packaging limit (4.7-5 kb), which motivates the use of small recombinant promoters.

    “The major restriction of the system is the small DNA packaging limit of 4.7 kb (Dong et al., 1996).”
    • Also stated in: § Abstract · plain
      “The major caveat of the AAV system is its limited packaging capacity of ~5 kb, providing the rationale for identifying small yet selective recombinant promoters.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dong et al., 1996 source

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

    Premisenone given

    Direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins.

    “However, direct gene transfer to oligodendrocytes is required for the majority of leukodystrophies with underlying mutations in genes encoding non-secreted oligodendroglial proteins.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    The Mbp promoters have been used independently to achieve reporter gene expression in the adult rodent CNS using AAV2, AAV8, rh39, rh20 and cy5.

    “The Mbp promoters have been used independently to achieve reporter gene expression in the adult rodent CNS using AAV2, AAV8, rh39, rh20 and cy5 (Chen et al., 1998; Lawlor et al., 2009).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Chen et al., 1998 source
    • cites Lawlor et al., 2009 source

  • May combine two claims.

    Interpretationown data

    The extremely short yet oligodendrocyte-specific human MAG promoter may facilitate modeling neurological diseases caused by oligodendrocyte pathology and has translational relevance for leukodystrophy gene therapy.

    “Broadly, the characterization of the extremely short yet oligodendrocyte-specific human MAG promoter may facilitate modeling neurological diseases caused by oligodendrocyte pathology and has translational relevance for leukodystrophy gene therapy.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    A recent pioneering study has confirmed very good vector spread following cy5-CAG-GFP delivery to the adult rodent brain, but the effects of cellular promoters were not examined.

    “While a recent pioneering study has confirmed very good vector spread following cy5-CAG-GFP delivery to the adult rodent brain the effects of cellular promoters were not examined (Lawlor et al., 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Lawlor et al., 2009 source

  • Classified as probably not a substantial claim.

    Premisecitation

    The 2,3-cyclic nucleotide 3-phosphodiesterase promoter has been reported to convey oligodendroglial-specific GFP reporter gene expression following lentiviral delivery to the neonatal mouse brain.

    “Recently, the 2,3-cyclic nucleotide 3-phosphodiesterase promoter, first described in transgenic mice (Gravel et al., 1998), has been reported to convey oligodendroglial-specific GFP reporter gene expression following lentiviral delivery to the neonatal mouse brain (Kagiava et al., 2014).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Gravel et al., 1998 source
    • cites Kagiava et al., 2014 source

  • Classified as probably not a substantial claim.

    Study design and conductown data

    Cell-type specificity of AAV-mediated transgene expression was quantified by counting at least 50 cells from each of three non-adjacent sections, totaling at least 150 GFP+ cells.

    “The percentage of GFP-expressing cells per cell-type was determined by counting at least 50 cells from each of three non-adjacent sections for a total of at least 150 GFP+ cells using the “cell counter” plugin for ImageJ version 1.45 k (NIH).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    AAV-mediated transgene expression has peaked to stable levels by 3 weeks after vector injection.

    “At 3 weeks following vector injection, when AAV-mediated transgene expression has peaked to stable levels (Klugmann et al., 2005a), animals were killed and the brains assessed by immunohistochemistry to detect GFP in immuno-identified neurons, oligodendrocytes, and astrocytes.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Klugmann et al., 2005a source

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

    Premiseown data

    The Mbp promoter exhibits mostly astroglial activity following injection at P0.

    “We have previously reported that the Mbp promoter exhibits mostly astroglial activity following injection at P0.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Self-complementary AAV vectors inherently possess only half the packaging capacity yet have a superior transduction profile compared with single-stranded vectors.

    “self-complementary (sc) AAV vectors that inherently possess only half the packaging capacity yet have a superior transduction profile compared with single-stranded vectors (McCarty, 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites McCarty, 2008 source

  • May combine two claims.

    Interpretationreasoning

    The MAG promoter has superior features considering its human origin, small size and oligodendroglial selectivity in adults and neonates.

    “In conclusion, the MAG promoter has superior features considering its human origin, small size and oligodendroglial selectivity in adults and neonates.”
    Quote in the paperSubstantial claim
33 of 33

  • The paper relies on this without support.

    The potential of the promoter-swapping strategy for oligodendrocyte-targeted AAV transgene expression has not been exploited.

    “The potential of this strategy has not been exploited.”

  • The paper relies on this without support.

    AAV is the preferred vector platform for CNS gene delivery due to minimal immune response, episomal localization, and long-term transgene expression.

    “Recombinant adeno-associated virus (AAV) is the preferred vector platform for gene delivery to the central nervous system (CNS) due to its minimal potential to elicit immune response, episomal localization of the vector genome and long-term transgene expression.”

  • The paper relies on this without support.

    There is virtually no size limitation for myelin gene promoters used in transgenic mouse lines.

    “There is virtually no size limitation for myelin gene promoters used in transgenic mouse lines.”

  • The paper relies on this without support.

    Only a few small cellular promoters have been described to reliably drive transgene expression in oligodendrocytes in genetically modified animals.

    “Only a few small cellular promoters have been described to reliably drive transgene expression in oligodendrocytes in genetically modified animals.”

  • The paper relies on this without support.

    To date there is no oligodendrocyte-specific alternative to the 1.3 and 1.9 kb Mbp promoter variants appropriate for AAV viral gene delivery.

    “To date there is no oligodendrocyte-specific alternative to the 1.3 and 1.9 kb Mbp promoter variants appropriate for AAV viral gene delivery.”

  • The paper relies on this without support.

    This is the first report on a MAG cis-acting element to drive transgene expression in vivo.

    “To our best knowledge, this is the first report on a MAG cis-acting element to drive transgene expression in vivo.”

  • The paper relies on this without support.

    AAV-mediated gene activation in the CNS takes several days as it requires uncoating, nuclear translocation and second-strand synthesis of the ssAAV genome.

    “AAV-mediated gene activation in the CNS takes several days as it requires uncoating, nuclear translocation and second-strand synthesis of the ssAAV genome.”
7 of 7

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?

  • A short ~300-bp fragment of the myelin-associated glycoprotein promoter can drive high levels of transgene expression in a chimeric AAV1/2 vector without occupying an impractical proportion of available packaging space.

    “von Jonquieres et al.24 recently published a very nice study demonstrating the ability of a short ∼300-bp fragment of the myelin-associated glycoprotein promoter to drive high levels of transgene expression in a chimeric AAV1/2 vector without occupying an impractical proportion of available packaging space”

    From Preclinical biodistribution, tropism, and efficacy of oligotropic AAV/Olig001 in a mouse model of congenital white matter disease (2021) · cites this paper as [24]

    The Methods (Plasmid Constructs; page unavailable) describe a 0.3-kb MAG promoter, and the Discussion (page unavailable) says its small size makes it suitable even for self-complementary AAV packaging. The Results (Specificity of MAG Promoter Driven cy5 Vectors; page unavailable) report GFP expression with that promoter, but the constructs tested in vivo were packaged in cy5 vectors, not chimeric AAV1/2. The paper establishes strong oligodendrocyte targeting, rather than quantifying the claimed high expression levels in AAV1/2. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • A prominent OL role in AMN pathogenesis is supported by the results of a gene-therapy study that used an adeno-associated vector carrying the human ABCD1 gene driven by the myelin-associated glycoprotein promoter.

    “A prominent OL role in AMN pathogenesis is supported by the results of a gene-therapy study that used an adeno-associated vector (AAV) carrying the human ABCD1 gene driven by the myelin-associated glycoprotein (MAG) promoter [289].”

    From Revisiting the Pathogenesis of X-Linked Adrenoleukodystrophy (2025) · cites this paper as [289]

    The Abstract and Materials and Methods (Plasmid Constructs; no page numbers provided) describe AAV vectors using the human MAG promoter to drive GFP expression in oligodendrocytes. They do not describe an AAV carrying human ABCD1 or report gene-therapy results in AMN, so the claimed support for an oligodendrocyte role in AMN pathogenesis is not established by this study. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

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.

  • Current-assembly coordinates cannot contain the stated 5 kb upstream region

    Minor

    The stated GRCh38 interval spans only 2,185 bp, so it cannot contain a 5 kb upstream promoter region, let alone that region plus exons and introns.

  • Neonatal MAG selectivity is reported as 90% instead of 82.2%

    Minor

    The discussion uses 90% for oligodendrocyte-targeted expression in neonates, but the neonatal experiment reports 82.2 ± 0.4%. This is the value used in the discussion's comparison with the Mbp promoter.

2 of 2

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Authors
von Jonquieres, G., Fröhlich, D., Klugmann, C. B., Wen, X., Harasta, A. E., Ramkumar, R., Spencer, Z. H. T., Housley, G. D., & Klugmann, M.
Affiliations
UNSW Sydney (AU)
Corresponding author
Klugmann, Matthias
ORCID
Venue
Frontiers in Molecular Neuroscience
Publisher
Frontiers Media SA
Published
2016-02-23
Volume
9
Pages
13-13
ISSN
1662-5099 (ISSN-L)
DOI
10.3389/fnmol.2016.00013
PMC
PMC4763065
OpenAlex
W2316908236
Semantic Scholar
761bda60da653ef4f5b10f2abcd78ff1a645f733
Cited by
60
References
47
Citations by year
2026: 3 · 2025: 11 · 2024: 8 · 2023: 2 · 2022: 8 · 2021: 8 · 2020: 6 · 2019: 6 · 2018: 2 · 2017: 3 · 2016: 2
FWCI
3.19
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Frontiers in Molecular Neuroscience (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Europe PMC (PubMed Central) (submittedVersion, cc-by)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Health and Medical Research Council (APP1050277)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
Keywords
adeno-associated virus vectors, leukodystrophy, ex vivo gene therapy, GFP reporter

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.

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