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Cited by 41Virus-based gene therapy researchRNA Interference and Gene DeliveryNeurogenetic and Muscular Disorders Research

Preclinical biodistribution, tropism, and efficacy of oligotropic AAV/Olig001 in a mouse model of congenital white matter disease

Open access
Francis, Jeremy S., Markov, Vladimir, Wojtas, Irenuez D., Gray, Steve, McCown, Thomas, Samulski, R. Jude, Figueroa, Marciano, Leone, Paola·2021·Molecular Therapy - Methods & Clinical Development
DOI 10.1016/j.omtm.2021.01.009PMC PMC7878967
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Abstract

Recent advances in adeno-associated viral (AAV) capsid variants with novel oligotropism require validation in models of disease in order to be viable candidates for white matter disease gene therapy. We present here an assessment of the biodistribution, tropism, and efficacy of a novel AAV capsid variant (AAV/ Olig001) in a model of Canavan disease. We first define a combination of dose and route of administration of an AAV/Olig001-GFP reporter conducive to widespread CNS oligodendrocyte transduction in acutely symptomatic animals that model the Canavan brain at time of diagnosis. Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements. Intracerebroventricular infusion into the cerebrospinal fluid (CSF) was identified as the most appropriate route of administration and employed for delivery of an AAV/Olig001 vector to reconstitute oligodendroglial aspartoacylase (ASPA) in adult Canavan mice, which resulted in a dose-dependent rescue of ASPA activity, motor function, and a near-total reduction in vacuolation. A head-to-head efficacy comparison with astrogliotropic AAV9 highlighted a significant advantage conferred by oligotropic AAV/Olig001 that was independent of overall transduction efficiency. These results support the continued development of AAV/Olig001 for advancement to clinical application to white matter disease.

AI snapshot

Written from the paper’s full text.

TL;DR

AAV/Olig001 retains oligotropism in symptomatic nur7 mice, with intracerebroventricular (i.c.v.) delivery giving the best spread and highest total transduction. In a head-to-head comparison, AAV/Olig001-ASPA produced greater dose-responsive rescue of motor function and vacuolation than AAV9-ASPA despite lower brain vector genome copy number.

Key findings

  • i.c.v. was the most effective route: total GFP-positive cells were 1,104,256 ± 106,817 for i.c.v. versus 841,366 ± 121,723 for intraparenchymal (IP), 815,487 ± 106,980 for intracisterna magna (ICM), and 742,143 ± 79,497 for intrathecal (IT).
  • AAV/Olig001 showed >70% oligotropism in all regions except cerebellum; subcortical white matter was >90% oligotropic across all routes, while cerebellum was only ~10% Olig2-positive and >80% NeuN-positive.
  • AAV/Olig001-GFP versus AAV9-GFP in striatum: 75% versus 19% oligodendroglial colabeling, and 4% versus 65% GFAP colabeling.
  • At the highest ASPA dose (2.5 × 10^11 vg), AAV/Olig001-ASPA-treated mice were indistinguishable from wild-type on rotarod, whereas AAV9-ASPA mice had reduced latency at 18 and 22 weeks; repeated-measures ANOVA versus sham was p = 0.028 for AAV/Olig001 and p = 0.551 for AAV9. Thalamic vacuolation was reduced 13-fold by AAV/Olig001 versus 12-fold by AAV9 at high dose, and whole-brain NAA was reduced 2.6-fold versus 2-fold.

Methods

  • Six-week-old nur7 mice received AAV/Olig001-GFP at 1 × 10^10, 1 × 10^11, or 5 × 10^11 vg via IP, IT, i.c.v., or ICM routes (n = 5 per dose/route); brains were analyzed by unbiased stereology using the optical fractionator for GFP-positive soma and by Olig2/NeuN/GFAP colabeling for tropism.
  • For efficacy, 6-week-old nur7 mice received i.c.v. AAV/Olig001-ASPA or AAV9-ASPA at 2.5 × 10^11, 7.5 × 10^10, or 2.5 × 10^10 vg (n = 12/group); outcomes included rotarod at 10, 14, 18, and 22 weeks, qPCR vector genome copies, western blot for ASPA, HPLC for whole-brain NAA, and H&E-based vacuole volume fraction.
  • BrdU labeling was used in 2- and 8-week-old wild-type and nur7 mice to assess proliferating oligodendrocyte progenitors/immature oligodendrocytes.

Limitations

  • The paper states that commercially available antibodies could not reliably detect ASPA transgene-positive cells in vivo, preventing side-by-side analysis of transgene distribution and tropism between AAV/Olig001-ASPA and AAV9-ASPA.
  • The paper notes that Olig2 can also be detected in developing white matter tract astrocytes, so the reported 31% transduction of resident SCWM oligodendrocytes may overstate oligotropism; it also states that efficacy data are preliminary and that the cerebellum was refractory to oligodendroglial transduction, with Purkinje neurons dominating.
  • Evident from the text: the study uses a constitutive CBh promoter active in all cell lineages, so non-oligodendrocyte expression is possible; the tropism comparison with AAV9 is described as “far from comprehensive,” and findings are from a single mouse model of Canavan disease.

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

Integrity signals

Registry

Crossref · 10.1016/j.omtm.2021.01.009

Cites

27 of 27 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

15 of 32 cited sources checked · 10 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    AAV/Olig001 utilizes a capsid with preferential tropism for the oligodendrocyte cell surface.

    “This engineered variant, AAV/Olig001, utilizes a capsid with preferential tropism for the oligodendrocyte cell surface”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [3] Powell, 2016, Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism, Gene Ther. source
  • Premisecitation

    AAV/Olig001 has been shown to partially rescue the phenotype in a mouse model of lysosomal storage disease.

    “AAV/Olig001 has been shown to partially rescue the phenotype in a mouse model of lysosomal storage disease”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [9] Karumuthil-Melethil, 2016, Intrathecal administration of AAV/GALC vectors in 10-11-day-old twitcher mice improves survival and is enhanced by bone marrow transplant, J. Neurosci. Res. source
  • Premisecitation

    nur7 mice present evidence of attempted remyelination in the brain in the form of increased markers of immature oligodendrocytes as late as several weeks of age.

    “nur7 mice present evidence of attempted remyelination in the brain in the form of increased markers of immature oligodendrocytes as late as several weeks of age”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [11] Traka, 2008, Nur7 is a nonsense mutation in the mouse aspartoacylase gene that causes spongy degeneration of the CNS, J. Neurosci. source
    • cites [12] Francis, 2012, Aspartoacylase supports oxidative energy metabolism during myelination, J. Cereb. Blood Flow Metab. source
  • Premisecitation

    The capsid-cell surface interaction is the primary determinant of AAV tropism.

    “Although it is possible to utilize promoter elements that limit transgene expression to specific cellular lineages, the capsid-cell surface interaction is the primary determinant of AAV tropism.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [1] Chao, 2000, Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors, Mol. Ther. source
    • cites [2] Davidsson, 2019, A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism, Proc. Natl. Acad. Sci. USA source

      In Results, ‘Validation of the BRAVE Screening Principle’ (page unavailable), removing a capsid motif that binds a cell-surface proteoglycan eliminated HEK293T-cell tropism, and a modified capsid restored it. This supports a role for capsid–cell-surface interactions, but the paper does not establish that they are the primary determinant of AAV tropism relative to other factors. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
  • Premisecitation

    AAV/Olig001 has been shown to transduce oligodendrocytes in vivo in healthy rodents and nonhuman primates with upward of 90% oligotropism.

    “A very limited number of studies have shown the ability of AAV/Olig001 to transduce oligodendrocytes in vivo in healthy rodents3,8 and nonhuman primates8 with upward of 90% oligotropism.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [3] Powell, 2016, Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism, Gene Ther. source
    • cites [8] Mandel, 2017, Novel oligodendroglial alpha synuclein viral vector models of multiple system atrophy: studies in rodents and nonhuman primates, Acta Neuropathol. Commun. source
  • May combine two claims. 49
  • Classified as probably not a substantial claim. 14

  • May combine two claims.

    Premisecitation

    The nur7 mouse is an (ENU)-induced mutant that has a nonsense point mutation in the ASPA gene, resulting in the absence of functional protein and chronically elevated CNS NAA.

    “The nur7 mouse is an (ENU)-induced mutant that has a nonsense point mutation in the ASPA gene, resulting in the absence of functional protein and chronically elevated CNS NAA.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Traka, 2008, Nur7 is a nonsense mutation in the mouse aspartoacylase gene that causes spongy degeneration of the CNS, J. Neurosci. source

      The Results section identifies nur7 as an ENU-induced nonsense mutation in Aspa and reports no detectable Aspa protein in mutant mice. It also reports elevated NAA in several CNS regions at postnatal day 21, but does not establish that NAA remains elevated chronically; the NAA analysis described in Methods used mice at that single age. Page numbers are unavailable in our copy. (LLM, conf=0.85)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    nur7 pathology is progressive, with deteriorating motor function and CNS cell loss manifest from 2 to 3 weeks of age onward.

    “nur7 pathology is progressive, with deteriorating motor function and CNS cell loss manifest from 2 to 3 weeks of age onward.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [11] Traka, 2008, Nur7 is a nonsense mutation in the mouse aspartoacylase gene that causes spongy degeneration of the CNS, J. Neurosci. source

      The Results sections on early pathology and disease progression report vacuoles at P14, oligodendrocyte loss in affected CNS regions at P21, and worsening pathology with age. They do not establish deteriorating motor function from 2–3 weeks onward: motor impairment was measured at P42–P56, while severe ataxia is described at later stages. Page numbers are unavailable in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    AAV/Olig001 can transduce neonatal oligodendrocytes in a mouse model of the congenital leukodystrophy Canavan disease, resulting in rescue of the congenital genetic defect and resistance to disease.

    “We have previously shown AAV/Olig001 capable of transducing neonatal oligodendrocytes in a mouse model of the congenital leukodystrophy Canavan disease, resulting in rescue of the congenital genetic defect and resistance to disease.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [4] Francis, 2016, N-acetylaspartate supports the energetic demands of developmental myelination via oligodendroglial aspartoacylase, Neurobiol. Dis. source

  • Classified as probably not a substantial claim.

    Premisecitation

    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”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [24] von Jonquieres, 2016, Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes, Front. Mol. Neurosci. source

      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

  • Classified as probably not a substantial claim.

    Premisecitation

    Chronically elevated NAA in Canavan disease is proposed to directly cause gross pathology and vacuolation.

    “Chronically elevated NAA in Canavan disease is proposed to directly cause gross pathology and vacuolation”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [22] Maier, 2015, N-Acetylaspartate Synthase Deficiency Corrects the Myelin Phenotype in a Canavan Disease Mouse Model But Does Not Affect Survival Time, J. Neurosci. source
    • cites [23] Guo, 2015, Ablating N-acetylaspartate prevents leukodystrophy in a Canavan disease model, Ann. Neurol. source

  • May combine two claims.

    Findingown data

    The cortex of ICM brains manifest 76% GFP colabeling with Olig2 and 17.4% with NeuN.

    “whereas the cortex of ICM brains manifest 76% GFP colabeling with Olig2 and 17.4% with NeuN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Nonoligodendrocyte transgene expression was attributable in the main to neuronal expression, with very few astrocytes observed expressing GFP in either of the 4 ROA cohorts (<5%).

    “Nonoligodendrocyte transgene expression was attributable in the main to neuronal expression, with very few astrocytes observed expressing GFP in either of the 4 ROA cohorts (<5%).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Approximately 75% of the difference in overall GFP-positive cell numbers between i.c.v. and IP cohorts (∼262,891) was accounted for by SCWM (35%) and striatal (36%) ROIs, which manifest >80% oligotropism in both ROA cohorts.

    “Approximately 75% of the difference in overall GFP-positive cell numbers between i.c.v. and IP cohorts (∼262,891) was accounted for by SCWM (35%) and striatal (36%) ROIs, which manifest >80% oligotropism in both ROA cohorts.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The i.c.v. ROA resulted in the highest number of total GFP-positive cells (sum of all ROI counts in individual brains), which were 1.3-fold more than the next-ranked ROA, IP (p = 0.0067).

    “The i.c.v. ROA resulted in the highest number of total GFP-positive cells (sum of all ROI counts in individual brains), which were 1.3-fold more than the next-ranked ROA, IP (p = 0.0067).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In IT ROA brains, 75.5% of cortical GFP-positive cells colabeled with Olig2 and 20.2% with NeuN.

    “In IT ROA brains, 75.5% of cortical GFP-positive cells colabeled with Olig2 and 20.2% with NeuN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The efficiency of transduction of the cerebellum was comparable across all IP, IT, and i.c.v. ROAs, but ICM administration resulted in the highest numbers of transduced cerebellar cells (ICM versus i.c.v., p = 0.045).

    “The efficiency of transduction of the cerebellum was comparable across all IP, IT, and i.c.v. ROAs (Figure 2D), but ICM administration resulted in the highest numbers of transduced cerebellar cells (ICM versus i.c.v., p = 0.045).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Intrasample variance (coefficient of error [CE]) for IP brains was ∼12% of total variance, whereas that for i.c.v. brains was ∼3%, meaning GFP-positive cells were more evenly distributed across all sections sampled in i.c.v. brains.

    “Intrasample variance (coefficient of error [CE]) for IP brains was ∼12% of total variance, whereas that for i.c.v. brains was ∼3%, meaning GFP-positive cells were more evenly distributed across all sections sampled in i.c.v. brains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Although IP and i.c.v. ROA brains were comparable in absolute numbers of cells transduced by AAV/Olig001-GFP in specific regions, the bulk of positive cell counts in IP brains was found in sections immediately adjacent to injection sites, whereas positive cells in i.c.v. brains were found relatively evenly distributed throughout all sections sampled.

    “Although IP and i.c.v. ROA brains were comparable in absolute numbers of cells transduced by AAV/Olig001-GFP in specific regions, the bulk of positive cell counts in IP brains was found in sections immediately adjacent to injection sites, whereas positive cells in i.c.v. brains were found relatively evenly distributed throughout all sections sampled”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    IT and ICM ROA were both disappointingly inefficient at transducing SCWM cells.

    “IT and ICM ROA were both disappointingly inefficient at transducing SCWM cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The i.c.v. ROA efficiently transduced cells in the striatum, resulting in higher numbers of striatal GFP-positive soma than any other ROA.

    “The i.c.v. ROA efficiently transduced cells in the striatum (Figure 2C), resulting in higher numbers of striatal GFP-positive soma than any other ROA”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The difference in oligotropism manifest among the 4 different ROAs was small, but the IP ROA did present with a significant increase in NeuN colabeling and slight but significant reductions in Olig2-colabeling relative to the other 3 ROAs.

    “The difference in oligotropism manifest among the 4 different ROAs was small, but the IP ROA did present with a significant increase in NeuN colabeling (p = 0.0043 versus IT; p = 0.0119 versus i.c.v.; p = 0.00059 versus ICM) that coincided with slight but significant reductions in Olig2-colabeling relative to the other 3 ROAs (p = 0.026 versus IT; p = 0.048 versus i.c.v.; p = 0.0085 versus ICM)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Numbers of cells in IP ROA brains were not significantly increased over either ICM (p = 0.730) or IT numbers (p = 0.165).

    “Numbers of cells in IP ROA brains were not significantly increased over either ICM (p = 0.730) or IT numbers (p = 0.165)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    i.c.v.- and IP-transduced brains gave the highest and second-highest numbers of transduced white matter tract cells, respectively.

    “i.c.v.- and IP-transduced brains gave the highest and second-highest numbers of transduced white matter tract cells, respectively”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    On the whole, all four ROAs generated comparable results, with inherent AAV/Olig001 oligotropism largely intact.

    “On the whole, all four ROAs generated comparable results, with inherent AAV/Olig001 oligotropism largely intact.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cortical GFP-positive cell numbers in i.c.v. brains were identical to that in IP brains, but vg copy number was significantly higher (p = 0.05).

    “Similarly, cortical GFP-positive cell numbers in i.c.v. brains were identical to that in IP brains, but vg copy number was significantly higher (p = 0.05)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the cortex, nonsignificant differences in total GFP-positive cells via all ROAs contrasted with significant differences in vg copy number between some ROAs.

    “In the cortex, nonsignificant differences in total GFP-positive cells via all ROAs (Figure 2A) contrasted with significant differences in vg copy number between some ROAs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Marked differences in transduction of cells within SCWM of the corpus callosum and external capsule were evident, with both the IT and ICM ROA being relatively poor in this ROI.

    “marked differences in transduction of cells within SCWM of the corpus callosum and external capsule were evident with both the IT and ICM ROA being relatively poor in in this ROI.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    For all ROAs, an increase in transduced cells was evident when dose was increased from 1 × 1010 to 1 × 1011 doses, but the highest 5 × 1011 dose resulted in only modest additional increases for all ROAs, indicating a ceiling effect in most ROIs.

    “For all ROAs, an increase in transduced cells was evident when dose was increased from 1 × 1010 to 1 × 1011 doses in all ROAs, but the highest 5 × 1011 dose resulted in only modest additional increases for all ROAs, indicating a ceiling effect in most ROIs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements.

    “Administration of AAV/Olig001-GFP resulted in >70% oligotropism in all regions of interest except the cerebellum without the need for lineage-specific expression elements.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cortical GFP-positive cell numbers in IT brains were not significantly different from the same in IP brains, but vg per milligram of cortical tissue in IT brains was significantly greater than vg per milligram of cortical tissue in IP brains (p = 0.042).

    “Cortical GFP-positive cell numbers in IT brains were not significantly different from the same in IP brains, but vg per milligram of cortical tissue in IT brains was significantly greater than vg per milligram of cortical tissue in IP brains (p = 0.042)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The average 1.9 × 104 GFP-positive SCWM cells in the ICM group were 14-fold less than i.c.v. brains (p = 0.000083) and in the IT group 4-fold less (p = 0.0001).

    “The average 1.9 × 104 GFP-positive SCWM cells in the ICM group were 14-fold less than i.c.v. brains (p = 0.000083) and in the IT group 4-fold less (p = 0.0001).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the cortex, 62.3% of all GFP-positive cells colabeled with Olig2 and 35.1% with NeuN in IP brains.

    “In the cortex, 62.3% of all GFP-positive cells colabeled with Olig2 and 35.1% with NeuN in IP brains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    i.c.v. ROA brains presented with 70.8% oligotropism and 23.6% neurotropism in the cortex.

    “i.c.v. ROA brains presented with 70.8% oligotropism and 23.6% neurotropism in the cortex”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    All 4 of IP, IT, i.c.v., and ICM ROAs resulted in >90% oligotropism, <6% neurotropism, with no significant differences in percent colabeling with either antigen.

    “All 4 of IP, IT, i.c.v., and ICM ROAs resulted in >90% oligotropism, <6% neurotropism, with no significant differences in percent colabeling with either antigen”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At 18 and 22 weeks, AAV/Olig001-ASPA mean latencies to fall were significantly greater than AAV9-ASPA animals (p = 0.00018 and p = 0.027, respectively).

    “At the two latter time points (18 and 22 weeks), when sham nur7 motor function was severely compromised, AAV/Olig001-ASPA mean latencies to fall were significantly greater than AAV9-ASPA animals (p = 0.00018 and p = 0.027, respectively).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AAV/Olig001-ASPA-treated animals were indistinguishable from wild-type reference controls at all ages, whereas AAV9-ASPA-treated animals presented significantly reduced latency to fall relative to wild-type at 18 and 22 weeks of age.

    “However, AAV/Olig001-ASPA-treated animals were indistinguishable from wild-type reference controls at all ages, whereas AAV9-ASPA-treated animals presented significantly reduced latency to fall relative to wild-type at 18 and 22 weeks of age”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Counts of BrdU-positive cells in the corpus callosum and external capsule of genotype cohorts at each age revealed a significant 1.8-fold increase in BrdU-positive cells in 2-week-old nur7 brains relative to wild type (p = 0.029) and a 1.6-fold increase in nur7 brains at 8 weeks (p = 0.034).

    “Counts of BrdU-positive cells in the corpus callosum and external capsule of genotype cohorts at each age revealed a significant 1.8-fold increase in BrdU-positive cells in 2-week-old nur7 brains relative to wild type (p = 0.029) and a 1.6-fold increase in nur7 brains at 8 weeks (p = 0.034).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cerebellar transduction was dominated by large Purkinje neurons in the granule cell layer, with relatively sporadic oligodendrocyte transgene expression in white matter tracts.

    “Cerebellar transduction was dominated by large Purkinje neurons in the granule cell layer, with relatively sporadic oligodendrocyte transgene expression in white matter tracts.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cortical transduction was spread from 18% to 25%, with the ICM ROA resulting in the highest levels of oligodendrocyte transduction.

    “Cortical transduction was spread from 18% to 25%, with the ICM ROA resulting in the highest levels of oligodendrocyte transduction.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AAV/Olig001-GFP promoted oligodendroglial expression of GFP in 75% of all transgene-positive cells, whereas AAV9-GFP promoted oligodendroglial GFP expression in only 19% of all transgene-positive cells, a nearly 4-fold difference (p = 1.64 × 10−5).

    “Although AAV/Olig001-GFP promoted oligodendroglial expression of GFP in 75% of all transgene-positive cells, AAV9-GFP promoted oligodendroglial GFP expression in only 19% of all transgene-positive cells, a nearly 4-fold difference (p = 1.64 × 10−5).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The cerebellum presented with starkly contrasting tropism for all 4 ROAs, with a mere 10% of all GFP-positive cells colabeling with Olig2 and over 80% colabeling with NeuN.

    “The cerebellum presented with starkly contrasting tropism for all 4 ROAs, with a mere 10% of all GFP-positive cells colabeling with Olig2 and over 80% colabeling with NeuN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The percent resident oligodendrocytes transduced was dose responsive for each ROA but with the increase from 1 × 1011 to 5 × 1011 dose being very modest.

    “The percent resident oligodendrocytes transduced was dose responsive for each ROA but with the increase from 1 × 1011 to 5 × 1011 dose being very modest.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    AAV/Olig001-ASPA brains manifest greater reduction in NAA than AAV9-ASPA, but the difference failed to reach statistical significance at any dose.

    “AAV/Olig001-ASPA brains manifest the greater reduction in NAA, but this reduction failed to reach statistical significance at any dose”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Stereological estimates of Olig2-positive cells in 8-week-old naive nur7 mice were mean 183,095 in cortex, 913,428 in SCWM, 136,252 in striatum, and 344,651 in cerebellum.

    “Mean: 183,095 | mean: 913,428 | mean: 136,252 | mean: 344,651”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Striatal tropism was also comparable for all 4 ROAs, with >80% of all GFP-positive cells colabeling with Olig2 and <20% with NeuN.

    “Striatal tropism was also comparable for all 4 ROAs, with >80% of all GFP-positive cells colabeling with Olig2 and <20% with NeuN.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At lowest dose (2.5 × 10^10), only AAV/Olig001-ASPA treatment resulted in significantly increased latency to fall, and only at 18- and 22-week time points.

    “At the lowest dose administered (2.5 × 1010), only AAV/Olig001-ASPA treatment resulted in significantly increased latency to fall and only at the 18- and 22-week time points”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AAV/Olig001 was still able to improve profoundly gross pathology in thalamus, midbrain, and cerebellum and to a significantly greater degree than AAV9.

    “it was still able to improve profoundly gross pathology in these regions and to a significantly greater degree than seen with AAV9 (Figure 7C)”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    In humans, Canavan disease manifests in early postnatal life, with diagnosis typically occurring after key neurodevelopmental milestones have been missed.

    “In humans, Canavan disease manifests in early postnatal life, with diagnosis typically occurring after key neurodevelopmental milestones have been missed”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Janson, 2006, Natural history of Canavan disease revealed by proton magnetic resonance spectroscopy (1H-MRS) and diffusion-weighted MRI, Neuropediatrics source

  • May combine two claims.

    Interpretationown data

    The vg difference suggests superior cortical diffusion via the IP ROA, despite resulting in approximately one-half the number of available vgs.

    “suggesting superior cortical diffusion via the IP ROA, despite resulting in approximately one-half the number of available vgs.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Unbiased stereology was used to assess effects of dose and route of administration on vector biodistribution and tropism of an AAV/Olig001-GFP reporter vector.

    “Unbiased stereology was used to assess effects of dose and route of administration (ROA) on vector biodistribution and tropism of an AAV/Olig001-GFP reporter vector”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Canavan disease is caused by inherited mutations in the gene encoding for the enzyme aspartoacylase (ASPA), which results in the accumulation of its substrate, N-acetylaspartate (NAA).

    “Canavan disease is caused by inherited mutations in the gene encoding for the enzyme aspartoacylase (ASPA)10 which results in the accumulation of its substrate, N-acetylaspartate (NAA).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Kaul, 1993, Cloning of the human aspartoacylase cDNA and a common missense mutation in Canavan disease, Nat. Genet. source

  • May combine two claims.

    Study design and conductreasoning

    These estimates of Olig2 cell number are absolute and independent of volume density, meaning bias in possible differences in tissue volume produced by GFP staining in AAV/Olig001-GFP cohorts is avoided.

    “These estimates of Olig2 cell number are absolute and independent of volume density, meaning bias in possible differences in tissue volume produced by GFP staining in AAV/Olig001-GFP cohorts is avoided.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    SCWM in Canavan patients presents with severe abnormalities, such as a swollen aspect, and progressive fractional anisotropic abnormalities, as documented by diffusion tensor imaging (DTI).

    “SCWM in Canavan patients presents with severe abnormalities, such as a swollen aspect, and progressive fractional anisotropic abnormalities, as documented by diffusion tensor imaging (DTI).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Janson, 2006, Natural history of Canavan disease revealed by proton magnetic resonance spectroscopy (1H-MRS) and diffusion-weighted MRI, Neuropediatrics source

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

    Study design and conductnone given

    Both serotypes were self-complimentary systems, able to package pseudo double-stranded genomes.

    “and both were self-complimentary systems, able to package pseudo double-stranded genomes”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Commercially available antibodies were unable to reliably detect ASPA transgene-positive cells in vivo, precluding side-by-side analysis.

    “Unfortunately, commercially available antibodies were unable to reliably detect ASPA transgene-positive cells in vivo, precluding a side-by-side analysis of transgene distribution and tropism of AAV/Olig001-ASPA and AAV9-ASPA.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Numbers of recombinant vgs were performed by real-time qPCR using an in-house-designed and -validated TaqMan probe/primer set targeted to the BGH poly(A) sequence of vgs.

    “Numbers of recombinant vgs in transduced animals were performed by real-time qPCR using an in-house-designed and -validated TaqMan probe/primer set targeted to the BGH poly(A) sequence of vgs.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All of AAV/Olig001-GFP, AAV/Olig001-ASPA, and AAV9-ASPA contained an identical BGH poly(A) sequence.

    “All of AAV/Olig001-GFP, AAV/Olig001-ASPA, and AAV9-ASPA contained an identical BGH poly(A) sequence.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All procedures involving animals were conducted under approved institutional guidelines.

    “all procedures involving animals were conducted under approved institutional guidelines”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    A 15% threshold for the CE of intrasample variance contribution to total variance was adhered to in order to reduce technical noise masking true biological variance between individual brains.

    “A 15% threshold for the CE of intrasample variance contribution to total variance (coefficient of variation [CV]) was adhered to in order to reduce technical noise masking true biological variance between individual brains and controlling for the relative SD of individual datasets.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The AAV/Olig001 capsid likely has a natural affinity for Purkinje cells or is at least bound in significant numbers to these cells.

    “It is likely that the AAV/Olig001 capsid has a natural affinity for Purkinje cells or is at least bound in significant numbers to these cells”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Both AAV/Olig001-ASPA and AAV9-ASPA contained an identical codon-optimized, 941-bp human ASPA CDS.

    “Both AAV/Olig001-ASPA and AAV9-ASPA contained an identical codon-optimized, 941-bp human ASPA CDS.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    Estimates of GFP-positive cell number were generated by unbiased stereology using the optical fractionator method.

    “Estimates of a GFP-positive cell number in all dose and ROA cohorts were generated by unbiased stereology using the optical fractionator method.27”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [27] West, 1991, Unbiased stereological estimation of the total number of neurons in thesubdivisions of the rat hippocampus using the optical fractionator, Anat. Rec. source

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

    Study design and conductnone given

    All treatment and control cohorts were n = 12 (50/50 gender split).

    “All treatment and control cohorts were n = 12 (50/50 gender split).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Stereological estimates were generated by observers blinded to treatment.

    “Stereological estimates were generated by observers blinded to treatment.”
    Quote in the paperSubstantial claim
59 of 59

  • The paper relies on this without support.

    Oligodendrocytes remain refractory to gene delivery by available AAV serotypes, restricting potential for white matter diseases.

    “but oligodendrocytes remain somewhat refractory to gene delivery by available serotypes, thereby restricting potential for white matter diseases.”

  • The paper relies on this without support.

    Most native AAV serotypes are inherently neurotropic in the main.

    “Most native AAV serotypes are inherently neurotropic in the main”

  • The paper relies on this without support.

    No comprehensive analysis of the potential therapeutic benefits of AAV/Olig001 tropism has been undertaken in a bona fide model of symptomatic congenital white matter disease.

    “but no comprehensive analysis of the potential therapeutic benefits of AAV/Olig001 tropism has been undertaken in a bona fide model of symptomatic congenital white matter disease.”

  • The paper relies on this without support.

    White matter has a significant capacity for remyelination, even in the adult brain.

    “Given that white matter has a significant capacity for remyelination, even in the adult brain”

  • The paper relies on this without support.

    The 6-week-old nur7 mouse models the Canavan brain at time of diagnosis and is representative of the clinical condition at diagnosis.

    “model the Canavan brain at time of diagnosis”
    “a stage of the phenotype representative of the clinical condition at the time of diagnosis.”

  • The paper relies on this without support.

    Oligodendroglial transduction by currently available viral vectors is inefficient, representing a significant unmet need for white matter disease gene therapy.

    “The relative inefficiency of oligodendroglial transduction by currently available viral vectors is a significant unmet need for white matter disease gene therapy.”

  • The paper relies on this without support.

    NAA is naturally exported to oligodendrocytes.

    “Given that NAA is naturally exported to oligodendrocytes”

  • The paper relies on this without support.

    Vacuolation is a prominent pathological hallmark of Canavan disease.

    “Vacuolation is a prominent pathological hallmark of Canavan disease”
8 of 8

Claims other papers make about this one

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

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.

  • The initial dose list repeats the lowest dose

    Minor

    The Results initially list three doses but repeat 1 × 1010 as the third. The subsequent Results and Methods identify the highest dose as 5 × 1011.

  • Cerebellar transduction percentage does not match its counts

    Minor

    Using Table 2's resident cerebellar Olig2 population of 344,651, the 1,225.9 colabeled cells shown here give about 0.36%, rather than 3.56%.

    Reported 3.56 · recomputed 0.355693

  • ICM is called the highest cortical-transduction route despite Table 3

    Minor

    Table 3 reports 25.20% cortical resident-cell transduction for IT at the highest dose, exceeding the 23.10% reported for ICM at that dose.

  • Highest-dose AAV9 is not significant at every rotarod time point

    Minor

    The text says both treatments significantly improved latency versus sham at all four time points. Table 4 gives p = 0.055 for highest-dose AAV9 at 10 weeks, above the paper's stated α = 0.05 threshold.

  • A colabeled-cell count exceeds its total GFP-cell count

    Minor

    GFP/Olig2-colabeled cells must be a subset of total GFP-positive cells, but this Table 3 row lists more than 31 million colabeled cells and only 513,476.7 total GFP-positive cells.

  • Sham-versus-wild-type p values disagree within Table 4

    Minor

    The sham row's p values versus wild type differ from the wild-type row's p values versus sham at every age, although these describe the same pairwise comparisons.

6 of 6

Cites

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Cited by

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Related

Authors
Francis, J. S., Markov, V., Wojtas, I. D., Gray, S., McCown, T., Samulski, R. J., Figueroa, M., & Leone, P.
Affiliations
  • Rowan University (US)
  • The University of Texas Southwestern Medical Center (US)
  • University of North Carolina at Chapel Hill (US)
  • AskBio (United States) (US)
Corresponding author
Leone, Paola
ORCID
Venue
Molecular Therapy - Methods & Clinical Development
Publisher
Elsevier BV
Published
2021-03
Volume
20
Pages
520-534
ISSN
2329-0501 (ISSN-L)
DOI
10.1016/j.omtm.2021.01.009
PMC
PMC7878967
OpenAlex
W3125720600
Semantic Scholar
b2062001627ecb2a13fafa88be37abf088fa713c
Cited by
41
References
27
Citations by year
2026: 5 · 2025: 4 · 2024: 9 · 2023: 9 · 2022: 9 · 2021: 5
FWCI
4.04
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • PubMed Central (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Pfizer
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
  • Health Sciences › Medicine › Genetics › Neurogenetic and Muscular Disorders Research
Keywords
Canavan disease, white matter disease, aspartoacylase, intracerebroventricular administration, tissue distribution, gene therapy, AAV9, vacuolization, motor function

About these checks

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

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

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

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

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

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