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Cited by 21Virus-based gene therapy researchRNA Interference and Gene DeliveryNeuroscience and Neuropharmacology Research

An innate immune response to adeno-associated virus genomes decreases cortical dendritic complexity and disrupts synaptic transmission

Open access
Suriano, Christos M., Kumar, Neerav, Verpeut, Jessica L., Ma, Jie, Jung, Caroline, Dunn, Connor E., Carvajal, Brigett V., Nguyen, Ai Vy +1 more·2024·Molecular Therapy
DOI 10.1016/j.ymthe.2024.03.036PMC PMC11184335
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Abstract

Recombinant adeno-associated viruses (AAVs) allow rapid and efficient gene delivery to the nervous system, are widely used in neuroscience research, and are the basis of FDA-approved neuron-targeting gene therapies. Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex. Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers, is not restricted to a particular capsid serotype, transgene, promoter, or production facility, and cannot be explained by responses to surgery or transgene expression. AAV-associated dendritic loss is accompanied by a decrease in the frequency and amplitude of miniature excitatory postsynaptic currents and an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors. The AAV genome is rich in unmethylated CpG DNA, which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9), and acutely blocking TLR9 preserves dendritic complexity and AMPA receptor subunit composition in AAV-injected mice. These results reveal unexpected impacts of an immune response to the AAV genome on neuronal structure and function and identify approaches to improve the safety and efficacy of AAV-mediated gene delivery in the nervous system.

AI snapshot

Written from the paper’s full text.

TL;DR

AAV-mediated gene delivery in mouse somatosensory cortex triggers an innate immune response to the AAV genome that reduces dendritic complexity and disrupts excitatory synaptic transmission. Blocking TLR9 around injection preserves dendritic arbors and AMPA receptor subunit composition, implicating unmethylated CpG DNA in the AAV genome as a key trigger.

Key findings

  • AAV injection reduced total dendritic length and Sholl intersections in ipsilateral S1 at 21 dpi; a lower dose of AAV8-hSyn-mCherry (2 × 10⁹ vg) was indistinguishable from untreated controls, while AAV1-pCAG-FLEX-EGFP (4.2 × 10⁹ vg) caused similar loss despite no detectable EGFP expression. Dendritic loss was not restricted to capsid serotype, transgene, promoter, or production facility.
  • Dendritic loss began by 4 dpi and progressed by 21 dpi; by 70 dpi ipsilateral and contralateral hemispheres were indistinguishable, but contralateral dendrites also showed significant loss versus 4 and 21 dpi, while untreated 18–20-week-old controls showed no significant loss. AAV injection also modestly reduced cortical thickness from 4 to 21 dpi, reduced cell density from 4 to 21 dpi and at 70 dpi, and increased caspase-3 at 21 dpi.
  • AAV1-pCAG-FLEX-EGFP reduced mEPSC frequency, increased inter-event interval, and decreased mEPSC amplitude. NASPM blocked 5.1% of sEPSCs in untreated controls versus 21.8% in AAV-injected hemispheres, a more than 4-fold increase in Ca²⁺-permeable AMPA receptor contribution, and GluA2 levels were reduced relative to GluA1.
  • AAV upregulated H2-K and H2-D at 21 dpi and multiple cytokines/chemokines including IFN-γ, CD54, C5a, CXCL9, CXCL10, CXCL13, CCL2, and CCL5. The TLR9 antagonist ODN 2088 prevented AAV-induced dendritic loss and abolished the NASPM effect on sEPSC amplitude.

Methods

  • Male C57BL/6J mice, 8–10 weeks old at injection, received unilateral stereotaxic injections of AAV8-hSyn-mCherry, AAV1-pCAG-FLEX-EGFP, vehicle, or sham surgery into S1, motor cortex, and/or cerebellum; tissues were collected at 4, 21, or 70 dpi.
  • Dendritic morphology was assessed by DiOlistic labeling, blinded tracing, Sholl analysis, and Strahler order; cortical thickness and DAPI+ cell density were measured; synaptic transmission was assessed by whole-cell patch clamp for mEPSCs and sEPSCs with NASPM; immune readouts included RT-qPCR, western blotting, and cytokine arrays.
  • Data were analyzed with Shapiro-Wilk normality tests, Grubb’s outlier tests, two-tailed unpaired t tests, and Kolmogorov-Smirnov tests for cumulative electrophysiology curves; dendritic data were often expressed as ipsilateral/contralateral ratios within animals.

Limitations

  • Stated: it remains unknown whether AAV-induced dendritic loss occurs in non-human primates or humans, and species differences in TLR responses may limit translation; future studies are needed to define the individual roles of the many upregulated immune proteins.
  • Stated: the authors cannot rule out that some analyzed neurons were transduced but expressed undetectably low mCherry, and it is likely many non-transduced neurons were included, suggesting the effects are unlikely to be cell autonomous.
  • Evident: the study is limited to male C57BL/6J mice, specific AAV doses/routes/ages, and pharmacological TLR9 blockade rather than genetic TLR9 loss-of-function; dendritic sampling may underestimate loss because sectioning can truncate dendrites, as the authors note.

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

Integrity signals

Registry

Crossref · 10.1016/j.ymthe.2024.03.036

Cites

99 of 99 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 84 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers.

    “Dendritic loss is apparent 3 weeks after injection of experimentally relevant viral titers”
    Quote in the paperSubstantial claim
  • Findingown data

    AAV-associated dendritic loss is accompanied by a decrease in the frequency of miniature excitatory postsynaptic currents.

    “A decrease in the frequency and amplitude of miniature excitatory postsynaptic currents”
    Quote in the paperSubstantial claim
  • Findingown data

    AAV-mediated gene delivery reduces dendritic length and complexity in mouse somatosensory cortex.

    “Here we find that an innate immune response to the AAV genome reduces dendritic length and complexity and disrupts synaptic transmission in mouse somatosensory cortex.”
    Quote in the paperSubstantial claim
  • Findingown data

    AAV-associated dendritic loss is accompanied by an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors.

    “an increase in the proportion of GluA2-lacking, calcium-permeable AMPA receptors”
    Quote in the paperSubstantial claim
  • Findingown data

    AAV-associated dendritic loss is accompanied by a decrease in the amplitude of miniature excitatory postsynaptic currents.

    “A decrease in the frequency and amplitude of miniature excitatory postsynaptic currents”
    Quote in the paperSubstantial claim
  • The quote only partly states this claim. 1
  • May combine two claims. 40
  • Classified as probably not a substantial claim. 39

  • The quote only partly states this claim.

    Findingown data

    AAV-mediated gene delivery disrupts synaptic transmission in mouse somatosensory cortex.

    “AAV1-pCAG-FLEX-EGFP disrupts synaptic transmission”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    By 70 dpi, dendritic length and Sholl intersections between ipsilateral and contralateral hemispheres are indistinguishable.

    “By 70 dpi, dendritic length and Sholl intersections between ipsilateral and contralateral hemispheres are indistinguishable”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In the non-injected contralateral hemispheres, total dendritic length and Sholl intersections are indistinguishable from untreated controls.

    “In the non-injected contralateral hemispheres, total dendritic length and Sholl intersections are indistinguishable from untreated controls”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both AAV8-hSyn-mCherry and AAV1-pCAG-FLEX-EGFP (UPenn) significantly upregulate H2-K and H2-D relative to untreated and vehicle controls at 21 dpi.

    “both AAV8-hSyn-mCherry (1.2 × 1011 vg) and AAV1-pCAG-FLEX-EGFP (UPenn) (4.2 × 109 vg) significantly upregulate H2-K and H2-D relative to untreated and vehicle controls at 21 dpi”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AAV1-pCAG-FLEX-EGFP still causes a significant reduction in total dendritic length and Sholl intersections, closely matching what is seen after AAV8-hSyn-mCherry.

    “AAV1-pCAG-FLEX-EGFP still causes a significant reduction in total dendritic length and Sholl intersections, closely matching what is seen after AAV8-hSyn-mCherry”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Only 4% of cells from the injected ipsilateral hemisphere reach SN4 after AAV8-hSyn-mCherry, and 7% after AAV1-pCAG-FLEX-EGFP (UPenn).

    “only 4% (after AAV8-hSyn-mCherry) or 7% (after AAV1-pCAG-FLEX-EGFP (UPenn)) reach SN4”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Untreated animals at 18-20 weeks old showed no significant loss of dendritic length or Sholl intersections relative to younger adults.

    “Untreated animals at 18–20 weeks old showed no significant loss of dendritic length or Sholl intersections relative to younger adults”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fyn is persistently upregulated by AAV8-hSyn-mCherry but not AAV1-pCAG-FLEX-EGFP (UPenn) at 21 dpi.

    “Fyn is persistently upregulated by AAV8-hSyn-mCherry but not AAV1-pCAG-FLEX-EGFP (UPenn)”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Dendritic complexity in contralateral S1 at 21 dpi is indistinguishable from untreated controls, despite the presence of mCherry that is trafficked from the injected hemisphere.

    “Third, dendritic complexity in contralateral S1 at 21 dpi is indistinguishable from untreated controls, despite the presence of mCherry that is trafficked from the injected hemisphere.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Blocking immunodetection of the AAV genome via TLR9 preserves dendritic arbors and maintains normal AMPA receptor responses in AAV-injected animals.

    “and blocking immunodetection of the AAV genome via TLR9 preserves dendritic arbors and maintains normal AMPA receptor responses in AAV-injected animals.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In AAV8-hSyn-mCherry-injected animals, cells that expressed readily detectable mCherry were not traced, but significant dendritic loss was still observed.

    “Second, in AAV8-hSyn-mCherry-injected animals, cells that expressed readily detectable mCherry were not traced (see materials and methods), but significant dendritic loss was still observed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    An immune response to the AAV genome significantly reduces dendritic complexity and disrupts synaptic transmission in adult rodent cortex.

    “Here, we show that an immune response to the AAV genome significantly reduces dendritic complexity and disrupts synaptic transmission in adult rodent cortex.”
    • Also stated in: § Discussion · plain
      “These results reveal that an innate immune response to AAV’s genome significantly impacts circuit structure and function”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Transgene expression is seen in fibers in contralateral S1, which does not show upregulation of immune genes or changes in dendritic structure at 21 dpi.

    “We see transgene expression in fibers in contralateral S1, which does not show upregulation of immune genes or changes in dendritic structure at 21 dpi”
    Quote in the paperSubstantial claim

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

    Findingown data

    There was no detectable mCherry mRNA in untreated or sham surgery samples.

    “There was no detectable mCherry mRNA in untreated or sham surgery samples, so the lowest possible CT values (50) were assigned to these samples.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    H2-K and H2-D limit the levels of Ca2+-permeable AMPA receptors in the lateral geniculate nucleus.

    “limit the levels of Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the lateral geniculate nucleus.21,22,26,27,28,29”
    Quote in the paperNot a substantial claim0 of 6 sources checked
    • cites [21] Huh, 2000, Functional requirement for class I MHC in CNS development and plasticity, Science source

      Not checked in the free run.

    • cites [22] Lee, 2014, Synapse elimination and learning rules co-regulated by MHC class I H2-D b, Nature source

      Not checked in the free run.

    • cites [26] Dixon-Salazar, 2014, MHC class I limits hippocampal synapse density by inhibiting neuronal insulin receptor signaling, J. Neurosci. source

      Not checked in the free run.

    • cites [27] Glynn, 2011, MHCI negatively regulates synapse density during the establishment of cortical connections, Nat. Neurosci. source

      Not checked in the free run.

    • cites [28] Hu, 2020, MiR34a Regulates Neuronal MHC Class I Molecules and Promotes Primary Hippocampal Neuron Dendritic Growth and Branching, Front. Cell. Neurosci. source

      Not checked in the free run.

    • cites [29] Shen, 2019, MHC Class I Molecules and PirB Shape Neuronal Morphology by Affecting the Dendritic Arborization of Cortical Neurons, Neurochem. Res. source

      Not checked in the free run.

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

    Premisecitation

    H2-K and H2-D negatively regulate dendritic complexity and the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal and cortical neurons.

    “H2-K and H2-D also negatively regulate dendritic complexity and the frequency and amplitude of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal and cortical neurons”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [21] Huh, 2000, Functional requirement for class I MHC in CNS development and plasticity, Science source

      Not checked in the free run.

    • cites [22] Lee, 2014, Synapse elimination and learning rules co-regulated by MHC class I H2-D b, Nature source

      Not checked in the free run.

    • cites [26] Dixon-Salazar, 2014, MHC class I limits hippocampal synapse density by inhibiting neuronal insulin receptor signaling, J. Neurosci. source

      Not checked in the free run.

    • cites [27] Glynn, 2011, MHCI negatively regulates synapse density during the establishment of cortical connections, Nat. Neurosci. source

      Not checked in the free run.

    • cites [28] Hu, 2020, MiR34a Regulates Neuronal MHC Class I Molecules and Promotes Primary Hippocampal Neuron Dendritic Growth and Branching, Front. Cell. Neurosci. source

      Not checked in the free run.

    • cites [29] Shen, 2019, MHC Class I Molecules and PirB Shape Neuronal Morphology by Affecting the Dendritic Arborization of Cortical Neurons, Neurochem. Res. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Dendritic simplification is not caused by surgery or vehicle injection but is specifically associated with the presence of AAV.

    “Dendritic loss in AAV-injected but not vehicle-injected animals suggests that dendritic simplification is not caused by surgery or vehicle injection but rather is specifically associated with the presence of AAV.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    In the periphery, AAV-mediated gene delivery can elicit humoral, cellular, and innate immune responses to features including the viral capsid, expressed transgene, and impurities from the viral preparation.

    “In the periphery, AAV-mediated gene delivery can elicit humoral, cellular, and innate immune responses to features including the viral capsid, expressed transgene, and impurities from the viral preparation,6,7”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] Ertl, 2022, Immunogenicity and toxicity of AAV gene therapy, Front. Immunol. source

      Not checked in the free run.

    • cites [7] Wright, 2014, Product-Related Impurities in Clinical-Grade Recombinant AAV Vectors: Characterization and Risk Assessment, Biomedicines source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    AAV-mediated gene delivery causes a decrease in synapse number that parallels the loss of dendritic length and complexity and disrupts synaptic transmission at remaining synapses.

    “AAV-mediated gene delivery causes a decrease in synapse number that parallels the loss of dendritic length and complexity and disrupts synaptic transmission at remaining synapses”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Proteins of the complement cascade (including C1q, C3, and C4) and members of MHCI (including H2-K and H2-D) contribute to synapse elimination in the developing visual system.

    “proteins of the complement cascade (including C1q, C3, and C4) and members of MHCI (including H2-K and H2-D) contribute to synapse elimination in the developing visual system.20,21,22,23,24,25”
    Quote in the paperSubstantial claim0 of 6 sources checked
    • cites [20] Datwani, 2009, Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity, Neuron source

      Not checked in the free run.

    • cites [21] Huh, 2000, Functional requirement for class I MHC in CNS development and plasticity, Science source

      Not checked in the free run.

    • cites [22] Lee, 2014, Synapse elimination and learning rules co-regulated by MHC class I H2-D b, Nature source

      Not checked in the free run.

    • cites [23] Schafer, 2012, Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner, Neuron source

      Not checked in the free run.

    • cites [24] Stevens, 2007, The classical complement cascade mediates CNS synapse elimination, Cell source

      Not checked in the free run.

    • cites [25] Yilmaz, 2021, Overexpression of schizophrenia susceptibility factor human complement C4A promotes excessive synaptic loss and behavioral changes in mice, Nat. Neurosci. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    The smaller amplitude of mEPSCs recorded in neurons from AAV-injected animals is unlikely to be caused by smaller dendritic arbors but could occur if there was a change in AMPA receptor subunit composition.

    “The smaller amplitude of mEPSCs recorded in neurons from AAV-injected animals are unlikely to be caused by smaller dendritic arbors but could occur if there was a change in AMPA receptor subunit composition.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Dendritic loss is not specific to a particular capsid serotype, promoter, encoded transgene, or production facility but is triggered by AAV-based gene delivery systems that differ in all these parameters.

    “dendritic loss does not require detectable transgene expression and is not specific to a particular capsid serotype, promoter, encoded transgene, or production facility but is triggered by AAV-based gene delivery systems that differ in all these parameters.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    AAV-mediated gene delivery can cause modest cortical thinning and cell death in mouse cortex.

    “AAV-mediated gene delivery can cause modest cortical thinning and cell death in mouse cortex”
    Quote in the paperSubstantial claim

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

    Premisecitation

    ITR-driven toxicity does not require Sting, a TLR9-independent pathway for detecting foreign DNA.

    “This ITR-driven toxicity does not require Sting, a TLR9-independent pathway for detecting foreign DNA.63”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] Johnston, 2021, AAV ablates neurogenesis in the adult murine hippocampus, eLife source

      Not checked in the free run.

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

    Study design and conductown data

    No-primary controls show lack of non-specific labeling.

    “No-primary controls show lack of non-specific labeling”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    AAV-mediated gene delivery into the CNS of pigs or non-human primates causes dorsal root ganglion toxicity that is alleviated by reducing or deleting transgene expression.

    “For example, AAV-mediated gene delivery into the CNS of pigs or non-human primates causes dorsal root ganglion toxicity that is alleviated by reducing or deleting transgene expression.38,39,66”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [38] Hordeaux, 2018, Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Human Alpha-L-Iduronidase in Rhesus Macaques, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

    • cites [39] Hordeaux, 2020, MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates, Sci. Transl. Med. source

      Not checked in the free run.

    • cites [66] Buss, 2022, Characterization of AAV-mediated dorsal root ganglionopathy, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

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

    Premisecitation

    Previous studies showed upregulation of MHCI protein 4 days after striatal injection of AAV9 or AAV2 in rats and upregulation of MHCI mRNA in mouse cortical microglia 3 days post AAV injection.

    “These findings from mouse cortex align well with previous studies showing upregulation of MHCI protein 4 days after striatal injection of AAV9 or AAV2 in rats80,81 and upregulation of MHCI mRNA in mouse cortical microglia 3 days post AAV injection.82”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [80] Peden, 2009, Striatal readministration of rAAV vectors reveals an immune response against AAV2 capsids that can be circumvented, Mol. Ther. source

      Not checked in the free run.

    • cites [81] Yang, 2013, Pre-immunization with an intramuscular injection of AAV9-human erythropoietin vectors reduces the vector-mediated transduction following re-administration in rat brain, PLoS One

      Not checked in the free run.

    • cites [82] Brown, 2021, Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing, bioRxiv

      Not checked in the free run.

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

    Premisecitation

    Overexpression of SMN1 in a spinal muscular atrophy mouse model causes a toxic gain of function.

    “Additionally, overexpression of SMN1 in a spinal muscular atrophy mouse model causes a toxic gain of function.67”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [67] Van Alstyne, 2021, Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit, Nat. Neurosci. source

      Not checked in the free run.

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

    Premisecitation

    Broadly active cis-regulatory elements are correlated with dose-dependent, cell-type-specific toxicity after subretinal injection of AAV8, AAV5, or AAV2 in mice, and toxicity is still observed with AAVs that encode no transgene.

    “Broadly active cis-regulatory elements are correlated with dose-dependent, cell-type-specific toxicity after subretinal injection of AAV8, AAV5, or AAV2 in mice, and toxicity is still observed with AAVs that encode no transgene.36”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [36] Xiong, 2019, AAV cis-regulatory sequences are correlated with ocular toxicity, Proc. Natl. Acad. Sci. USA source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Transgene expression is neither necessary nor sufficient for AAV-associated dendritic loss.

    “Together, these findings demonstrate that transgene expression is neither necessary nor sufficient for AAV-associated dendritic loss.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All data collection and analysis were performed by observers who were blinded to treatment.

    “All data collection and analysis were performed by observers who were blinded to treatment.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Electroporating only AAV's single-stranded DNA inverted terminal repeats (ITRs) into progenitor cells in vitro recapitulates dose-dependent loss of neural progenitor cells and immature adult-born dentate granule cells following injection of AAV into dentate gyrus.

    “A recent study showed that electroporating only AAV’s single-stranded DNA inverted terminal repeats (ITRs) into progenitor cells in vitro recapitulates dose-dependent loss of neural progenitor cells and immature adult-born dentate granule cells following injection of AAV into dentate gyrus.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [63] Johnston, 2021, AAV ablates neurogenesis in the adult murine hippocampus, eLife source

      Not checked in the free run.

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

    Premisecitation

    AAV-mediated gene delivery has been associated with neurotoxicity in specific cell populations, some of which can be attributed to the overexpressed transgene.

    “Despite an overall strong safety profile, AAV-mediated gene delivery has been associated with neurotoxicity in specific cell populations, some of which can be attributed to the overexpressed transgene.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [38] Hordeaux, 2018, Toxicology Study of Intra-Cisterna Magna Adeno-Associated Virus 9 Expressing Human Alpha-L-Iduronidase in Rhesus Macaques, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

    • cites [39] Hordeaux, 2020, MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates, Sci. Transl. Med. source

      Not checked in the free run.

    • cites [66] Buss, 2022, Characterization of AAV-mediated dorsal root ganglionopathy, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

    • cites [67] Van Alstyne, 2021, Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit, Nat. Neurosci. source

      Not checked in the free run.

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

    Premisecitation

    A growing number of immune proteins have been identified that play central roles in neuronal homeostasis, where they negatively regulate neurite length, dendritic complexity, and synaptic transmission, and many are upregulated by TLR9.

    “A growing number of immune proteins have been identified that play central roles in neuronal homeostasis, where they negatively regulate neurite length, dendritic complexity, and synaptic transmission,20,21,22,23,24,25,26,27,28,29,45,46,48,72,73,74,75,76,77,78,79 and many are upregulated by TLR9.”
    Quote in the paperNot a substantial claim0 of 10 sources checked
    • cites [20] Datwani, 2009, Classical MHCI molecules regulate retinogeniculate refinement and limit ocular dominance plasticity, Neuron source

      Not checked in the free run.

    • cites [21] Huh, 2000, Functional requirement for class I MHC in CNS development and plasticity, Science source

      Not checked in the free run.

    • cites [22] Lee, 2014, Synapse elimination and learning rules co-regulated by MHC class I H2-D b, Nature source

      Not checked in the free run.

    • cites [23] Schafer, 2012, Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner, Neuron source

      Not checked in the free run.

    • cites [24] Stevens, 2007, The classical complement cascade mediates CNS synapse elimination, Cell source

      Not checked in the free run.

    • cites [25] Yilmaz, 2021, Overexpression of schizophrenia susceptibility factor human complement C4A promotes excessive synaptic loss and behavioral changes in mice, Nat. Neurosci. source

      Not checked in the free run.

    • cites [26] Dixon-Salazar, 2014, MHC class I limits hippocampal synapse density by inhibiting neuronal insulin receptor signaling, J. Neurosci. source

      Not checked in the free run.

    • cites [27] Glynn, 2011, MHCI negatively regulates synapse density during the establishment of cortical connections, Nat. Neurosci. source

      Not checked in the free run.

    • cites [28] Hu, 2020, MiR34a Regulates Neuronal MHC Class I Molecules and Promotes Primary Hippocampal Neuron Dendritic Growth and Branching, Front. Cell. Neurosci. source

      Not checked in the free run.

    • cites [29] Shen, 2019, MHC Class I Molecules and PirB Shape Neuronal Morphology by Affecting the Dendritic Arborization of Cortical Neurons, Neurochem. Res. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    AAV-mediated gene delivery is associated with significant loss of dendritic complexity, changes in the functional properties of synapses, and upregulation of many immune proteins that maintain normal neuronal structure and function in the healthy nervous system.

    “The current results show that AAV-mediated gene delivery is associated with significant loss of dendritic complexity, changes in the functional properties of synapses, and upregulation of many immune proteins that maintain normal neuronal structure and function in the healthy nervous system”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductreasoning

    Animals that received AAV2/1-pCAG-FLEX-EGFP-WPRE did not receive CRE recombinase and therefore should not express EGFP.

    “Animals that received the AAV2/1-pCAG-FLEX-EGFP-WPRE virus did not receive any form of CRE recombinase and therefore should not express EGFP.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    A Shapiro-Wilk test was performed to ensure data were normally distributed.

    “For all datasets, a Shapiro-Wilk test was performed to ensure data were normally distributed, and a two-tailed Grubb’s test was used to identify outliers.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All SO analyses were performed by investigators blinded to condition.

    “All SO analyses were performed by investigators blinded to condition.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Rabbits and rats are more sensitive than monkeys to the TLR3 agonist rintatolimod, while mice are far more tolerant than humans to several TLR agonists, including the TLR4 ligand endotoxin (lipopolysaccharide).

    “For example, rabbits and rats are more sensitive than monkeys to the TLR3 agonist rintatolimod,83 while mice are far more tolerant than humans to several TLR agonists, including the TLR4 ligand endotoxin84 (lipopolysaccharide).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [83] Mitchell, 2014, Discordant biological and toxicological species responses to TLR3 activation, Am. J. Pathol. source

      Not checked in the free run.

    • cites [84] Copeland, 2005, Acute inflammatory response to endotoxin in mice and humans, Clin. Diagn. Lab. Immunol.

      Not checked in the free run.

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

    Study design and conductown data

    A subset of experiments included a washout period to confirm that sEPSC amplitude recovered after removing NASPM.

    “A subset of experiments also included a washout period to confirm that sEPSC amplitude recovered after removing NASPM.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    A synthetic TLR9 agonist causes neurite loss in organotypic cortico-hippocampal slices, which lack a source of infiltrating immune cells.

    “However, a synthetic TLR9 agonist causes neurite loss in organotypic cortico-hippocampal slices,87 which lack a source of infiltrating immune cells”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [87] Iliev, 2004, Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9), FASEB J. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Remote injection can prevent dendritic damage while permitting local transgene expression in projection fibers.

    “suggesting that remote injection can prevent dendritic damage while permitting local transgene expression in projection fibers.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The neuronal effects of AAV are caused by unmethylated CpG motifs, which are TLR9 ligands.

    “suggesting they are caused by unmethylated CpG motifs, which are TLR9 ligands.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Virus preps were treated with turbonuclease to minimize viral, host, and plasmid DNA impurities.

    “Virus preps were treated with turbonuclease to minimize viral, host, and plasmid DNA impurities.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    An investigator blinded to treatment traced cells, quantified total dendritic length and total nodes, and performed Sholl analyses on labeled neurons.

    “An investigator blinded to treatment traced cells, quantified total dendritic length and total nodes, and performed Sholl analyses on labeled neurons using the Simple Neurite Tracer Plugin in ImageJ FIJI.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    In current studies using AAV8-mCherry, DiO-labeled neurons that did not show detectable mCherry expression were exclusively imaged.

    “In the current studies using AAV8-mCherry, we exclusively imaged DiO-labeled neurons that did not show detectable mCherry expression”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationnone given

    The appropriate multiplicity of infection will vary depending on each unique combination of capsid serotype, promoter, encoded transgene, model, and route of administration.

    “The appropriate multiplicity of infection will vary depending on each unique combination of capsid serotype, promoter, encoded transgene, model, and route of administration.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Virus preps were endotoxin negative.

    “Viruses were prepared using endotoxin-free plasmid prep kits, and virus preps were endotoxin negative.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    A two-tailed Grubb's test was used to identify outliers.

    “For all datasets, a Shapiro-Wilk test was performed to ensure data were normally distributed, and a two-tailed Grubb’s test was used to identify outliers.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Three investigators blinded to treatment counted DAPI+ cells within a consistently sized boxed area using ImageJ in layers I, II/III, IV, V, and VI.

    “Three investigators blinded to treatment counted DAPI+ cells within a consistently sized boxed area using ImageJ in layers I, II/III, IV, V, and VI.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Cell death associated with high concentrations of AAV was partially attenuated by repeated treatment with antibodies against cytotoxic T lymphocytes.

    “Cell death associated with high concentrations of AAV was partially attenuated by repeated treatment with antibodies against cytotoxic T lymphocytes.60”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Guo, 2023, High-titer AAV disrupts cerebrovascular integrity and induces lymphocyte infiltration in adult mouse brain, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

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

    Premisecitation

    18s rRNA was the least affected among tested genes after viral infection.

    “Kuchipudi et al.97 found that 18s rRNA was the least affected among tested genes, and we therefore used 18s rRNA as a reference gene for this study.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kuchipudi et al.97 Kuchipudi, 2012, 18S rRNA is a reliable normalisation gene for real time PCR based on influenza virus infected cells, Virol. J. source

      Not checked in the free run.

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

    Study design and conductreasoning

    To reduce type I errors arising from multiple comparisons, only virus-untreated, virus-sham, and sham-untreated comparisons were performed.

    “To reduce type I errors arising from multiple comparisons, only virus-untreated, virus-sham, and sham-untreated comparisons were performed.”
    Quote in the paperNot a substantial claim

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

    Premisereasoning

    ODN 2088 and many other TLR9 inhibitors are short oligonucleotides, they can be introduced into host cells as part of the viral genome, in cis.

    “Because ODN 2088 and many other TLR9 inhibitors are short oligonucleotides, they can be introduced into host cells as part of the viral genome, in cis, an approach that has recently been used to reduce or delay AAV-induced toxicity in the retina in pigs and macaques, respectively.61”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [61] Chan, 2021, Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses, Sci. Transl. Med. source

      Not checked in the free run.

    • Rests on an assumption: ODN 2088 and many other TLR9 inhibitors are short oligonucleotides that can be introduced into host cells as part of the viral genome, in cis.

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

    Study design and conductown data

    No-primary controls show lack of non-specific secondary antibody binding in lanes containing protein samples for all of the secondary antibodies used.

    “No-primary controls show lack of non-specific secondary antibody binding in lanes containing protein samples for all of the secondary antibodies used (Figure S6B).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Prominent differences in TLR responses exist across mammals, including differences in the timing and magnitude of TLR-mediated signaling.

    “Prominent differences in TLR responses exist across mammals, including differences in the timing and magnitude of TLR-mediated signaling.”
    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

    Multiple exposures were taken, and appropriate exposures were used to quantify densitometry within the linear range of assay sensitivity.

    “Multiple exposures were taken, and appropriate exposures were used to quantify densitometry within the linear range of assay sensitivity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Injection of a large volume of concentrated AAV (1 μL of 1 × 1013 vg per mL) can cause infiltration of cytotoxic T cells and associated neurotoxicity, changes that are averted by injecting the same volume of a lower titer virus (1 × 1011 vg per mL).

    “For example, injection of a large volume of concentrated AAV (1 μL of 1 × 1013 vg per mL) can cause infiltration of cytotoxic T cells and associated neurotoxicity, changes that are averted by injecting the same volume of a lower titer virus (1 × 1011 vg per mL).60”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Guo, 2023, High-titer AAV disrupts cerebrovascular integrity and induces lymphocyte infiltration in adult mouse brain, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

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

    Study design and conductown data

    Lack of EGFP was confirmed by confocal immunofluorescence microscopy and immunoblotting.

    “Lack of EGFP was confirmed by both confocal immunofluorescence microscopy (Figures 1B and S1G) and by using immunoblotting to amplify EGFP signals (Figure 4A).”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    Cells selected for tracing were randomized into a cohort and labeled before being uploaded to a single folder for blinded investigators.

    “The contralateral and ipsilateral cells that were selected for tracing were randomized into a cohort and labeled as “Cell 1,” “Cell 2,” and so forth before being uploaded to a single folder for blinded investigators.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Lack of bands in negative control lanes indicates absence of primer dimers.

    “Lack of bands in these lanes indicates absence of primer dimers (Figure S6A).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductown data

    Notching fixed sections does not alter dendritic complexity because both vehicle control and AAV8-hSyn-mCherry 1:20 titer cohorts were notched, and dendritic complexity is not different from untreated controls in these groups.

    “Notching fixed sections does not alter dendritic complexity because both vehicle control and AAV8-hSyn-mCherry 1:20 titer cohorts were notched, and dendritic complexity is not different from untreated controls in these groups.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    These studies reveal unexpected effects of AAV-mediated gene delivery on neuronal structure and function and identify the immune response to the AAV genome as the triggering event.

    “Together, these studies reveal unexpected effects of AAV-mediated gene delivery on neuronal structure and function and identify the immune response to the AAV genome as the triggering event.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    Melt curves were calculated to establish the quality of the data, and any flagged data were omitted.

    “Melt curves were calculated to establish the quality of the data, and any flagged data were omitted.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Intraparenchymal administration of high doses of AAV9 causes significant neuronal death and titer-dependent infiltration of peripheral immune cells.

    “A recent study found that intraparenchymal administration of high doses of AAV9, which causes significant neuronal death, causes titer-dependent infiltration of peripheral immune cells.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [60] Guo, 2023, High-titer AAV disrupts cerebrovascular integrity and induces lymphocyte infiltration in adult mouse brain, Mol. Ther. Methods Clin. Dev. source

      Not checked in the free run.

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

    Study design and conductreasoning

    One-tailed t tests were used to analyze exogenous mCherry mRNA levels because changes in only one direction (rises) are possible from baseline.

    “One-tailed t tests were used to analyze exogenous mCherry mRNA levels because changes in only one direction (rises) are possible from baseline.”
    Quote in the paperNot a substantial claim
66 of 66

  • The paper relies on this without support.

    The AAV genome is rich in unmethylated CpG DNA.

    “The AAV genome is rich in unmethylated CpG DNA”
    “One feature that is shared by essentially all AAVs is a genome that is rich in unmethylated CpG DNA motifs”

  • The paper relies on this without support.

    Unmethylated CpG DNA is recognized by TLR9.

    “which is recognized by the innate immunoreceptor Toll-like receptor 9 (TLR9)”
    “which are detected by the innate immunoreceptor TLR9”

  • The paper relies on this without support.

    The AAV genome is not rapidly cleared by the immune response in the CNS.

    “Although the AAV genome is not rapidly cleared by the immune response in the CNS”

  • The paper relies on this without support.

    Unmethylated CpG motifs are a core feature of the AAV genome.

    “Since unmethylated CpGs are a core feature of the AAV genome”

  • The paper relies on this without support.

    TLR9 can detect unmethylated CpG DNA from diverse sources.

    “Because TLR9 can detect unmethylated CpG DNA from diverse sources”

  • The paper relies on this without support.

    Dendritic morphology powerfully influences integration and other emergent properties that arise due to the electrical separation of synapses.

    “Dendritic morphology powerfully influences integration and other emergent properties that arise due to the electrical separation of synapses”

  • The paper relies on this without support.

    AAV genomes are present in both experimental and control conditions when AAVs encoding fluorescent tracers or unexpressed transgenes are used as negative controls.

    “since AAV genomes are present in both experimental and control conditions.”

  • The paper relies on this without support.

    Many of the deleterious and efficacy-limiting immune responses to AAV in clinical trials were not predicted by preclinical work in animal models.

    “Notably, many of the deleterious and efficacy-limiting immune responses to AAV in clinical trials were not predicted by preclinical work in animal models.”

  • The paper relies on this without support.

    A lower number of viral genomes may, in some cases, be insufficient to drive robust transgene expression.

    “However, a lower number of vg may, in some cases, be insufficient to drive robust transgene expression.”

  • The paper relies on this without support.

    ODN 2088 and many other TLR9 inhibitors are short oligonucleotides that can be introduced into host cells as part of the viral genome, in cis.

    “Because ODN 2088 and many other TLR9 inhibitors are short oligonucleotides, they can be introduced into host cells as part of the viral genome, in cis”

  • The authors state this as an assumption.

    The data have equal variance across groups.

    “Two-tailed unpaired t tests assuming equal variance were used for all comparisons.”
11 of 11

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?

  • TLR9-dependent reduction of dendritic length and complexity and disruption of synaptic transmission occurred in mouse somatosensory cortex after AAV gene transfer, accompanied by changes in AMPA receptor subunit composition and prevented by TLR9 blockade.

    “Another recent investigation reports TLR9-dependent reduction of dendritic length and complexity and disruption of synaptic transmission in mouse somatosensory cortex in response to AAV gene transfer, which was accompanied by changes in AMPA receptor subunit composition but could be prevented by TLR9 blockade.”

    From Innate immune sensing of adeno-associated virus vectors (2024) · cites this paper as [28]

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.

  • Statistical methods say all comparisons used two-tailed t tests, then specify one-tailed tests

    Minor

    The statement that two-tailed t tests were used for all comparisons conflicts with the later statement that exogenous mCherry mRNA was analyzed using one-tailed t tests.

1 of 1

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Related

Authors
Suriano, C. M., Kumar, N., Verpeut, J. L., Ma, J., Jung, C., Dunn, C. E., Carvajal, B. V., Nguyen, A. V., & Boulanger, L. M.
Affiliations
  • Montclair State University (US)
  • Princeton University (US)
Corresponding author
  • Suriano, Christos M.
  • Boulanger, Lisa M.
ORCID
Venue
Molecular Therapy
Publisher
Elsevier BV
Published
2024-06
Volume
32
Issue
6
Pages
1721-1738
ISSN
1525-0016 (ISSN-L)
DOI
10.1016/j.ymthe.2024.03.036
PMC
PMC11184335
OpenAlex
W4393371324
Semantic Scholar
28bb6db576f6a3ec0eb56b8486599bbfdde156d8
Cited by
21
References
99
Citations by year
2026: 9 · 2025: 5 · 2024: 6 · 2022: 1
FWCI
5.29
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
Funders
  • Princeton University
  • Exploratory Research Center on Life and Living Systems, National Institutes of Natural Sciences
  • National Institutes of Health (T32MH065214-11, TL1TR003019, T32GM007388)
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 › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
Keywords
adeno-associated virus, innate immune response, dendritic complexity, synaptic transmission, TLR9, CpG DNA, AMPA receptors, calcium-permeable AMPA receptors, GluA2, somatosensory cortex, neuroimmunology
MeSH terms
Dependovirus, Animals, Dendrites, Genetic Vectors, Immunity, Innate, Synaptic Transmission, Somatosensory Cortex, Genome, Viral, Receptors, AMPA, Toll-Like Receptor 9, Mice

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 2026-10-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-06.

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