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Cited by 28CRISPR and Genetic EngineeringOcular Diseases and Behçet’s SyndromeOcular Infections and Treatments

Investigation of Cas9 Antibodies in the Human Eye

Open access
Toral, Marcus A., Charlesworth, Carsten T., Ng, Benjamin, Chemudupati, Teja, Homma, Shota, Nakauchi, Hiromitsu, Bassuk, Alexander G., Porteus, Matthew H. +1 more·2022·Nature Communications
DOI 10.1038/s41467-022-28674-1PMC PMC8881612
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Abstract

Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR–Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR–Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.

AI snapshot

Written from the paper’s full text.

TL;DR

Preexisting Cas9-reactive antibodies are highly prevalent in human serum but generally not detectable in vitreous fluid, except after severe ocular pathology or intraocular bacterial exposure. The authors conclude that preexisting Cas9 immunity may pose a lower risk in the human eye than systemically, though serum antibody levels may predict intraocular antibody development.

Key findings

  • In paired human serum/vitreous samples (n = 13), serum was 100% positive for α-SaCas9 and α-SpCas9, while vitreous fluid was positive in only 15% (2/13) for each; both positive vitreous samples came from the same patient with choroidal melanoma. In a validation vitreous cohort (n = 36), 0/36 were positive for α-SaCas9 and 1/36 for α-SpCas9, the latter from a patient with *S. pyogenes* endophthalmitis.
  • Vitreous immunoglobulin isotyping (n = 26) detected all subclasses except possibly IgM, with an aggregate vitreous-to-serum antibody ratio of approximately 1:143; IgG4 comprised ~20% of total vitreous antibodies and had a vitreous-to-serum ratio of 1:13.
  • In immunized mice, vitreous fluid antibodies developed in 40% for ovalbumin, 44% for SpCas9, and 10% for SaCas9; higher serum antibody levels trended with higher vitreous fluid levels of the same antibody in both humans and mice.
  • α-Tetanus was highly prevalent in vitreous fluid (100% of paired samples, 92% of validation samples), contrasting with low intraocular α-Cas9 prevalence.

Methods

  • Human vitreous fluid and serum biopsies were collected from adults undergoing vitreoretinal surgery: cohort 1 paired serum/vitreous (n = 13), cohort 2 unpaired vitreous validation (n = 36), and an isotyping subset (n = 26). Antibodies were measured by ELISA at 1:50 dilution, with positivity defined as mean α-hemoglobin A450 plus three standard deviations.
  • Mice (8-week-old male C57BL/6J) were immunized intramuscularly with ovalbumin, SpCas9, or SaCas9 in Freund’s adjuvant (50 µg total per mouse, n = 10 per group; SpCas9 group analyzed n = 9). Serum was collected at weeks 0, 2, 4, and 6; vitreous fluid was collected at week 6 and analyzed by ELISA.
  • Statistical analysis used Anderson-Darling tests for normality, parametric unpaired two-tailed Student’s t-tests or non-parametric two-tailed Mann–Whitney U-tests, with p < 0.05 considered significant; immunoglobulin isotyping used five-parameter logistic regression.

Limitations

  • Stated: human subjects had varying vitreoretinopathies because vitreous fluid was obtained during surgery; the vitreous positivity threshold may be biologically arbitrary; ELISA may miss α-Cas9 below detection; and the clinical impact of low vitreous antibody levels is unclear.
  • Stated: the paired human cohort was small (n = 13) and older (mean age ~66 years, s.d. 13.9), which may explain the unusually high 100% serum α-Cas9 prevalence compared with prior reports; mouse studies had limited sample sizes and one mouse may have had preexisting SaCas9 immunity.
  • Evident from the text: the study is cross-sectional and does not test whether intraocular Cas9 exposure from CRISPR–Cas9 therapy itself increases intraocular α-Cas9 or causes inflammation; mouse vitreous volume limits allowed only two antibody types to be tested in vitreous fluid.

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

Integrity signals

Registry

Crossref · 10.1038/s41467-022-28674-1

Cites

44 of 44 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 58 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    In mice, immunization against ovalbumin, SpCas9, or SaCas9 produced robust, specific, and reliable serum antibody responses by 6 weeks post-injection.

    “There was robust, specific, and reliable immunization of mice against each protein by 6-weeks post-injection (Fig. 2b).”
    Quote in the paperSubstantial claim
  • Findingown data

    SaCas9 immunization resulted in lower serum antibody levels compared with ovalbumin and SpCas9 in mice.

    “Notably, SaCas9 immunization resulted in lower serum antibody levels compared with ovalbumin and SpCas9 in mice.”
    Quote in the paperNot a substantial claim
  • Findingown data

    In mice, higher serum antibody levels indicated higher vitreous fluid antibody levels across all immunization categories.

    “Like the paired human samples, we observed a trend where higher serum antibody levels indicated higher vitreous fluid antibody levels across all immunization categories (Fig. 2f).”
    Quote in the paperNot a substantial claim
  • Findingown data

    Human vitreous fluid contains all immunoglobulin subclasses except possibly IgM.

    “Our analysis detected vitreous antibodies at different concentrations based on subclass, detecting all subclasses except possibly IgM (Fig. 1b).”
    • Also stated in: § Introduction · hedged
      “In human vitreous fluid biopsies, we identify all subclasses of antibodies except for possibly IgM.”
    Quote in the paperSubstantial claim
  • Findingown data

    In mice, the weakest immunization was against SaCas9, with antibody signals less than half of those for ovalbumin or SpCas9 by 6 weeks post-immunization.

    “We observed that mice showed the weakest immunization against SaCas9, as demonstrated by antibody signals less than half of those seen for ovalbumin or SpCas9 by 6-weeks post-immunization (Fig. 2b).”
    Quote in the paperSubstantial claim
  • May combine two claims. 29
  • The quote only partly states this claim. 2
  • Classified as probably not a substantial claim. 23

  • May combine two claims.

    Findingown data

    At least one mouse showed unusually high serum α-SaCas9 signal at week 0 prior to immunization and generated a particularly rapid and robust antibody response.

    “we observed that at least one mouse showed unusually high serum α-SaCas9 signal at the week 0 timepoint (prior to immunization) and generated a particularly rapid and robust antibody response (data not shown).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    IgG4 antibodies comprised ~20% of total vitreous fluid antibodies, a higher fraction than typically observed in serum.

    “Notably, we observed higher vitreous fluid IgG4 levels than expected, finding that IgG4 antibodies comprised ~20% of the total vitreous fluid antibodies, a much higher fraction than typically observed in the serum.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    In a validation cohort of human vitreous fluid biopsies (n=36), no samples were positive for α-SaCas9 and only one sample, from a subject with S. pyogenes endophthalmitis, was positive for α-SpCas9.

    “Here, we detected 33 vitreous fluid samples positive for α-tetanus, no samples positive for α-SaCas9, and only one sample (the infectious endophthalmitis case) positive for α-SpCas9.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In paired human vitreous fluid samples (n=13), only two were positive for α-SaCas9 and two for α-SpCas9.

    “However, only two vitreous fluid samples tested positive for α-SaCas9 and two for α-SpCas9, showing antibody levels only near or below the lowest α-tetanus samples.”
    Quote in the paperSubstantial claim

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

    Findingown data

    Vitreous-to-serum concentration ratios varied by immunoglobulin subclass, with IgG4 at 1:13 and IgM at 1:926.

    “IgG1 = 1:150 (range: 19.05 mg/dL, s.d. 4.93), IgG2 = 1:133 (range: 16.86 mg/dL, s.d. 4.43), IgG3 = 1:205 (range: 2.69 mg/dL, s.d. 0.71), IgG4 = 1:13”
    • Also stated in: § Characterization of human vitreous fluid antibodies · plain
      “IgM = 1:926 (range: 2.40 mg/dL, s.d. 0.45), IgA = 1:230 (range: 8.61 mg/dL, s.d. 1.98).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingreasoning

    α-tetanus, but not α-Cas9, was highly prevalent in vitreous fluid.

    “α-tetanus (a product of deliberate, repeated immunization resulting in high serum antibody concentrations) but not α-Cas9 were highly prevalent in vitreous fluid (Fig. 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Despite likely high sensitivity of our detection platform, we were unable to detect high frequencies of α-Cas9 in vitreous fluid of the eye despite detecting high frequencies of control α-tetanus in the eye.

    “despite the likely high sensitivity of our detection platform, we were still unable to detect high frequencies of α-Cas9 in vitreous fluid of the eye despite detecting high frequencies of control α-tetanus in the eye.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Two subjects in our study (cases #11 and #12) were diagnosed with cone-rod dystrophies, conditions similar to Leber congenital amaurosis, and tested negative for vitreous fluid α-SaCas9 despite demonstrating serum α-SaCas9.

    “two subjects in our study (cases #11 and #12; Supplementary Table 1) were diagnosed with cone-rod dystrophies, conditions similar to Leber congenital amaurosis (the focus of the ongoing aforementioned clinical trial which utilizes SaCas910), and tested negative for vitreous fluid α-SaCas9 despite demonstrating serum α-SaCas9.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [10] Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    These antibodies were typically not detectable in the human eye without prior ocular exposure to Cas9 antigen or severe ocular pathology, and even then, they were observed at relatively low levels.

    “we found that these antibodies were typically not detectable in the human eye without prior ocular exposure to Cas9 antigen (e.g., bacterial eye infection) or severe ocular pathology, and even then, they were observed at relatively low levels.”
    Quote in the paperSubstantial claim

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

    Interpretationown data

    This mouse may have had preexisting immunity to SaCas9, and preexisting Cas9 immunity led to an intensified immune reaction on repeat antigen exposure.

    “This indicates that this mouse may have had preexisting immunity to SaCas9 and that preexisting Cas9 immunity led to an intensified immune reaction on repeat antigen exposure, although no clinical evidence of intraocular inflammation was observed.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The eye lacks T-cells but contains antibodies, cytokines, and resident immune cells.

    “While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells.”
    • Also stated in: § Introduction · plain
      “While systemic immune cells such as T-cells are normally absent from the eye, other immune components—including antibodies, cytokines, and complement molecules—maintain a constant presence19,20.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [19] Song, J., Huang, Y. F., Zhang, W. J., Chen, X. F. & Guo, Y. M. Ocular diseases: immunological and molecular mechanisms. Int J. Ophthalmol. 9, 780–788 (2016).

      Not checked in the free run.

    • cites [20] Skeie, J. M., Roybal, C. N. & Mahajan, V. B. Proteomic insight into the molecular function of the vitreous. PLoS ONE 10, e0127567 (2015). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    In humans, the prevalence of preexisting α-Cas9 in the eye is low despite high prevalence of preexisting serum α-Cas9.

    “Taken together, these data indicate that in humans the prevalence of preexisting α-Cas9 in the eye is low despite high prevalence of preexisting serum α-Cas9.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Human vitreous fluid is thought to contain approximately 100-fold fewer antibodies than serum.

    “Human vitreous fluid is thought to contain approximately 100-fold fewer antibodies than serum and it is unknown whether α-Cas9 is represented21,33.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [21] Allansmith, M. R., Whitney, C. R., McClellan, B. H. & Newman, L. P. Immunoglobulins in the human eye. Location, type, and amount. Arch. Ophthalmol. 89, 36–45 (1973). source

      Not checked in the free run.

    • cites [33] Rose, G. E., Billington, B. M. & Chignell, A. H. Immunoglobulins in paired specimens of vitreous and subretinal fluids from patients with rhegmatogenous retinal detachment. Br. J. Ophthalmol. 74, 160–162 (1990). source

      Not checked in the free run.

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

    Interpretationreasoning

    This risk would likely depend on the amount of Cas9 being expressed by edited cells, the duration of expression needed to achieve therapeutic efficacy, and the potential for extracellular Cas9 leak from edited tissue.

    “This risk would likely depend on the amount of Cas9 being expressed by edited cells, the duration of expression needed to achieve therapeutic efficacy, and the potential for extracellular Cas9 leak from edited tissue.”
    Quote in the paperNot a substantial claim

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

    Interpretationreasoning

    The high prevalence of α-Cas9 observed in our paired cohort may be due to limited sample size (cohort 1, n = 13) or demographic differences between cohorts.

    “The high prevalence we observed here may be due to the limited sample size of our paired cohort (cohort 1, n = 13) or demographic differences between cohorts.”
    Quote in the paperNot a substantial claim

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

    Study design and conductown data

    The validity of antibody frequencies reported in our study is supported by the tractability of our system and patient-to-patient and clinical correlations in our data.

    “the validity of antibody frequencies reported in our study is supported by the tractability of our system”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Vitreous fluid makes up 80% of the human eye by volume.

    “This tissue makes up 80% of the human eye by volume and sequesters both intracellular and extracellular proteins shed from the retina and adjacent tissues20,26–28.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [20] Skeie, J. M., Roybal, C. N. & Mahajan, V. B. Proteomic insight into the molecular function of the vitreous. PLoS ONE 10, e0127567 (2015). source

      Not checked in the free run.

    • cites [26] Velez, G. et al. Precision medicine: personalized proteomics for the diagnosis and treatment of idiopathic inflammatory disease. JAMA Ophthalmol. 134, 444–448 (2016). source

      Not checked in the free run.

    • cites [27] Skeie, J. M. & Mahajan, V. B. Dissection of human vitreous body elements for proteomic analysis. J. Vis. Exp. https://doi.org/10.3791/2455 (2011). source

      Not checked in the free run.

    • cites [28] Murthy, K. R. et al. Proteomic analysis of human vitreous humor. Clin. Proteom. 11, 29 (2014). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    This bodes well for retinal CRISPR-Cas9 therapy, as those eligible also suffer from pre-treatment vitreoretinopathies.

    “This bodes well for retinal CRISPR–Cas9 therapy, as those eligible also suffer from pre-treatment vitreoretinopathies.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    It is possible that vitreous fluid samples contained α-Cas9 below detection capabilities.

    “it is possible that vitreous fluid samples contained α-Cas9 below detection capabilities.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Preexisting immunity indicates that Cas9 protein expressed by edited cells may lead to immunological activation, inflammation, and potentially killing of edited cells.

    “This preexisting immunity indicates that Cas9 protein expressed by edited cells may lead to immunological activation, inflammation, and potentially killing of edited cells—as has been demonstrated in vitro and in vivo4–6.”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites [4] Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019). source

      Not checked in the free run.

    • cites [5] Li, A. et al. AAV-CRISPR Gene editing is negated by pre-existing immunity to Cas9. Mol. Ther. 28, 1432–1441 (2020). source

      Not checked in the free run.

    • cites [6] Crudele, J. M. & Chamberlain, J. S. Cas9 immunity creates challenges for CRISPR gene editing therapies. Nat. Commun. 9, 3497 (2018). source

      Not checked in the free run.

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

    Premisecitation

    Intraocular inflammation remains a major cause of vision loss.

    “Yet, despite immune privilege, intraocular inflammation remains a major cause of vision loss—underscoring the limitations of our current understanding of the phenomenon.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [13] Rothova, A., Suttorp-van Schulten, M. S., Frits Treffers, W. & Kijlstra, A. Causes and frequency of blindness in patients with intraocular inflammatory disease. Br. J. Ophthalmol. 80, 332–336 (1996). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationreasoning

    Large discrepancies between studies regarding the prevalence of α-Cas9 may be due to relatively low levels of circulating α-Cas9 even in positive individuals, and differences in sensitivity between detection platforms used.

    “the large discrepancies between studies regarding the prevalence of α-Cas9 may be due to relatively low levels of circulating α-Cas9 even in positive individuals, and differences in sensitivity between detection platforms used.”
    Quote in the paperSubstantial claim

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

    Study design and conductreasoning

    The cutoffs for calling a vitreous fluid sample positive may be biologically arbitrary despite indicating statistical rarity.

    “despite indicating statistical rarity, these cutoffs may be biologically arbitrary.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The eye exhibits physical barriers and cell-bound and soluble immunosuppressive factors.

    “it has been shown that the eye exhibits physical barriers (e.g., blood-retina-barrier preventing free entry of systemic immune cells) as well as cell-bound and soluble immunosuppressive factors11,12.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [11] Zhou, R. & Caspi, R. R. Ocular immune privilege. F1000 Biol. Rep. 2, https://doi.org/10.3410/B2-3 (2010). source

      Not checked in the free run.

    • cites [12] Taylor, A. W. Ocular immune privilege. Eye (Lond.) 23, 1885–1889 (2009). source

      Not checked in the free run.

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

    Premisecitation

    The eye is currently at the forefront of genome editing, with a phase 1/2 clinical trial pioneering one of the first in vivo human trials for CRISPR-Cas9 therapy.

    “The eye is currently at the forefront of genome editing, with a phase 1/2 clinical trial pioneering one of the first in vivo human trials for CRISPR–Cas9 therapy (identifier: NCT03872479)10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Maeder, M. L. et al. Development of a gene-editing approach to restore vision loss in Leber congenital amaurosis type 10. Nat. Med. 25, 229–233 (2019). source

      Not checked in the free run.

  • May combine two claims.

    Interpretationcitation

    The prevalence of cellular immunity is closer to that which we detect for α-Cas9, indicating regular immunologic exposure.

    “this prevalence of cellular immunity is closer to that which we detect for α-Cas9, indicating regular immunologic exposure.”
    Quote in the paperSubstantial claim

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

    Interpretationreasoning

    The older age of our cohort may be associated with a waning immunity.

    “the older age of our cohort may be associated with a waning immunity.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisereasoning

    The eye is considered an immune privileged organ, and ocular tissues may be less likely to mount inflammatory reactions to gene therapies.

    “As the eye is considered an immune privileged organ, it is thought that ocular tissues may be less likely to mount inflammatory reactions to gene therapies.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Premisenone given

    Cas9 proteins function as endonucleases and are theoretically intracellular, unexposed to circulating antibodies.

    “Cas9 proteins, which function as endonucleases, are theoretically intracellular, unexposed to circulating antibodies.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    Inflammatory reactions against AAV vectors used to deliver gene therapy components have been described.

    “In the context of gene therapy, inflammatory reactions against adeno-associated viral (AAV) vectors used to deliver gene therapy components have been described.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [14] Chan, Y. K. et al. Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Sci. Transl. Med. 13, https://doi.org/10.1126/scitranslmed.abd3438 (2021). source

      Not checked in the free run.

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

    Interpretationcitation

    It is unclear at what level vitreous fluid antibodies become clinically impactful, though even low levels might exert functional consequences.

    “It is unclear at what level vitreous fluid antibodies become clinically impactful, though even low levels might exert functional consequences39.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [39] Andrzejewski, S., Moyle, P. M., Stringer, B. W., Steel, J. C. & Layton, C. J. Neutralisation of adeno-associated virus transduction by human vitreous humour. Gene Ther. 28, 242–255 (2021). source

      Not checked in the free run.

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

    Interpretationreasoning

    The older age of our cohort may explain the lower raw ELISA A450 values we observed compared with previous work.

    “This may explain the lower raw ELISA A450 values we observed compared with previous work (Table 1)3.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249–254 (2019). source

      Not checked in the free run.

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

    Interpretationreasoning

    The older age of our cohort may be associated with increased risk or increased lifetime exposure to S. aureus or S. pyogenes, potentially leading to a higher prevalence of preexisting antibodies against these microbes.

    “the older age of our cohort may also be associated with increased risk or increased lifetime exposure to S. aureus or S. pyogenes, potentially leading to a higher prevalence of preexisting antibodies against these microbes.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Some studies have found preexisting serum α-Cas9 to be present in only 2.5-10% of large human cohorts.

    “some studies have found preexisting serum α-Cas9 to be present in only 2.5–10% of large human cohorts1,2.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Simhadri, V. L. et al. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. Mol. Ther. Methods Clin. Dev. 10, 105–112 (2018). source

      Not checked in the free run.

    • cites [2] Ferdosi, S. R. et al. Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes. Nat. Commun. 10, 1842 (2019). source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    The relevance of α-Cas9 in serum compared to vitreous fluid may differ with regards to CRISPR-Cas9 gene therapy, reflecting the unique immunological environment and physiology of the eye.

    “The relevance of α-Cas9 in serum compared to vitreous fluid may differ with regards to CRISPR–Cas9 gene therapy, reflecting the unique immunological environment and physiology of the eye.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    Written informed consent was obtained from all participants.

    “Written informed consent was obtained from all participants, explaining the nature and purpose of the study.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    To check for normal distribution of ELISA data, we performed Anderson-Darling tests.

    “To check for normal distribution of ELISA data, we performed Anderson-Darling tests.”
    Quote in the paperSubstantial claim

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

    Study design and conductnone given

    All mouse experiments described were conducted in accordance with approved Stanford University IACUC protocols and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

    “All mouse experiments described were conducted in accordance with approved Stanford University IACUC protocols and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    For paired serum/vitreous biopsies (cohort 1), vitreous fluid and serum were collected within one hour of one another.

    “For paired serum/vitreous biopsies (cohort 1), vitreous fluid and serum were collected within one hour of one another.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    The study obtained Stanford University Institutional Review Board approval and adhered to the tenets set forth in the Declaration of Helsinki.

    “The study obtained Stanford University Institutional Review Board approval and adhered to the tenets set forth in the Declaration of Helsinki.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    Our data indicate the same is likely true for any systemically circulating antibody, though antibody subclass may also play a role.

    “Our data indicate the same is likely true for any systemically circulating antibody, though antibody subclass may also play a role.”
    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

    ELISAs were performed in experimental triplicates and the mean average measurements from those three experiments are shown.

    “ELISAs were performed in experimental triplicates and the mean average measurements from those three experiments are shown.”
    Quote in the paperNot a substantial claim
42 of 42

  • The paper relies on this without support.

    S. pyogenes intraocular infection exposes Cas9 protein to the eye, leading to detectable α-SpCas9 in vitreous fluid.

    “identification of α-SpCas9 in vitreous fluid from a subject diagnosed with S. pyogenes endophthalmitis shows that direct intraocular exposure to Cas9 may still increase α-Cas9 levels in vitreous fluid (Fig. 1f).”

  • The paper relies on this without support.

    Choroidal melanoma can cause severe blood-retina-barrier disruption and allow serum antibody leak into vitreous fluid.

    “This patient had been diagnosed with choroidal melanoma, a form of ocular cancer with the potential for major anatomical disruption to the eye and severe damage to the blood-retina-barrier with high potential for antibody leak from the serum into the vitreous fluid.”

  • The paper relies on this without support.

    Mouse ocular antibody responses to intramuscular immunization are informative for human ocular antibody dynamics.

    “The use of mice may provide an important in vivo approach as new CRISPR–Cas9 technology continues to make its way into human medical therapy.”

  • The paper relies on this without support.

    Sick cells that undergo editing may not survive and release cytosolic contents, allowing intracellular Cas9 to become exposed to intraocular antibodies.

    “Additionally, sick cells that undergo editing may not survive, ultimately releasing cytosolic contents.”

  • The paper relies on this without support.

    Systemic tissues are subject to regular surveillance by T-cells, unlike the eye.

    “Unlike in the eye, systemic tissues are subject to regular surveillance by T-cells.”

  • The paper relies on this without support.

    Surgical delivery of gene therapies in the eye may involve some degree of traumatic disruption to ocular anatomy.

    “While surgical delivery of gene therapies in the eye (particularly subretinal injection compared with intravitreal injections) may involve some degree of traumatic disruption to ocular anatomy”

  • The paper relies on this without support.

    ELISAs are sensitive.

    “while ELISAs are sensitive”
7 of 7

Claims other papers make about this one

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

  • In 13 patients who received vitreoretinal surgery with vitrectomy, all three serum samples were positive for SaCas9 and SpCas9, while only two vitreous fluid samples tested positive for SaCas9 and two others for SpCas9.

    “A study that compared the pre-existing anti-SaCas9 and anti-SpCas9 antibodies between serum and vitreous liquid from 13 patients who received vitreoretinal surgery with vitrectomy concluded that all the three serum samples were positive for SaCas9 and SpCas9, while only two vitreous fluid samples tested positive for SaCas9 and two others for SpCas9.”

    From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [26]

  • A study comparing serum and vitreous anti-Cas9 antibody levels in WT C57BL/6J mice immunized intramuscularly with Cas9 reported lower expression of antibodies in vitreous fluid compared to serum, implying preexisting immunity to Cas9 may present a lower risk in human eyes than systemically administered ones.

    “A study aimed at comparing the serum and vitreous anti-Cas9 antibody levels in wild-type (WT) C57BL/6J mice immunized intramuscularly wild-type (WT) C57BL/6J mice with Cas9 intramuscularly, and reported a lower expression of antibodies in vitreous fluid compared to the serum, implying that preexisting immunity to Cas9 may present a lower risk in human eyes than systemically administered ones [26].”

    From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [26]

  • Antibody levels were higher in serum than in vitreous liquid.

    “This study also showed that the antibodies' levels were higher in serum than in vitreous liquid [26].”

    From Immune responses to gene editing by viral and non-viral delivery vectors used in retinal gene therapy (2022) · cites this paper as [26]

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.

  • Blood collection procedure both prevents and requires clotting

    Minor

    The methods say blood was collected into EDTA tubes to prevent coagulation, then say the same blood was left to form clots before the serum was collected.

  • Table 1 assigns Charlesworth the wrong reference number

    Minor

    Table 1 labels the comparison study Charlesworth et al.4, but reference 4 is by Wagner; Charlesworth is reference 3.

2 of 2

Cites

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1 of the 45 references couldn’t be matched to a known paper.

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Related

Authors
Toral, M. A., Charlesworth, C. T., Ng, B., Chemudupati, T., Homma, S., Nakauchi, H., Bassuk, A. G., Porteus, M. H., & Mahajan, V. B.
Affiliations
  • University of Iowa (US)
  • Stanford University (US)
  • University of Oxford (GB)
  • Institute for Stem Cell Biology and Regenerative Medicine (US)
  • VA Palo Alto Health Care System (US)
Corresponding author
  • Porteus, Matthew H.
  • Mahajan, Vinit B.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2022-02-25
Volume
13
Issue
1
Pages
1053-1053
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-022-28674-1
PMC
PMC8881612
OpenAlex
W4214628568
Cited by
28
References
45
Citations by year
2026: 4 · 2025: 8 · 2024: 4 · 2023: 7 · 2022: 5
FWCI
1.96
Type
journal article
Language
en
Update notices
correction (2022-04-12)
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • U.S. Department of Health & Human Services | NIH | National Eye Institute (R01EY031952, R01EY030151, R01EY025225, R01EY024698, P30EY026877)
  • Research to Prevent Blindness
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Health Sciences › Medicine › Ophthalmology › Ocular Diseases and Behçet’s Syndrome
  • Health Sciences › Medicine › Ophthalmology › Ocular Infections and Treatments
Keywords
pre-existing immunity, ocular immunology, vitreous fluid, serum antibodies, Streptococcus pyogenes, intraocular infection, intraocular inflammation, vision loss, in vivo gene editing
MeSH terms
CRISPR-Associated Protein 9, Animals, Antibodies, Humans, Streptococcus pyogenes, T-Lymphocytes, Mice, CRISPR-Cas Systems

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.

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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