Cited by 230CRISPR and Genetic EngineeringVirus-based gene therapy researchRNA Interference and Gene Delivery

The DOI matches its Crossref record; 39 of the 40 references it cites were found in a registry or an index.

AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9

Open access

Li, Ang, Tanner, Mark R., Lee, Ciaran M., Hurley, Ayrea E., De Giorgi, Marco, Jarrett, Kelsey E., Davis, Timothy H., Doerfler, Alexandria M., Bao, Gang, Beeton, Christine, Lagor, William R.·2020·Molecular Therapy

DOI 10.1016/j.ymthe.2020.04.017PMC PMC7264438

Open this paper in CoChat — free account Open PDF

Abstract

Adeno-associated viral (AAV) vectors are a leading candidate for the delivery of CRISPR-Cas9 for therapeutic genome editing in vivo . However, AAV-based delivery involves persistent expression of the Cas9 nuclease, a bacterial protein. Recent studies indicate a high prevalence of neutralizing antibodies and T cells specific to the commonly used Cas9 orthologs from Streptococcus pyogenes (SpCas9) and Staphylococcus aureus (SaCas9) in humans. We tested in a mouse model whether pre-existing immunity to SaCas9 would pose a barrier to liver genome editing with AAV packaging CRISPR-Cas9. Although efficient genome editing occurred in mouse liver with pre-existing SaCas9 immunity, this was accompanied by an increased proportion of CD8 + T cells in the liver. This cytotoxic T cell response was characterized by hepatocyte apoptosis, loss of recombinant AAV genomes, and complete elimination of genome-edited cells, and was followed by compensatory liver regeneration. Our results raise important efficacy and safety concerns for CRISPR-Cas9-based in vivo genome editing in the liver.

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

Integrity signals

Registry

Crossref · 10.1016/j.ymthe.2020.04.017

Cites

39 of 39 references verified0 citation errors · 0 not checkable

Statistics

Graphical abstract CC BY-NC-ND · PubMed Central

Figure 1 Induction of Immune Memory against SaCas9 in C57BL/6 Mice (A) Mice were initially immunized with either purified ovalbumin (Ova) or SaCas9 protein. One week after immunization, mice were challenged with either ovalbumin or SaCas9, and ear thickness was measured after 24 h. (B) Histological analysis of the left ear of mice in (A) stained with hematoxylin and eosin. ∗p < 0.05, ∗∗p < 0.01; ns, not significant. Scale bar, 50 μm. (C) Representative H&E staining of mouse ears.CC BY-NC-ND · PubMed Central

Figure 2 AAV-CRISPR Gene Editing of Mice Immunized against SaCas9 Elicits a Robust CD8+ T Cell Response (A) Mice were immunized with either ovalbumin or SaCas9 protein 1 week prior to co-delivery of AAV vectors encoding a gRNA targeting Ldlr with SaCas9 and GFP. Tissues were harvested at 1, 2, 4, 6, and 12 weeks post-injection. (B) CD8+ T cells as a percentage of total lymphocytes in the liver were measured by flow cytometry. (C) CD8 mRNA expression between mice immunized against ovalbumin and SaCas9 measured by qPCR. (D) Representative TUNEL staining in mouse liver sections. (E) Serum alanine transaminase (ALT) activity levels indicating liver damage. (F) Representative Ki-67 staining of mouse liver sections. (G) Ki-67 mRNA expression in mouse liver. Group sizes in (C), (E), and (G) are n = 7, except for ovalbumin 6 weeks (n = 6) and SaCas9 1 week (n = 6).CC BY-NC-ND · PubMed Central

Figure 3 Pre-existing Immunity to SaCas9 Results in Elimination of Gene-Edited Hepatocytes following AAV Transduction (A) AAV genome copies of GFP transgene measured via qPCR. (B) Fold change of GFP genome copies relative to ovalbumin-treated mice. (C) AAV genome copies of SaCas9 transgene measured via qPCR. (D) Fold change of SaCas9 genome copies relative to ovalbumin-treated mice. (E) Representative western blots of LDLR, SaCas9 (hemagglutinin, HA-Tag), and GFP in mouse liver. Asterisk indicates non-specific band by the HA-Tag antibody. (F) Ldlr indels in mouse liver samples were measured via deep sequencing.CC BY-NC-ND · PubMed Central

Figure 4 CD8+ T Cell and Treg Responses in SaCas9-Immunized Mice after Treatment with Either AAV-GFP or AAV-CRISPR (A) Mice were immunized with SaCas9 protein 1 week prior to delivery of an AAV vector encoding a gRNA targeting Ldlr with SaCas9 or GFP. Tissues were harvested at 2 weeks post-injection. (B) T cells as a percentage of total lymphocytes in the liver were measured via flow cytometry. (C) CD8+ T cells as a percentage of T cells. (D) Percentage of memory CD8+ to total CD8+ T cells. (E) Percentage of activated CD8+ T cells to total CD8+ T cell population. (F) Percentage of activated memory CD8+ T cells to total CD8+ T cell population. (G) Tregs as a percentage of total lymphocytes in the liver measured via flow cytometry. n = 6 per condition.CC BY-NC-ND · PubMed Central

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

Claims other papers make about this one

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

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method
Livak, K. J., & Schmittgen, T. D. (2001) · Methods · doi:10.1006/meth.2001.1262 · Cited by 180,860
Mutation of the mouse klotho gene leads to a syndrome resembling ageing
Kuro-o, M., Matsumura, Y., Aizawa, H., Kawaguchi, H., Suga, T., Utsugi, T., Ohyama, Y., Kurabayashi, M., Kaname, T., Kume, E., et al. (1997) · Nature · doi:10.1038/36285 · Cited by 3,837
In vivo genome editing using Staphylococcus aureus Cas9
Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B., Shalem, O., Wu, X., Makarova, K. S., et al. (2015) · Nature · doi:10.1038/nature14299 · Cited by 2,687
Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovirus
Xiao, X., Li, J., & Samulski, R. J. (1998) · Journal of Virology · doi:10.1128/jvi.72.3.2224-2232.1998 · Cited by 1,381
In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy
Nelson, C. E., Hakim, C. H., Ousterout, D. G., Thakore, P. I., Moreb, E. A., Rivera, R. M. C., Madhavan, S., Pan, X., Ran, F. A., Yan, W. X., et al. (2016) · Science · doi:10.1126/science.aad5143 · Cited by 1,144
Identification of preexisting adaptive immunity to Cas9 proteins in humans
Charlesworth, C. T., Deshpande, P. S., Dever, D. P., Camarena, J., Lemgart, V. T., Cromer, M. K., Vakulskas, C. A., Collingwood, M. A., Zhang, L., Bode, N. M., et al. (2019) · Nature Medicine · doi:10.1038/s41591-018-0326-x · Cited by 1,093
In vivo gene editing in dystrophic mouse muscle and muscle stem cells
Tabebordbar, M., Zhu, K., Cheng, J. K. W., Chew, W. L., Widrick, J. J., Yan, W. X., Maesner, C., Wu, E. Y., Xiao, R., Ran, F. A., et al. (2016) · Science · doi:10.1126/science.aad5177 · Cited by 1,046
Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy
Long, C., Amoasii, L., Mireault, A. A., McAnally, J., Li, H. Z., Sanchez‐Ortiz, E., Bhattacharyya, S., Shelton, J. M., Bassel‐Duby, R., & Olson, E. N. (2015) · Science · doi:10.1126/science.aad5725 · Cited by 973
Epitope spreading in immune-mediated diseases: implications for immunotherapy
Vanderlugt, C. L., & Miller, S. D. (2002) · Nature reviews. Immunology · doi:10.1038/nri724 · Cited by 947
A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing
Finn, J. D., Smith, A. R., Patel, M. C., Shaw, L., Youniss, M. R., van Heteren, J., Dirstine, T., Ciullo, C., Lescarbeau, R., Seitzer, J., et al. (2018) · Cell Reports · doi:10.1016/j.celrep.2018.02.014 · Cited by 878
Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo
Yin, H., Song, C.-Q., Dorkin, J. R., Zhu, L. J., Li, Y., Wu, Q., Park, A., Yang, J., Suresh, S., Bizhanova, A., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3471 · Cited by 876
Cd8(+) T-Cell Responses to Adeno-Associated Virus Capsid in Humans
Mingozzi, F., Maus, M. V., Hui, D. J., Sabatino, D. E., Murphy, S. L., Rasko, J. E. J., Ragni, M. V., Manno, C. S., Sommer, J., Jiang, H., et al. (2007) · Nature Medicine · doi:10.1038/nm1549 · Cited by 748
CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences
Lin, Y., Cradick, T. J., Brown, M. T., Deshmukh, H., Ranjan, P., Sarode, N., Wile, B. M., Vertino, P. M., Stewart, F. J., & Bao, G. (2014) · Nucleic Acids Research · doi:10.1093/nar/gku402 · Cited by 634
Self-citation
A Multifunctional AAV-CRISPR-Cas9 and Its Host Response
Chew, W. L., Tabebordbar, M., Cheng, J. K. W., Mali, P., Wu, E. Y., Ng, A. H. M., Zhu, K., Wagers, A. J., & Church, G. M. (2016) · Nature Methods · doi:10.1038/nmeth.3993 · Cited by 617
A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice
Yang, Y., Wang, L., Bell, P., McMenamin, D., He, Z., White, J., Yu, H., Xu, C., Morizono, H., Musunuru, K., et al. (2016) · Nature Biotechnology · doi:10.1038/nbt.3469 · Cited by 556
Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing
Yin, H., Song, C.-Q., Suresh, S., Wu, Q., Walsh, S., Rhym, L. H., Mintzer, E., Bolukbasi, M. F., Zhu, L. J., Kauffman, K., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.4005 · Cited by 499
Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy
Nelson, C. E., Wu, Y., Gemberling, M. P., Oliver, M. L., Waller, M. A., Bohning, J. D., Robinson-Hamm, J. N., Bulaklak, K., Rivera, R. M. C., Collier, J. H., et al. (2019) · Nature Medicine · doi:10.1038/s41591-019-0344-3 · Cited by 444
High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population
Wagner, D. L., Amini, L., Wendering, D. J., Burkhardt, L.-M., Akyüz, L., Reinke, P., Volk, H.-D., & Schmueck-Henneresse, M. (2019) · Nature Medicine · doi:10.1038/s41591-018-0204-6 · Cited by 399
Pre-existing Anti–Adeno-Associated Virus Antibodies as a Challenge in AAV Gene Therapy
Louis Jeune, V., Joergensen, J. A., Hajjar, R. J., & Weber, T. (2013) · Human Gene Therapy Methods · doi:10.1089/hgtb.2012.243 · Cited by 358
Virus infection, antiviral immunity, and autoimmunity
Getts, D. R., Chastain, E. M. L., Terry, R. L., & Miller, S. D. (2013) · Immunological Reviews · doi:10.1111/imr.12091 · Cited by 299
Therapeutic levels of FVIII following a single peripheral vein administration of rAAV vector encoding a novel human factor VIII variant
McIntosh, J., Lenting, P. J., Rosales, C., Lee, D., Rabbanian, S., Raj, D., Patel, N., Tuddenham, E. G. D., Christophe, O. D., McVey, J. H., et al. (2013) · Blood · doi:10.1182/blood-2012-10-462200 · Cited by 278
A compact, high-accuracy Cas9 with a dinucleotide PAM for in vivo genome editing
Edraki, A., Mir, A., Ibraheim, R., Gainetdinov, I., Yoon, Y., Song, C.-Q., Cao, Y., Gallant, J., Xue, W., Rivera-Pérez, J. A., et al. (2019) · Molecular Cell · doi:10.1016/j.molcel.2018.12.003 · Cited by 265
Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population
Simhadri, V. L., McGill, J., McMahon, S., Wang, J., Jiang, H., & Sauna, Z. E. (2018) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2018.06.006 · Cited by 241
Multifunctional CRISPR-Cas9 with Engineered Immunosilenced Human T Cell Epitopes
Ferdosi, S. R., Ewaisha, R., Moghadam, F., Krishna, S., Park, J. G., Ebrahimkhani, M. R., Kiani, S., & Anderson, K. S. (2019) · Nature Communications · doi:10.1038/s41467-019-09693-x · Cited by 204
Delayed type hypersensitivity: Current theories with a historic perspective
Black, C. A. (1999) · Dermatology Online Journal · doi:10.5070/d32fw0g1xx · Cited by 202
Imaging of Effector Memory T Cells during a Delayed-Type Hypersensitivity Reaction and Suppression by Kv1.3 Channel Block
Matheu, M. P., Beeton, C., Garcia, A., Chi, V., Rangaraju, S., Safrina, O., Monaghan, K. P., Uemura, M. I., Li, D., Pal, S., et al. (2008) · Immunity · doi:10.1016/j.immuni.2008.07.015 · Cited by 196
Self-citation
RNA-guided transcriptional silencing in vivo with S. aureus CRISPR-Cas9 repressors
Thakore, P. I., Kwon, J. B., Nelson, C. E., Rouse, D. C., Gemberling, M. P., Oliver, M. L., & Gersbach, C. A. (2018) · Nature Communications · doi:10.1038/s41467-018-04048-4 · Cited by 181
Next-generation AAV vectors—Do not judge a virus (only) by its cover
Domenger, C., & Grimm, D. (2019) · Human Molecular Genetics · doi:10.1093/hmg/ddz148 · Cited by 147
A Self-Deleting AAV-CRISPR System for In Vivo Genome Editing
Li, A., Lee, C. M., Hurley, A. E., Jarrett, K. E., Giorgi, M. D., Lu, W., Balderrama, K. S., Doerfler, A. M., Deshmukh, H., Ray, A., et al. (2018) · Molecular Therapy — Methods & Clinical Development · doi:10.1016/j.omtm.2018.11.009 · Cited by 133
Self-citation
Immune-orthogonal orthologues of AAV capsids and of Cas9 circumvent the immune response to the administration of gene therapy
Moreno, A. M., Palmer, N. D., Alemán, F., Chen, G., Pla, A., Jiang, N. J., Chew, W. L., Law, M., & Mali, P. (2019) · Nature Biomedical Engineering · doi:10.1038/s41551-019-0431-2 · Cited by 132
Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease
Jarrett, K. E., Lee, C. M., Yeh, Y.-H., Hsu, R. H., Gupta, R., Zhang, M., Rodriguez, P. J., Lee, C. S., Gillard, B. K., Bissig, K., et al. (2017) · Scientific Reports · doi:10.1038/srep44624 · Cited by 100
Self-citation
Somatic Editing of Ldlr With Adeno-Associated Viral-CRISPR Is an Efficient Tool for Atherosclerosis Research
Jarrett, K. E., Lee, C. M., Giorgi, M. D., Hurley, A. E., Gillard, B. K., Doerfler, A. M., Li, A., Pownall, H. J., Bao, G., & Lagor, W. R. (2018) · Arteriosclerosis Thrombosis and Vascular Biology · doi:10.1161/atvbaha.118.311221 · Cited by 84
Self-citation
The Balance between CD8(+) T Cell-Mediated Clearance of AAV-Encoded Antigen in the Liver and Tolerance Is Dependent on the Vector Dose
Kumar, S. R. P., Hoffman, B. E., Terhorst, C., de Jong, Y. P., & Herzog, R. W. (2017) · Molecular Therapy · doi:10.1016/j.ymthe.2017.02.014 · Cited by 83
Staphylococcus aureus Cas9 is a multiple-turnover enzyme
Yourik, P., Fuchs, R. T., Mabuchi, M. Y., Curcuru, J. L., & Robb, G. B. (2018) · RNA · doi:10.1261/rna.067355.118 · Cited by 75
Nucleic acid cleavage with a hyperthermophilic Cas9 from an uncultured Ignavibacterium
Schmidt, S., Yu, F. B., Blainey, P. C., May, A. P., & Quake, S. R. (2019) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1904273116 · Cited by 29
Adeno-associated Viruses as Liver-Directed Gene Delivery Vehicles: Focus on Lipoprotein Metabolism
Lagor, W. R., Johnston, J., Lock, M. W., Vandenberghe, L. H., & Rader, D. J. (2013) · Methods in molecular biology · doi:10.1007/978-1-60327-369-5_13 · Cited by 28
Self-citation
Utility of Self-Destructing CRISPR/Cas Constructs for Targeted Gene Editing in the Retina
Fan, L., Hung, S. S. C., Khalid, M. K. N. M., Wang, J.-H., Chrysostomou, V., Wong, V. H. Y., Singh, V., Wing, K., Tu, L., Bender, J. A., et al. (2019) · Human Gene Therapy · doi:10.1089/hum.2019.021 · Cited by 24
Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Beeton, C., & Chandy, G. K. (2007) · Journal of Visualized Experiments · doi:10.3791/325 · Cited by 12
Self-citation
Induction and Monitoring of Adoptive Delayed-Type Hypersensitivity in Rats
Beeton, C., & Chandy, K. G. (2007) · Journal of Visualized Experiments · doi:10.3791/325-v · Cited by 8
Self-citation

Sorted by citations

Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy
Nelson, C. E., Wu, Y., Gemberling, M. P., Oliver, M. L., Waller, M. A., Bohning, J. D., Robinson-Hamm, J. N., Bulaklak, K., Rivera, R. M. C., Collier, J. H., et al. (2019) · Nature Medicine · doi:10.1038/s41591-019-0344-3 · Cited by 444
Immunogenicity of CAR T cells in cancer therapy
Wagner, D. L., Fritsche, E., Pulsipher, M. A., Ahmed, N. M., Hamieh, M., Hegde, M. G., Ruella, M., Savoldo, B., Shah, N. N., Turtle, C. J., et al. (2021) · Nature Reviews Clinical Oncology · doi:10.1038/s41571-021-00476-2 · Cited by 318
Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice
Kenjo, E., Hozumi, H., Makita, Y., Iwabuchi, K. A., Fujimoto, N., Matsumoto, S., Kimura, M., Amano, Y., Ifuku, M., Naoe, Y., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-26714-w · Cited by 313
Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges
Taha, E. A., Lee, J., & Hotta, A. (2022) · Journal of Controlled Release · doi:10.1016/j.jconrel.2022.01.013 · Cited by 259
Microneedle-assisted genome editing: A transdermal strategy of targeting NLRP3 by CRISPR-Cas9 for synergistic therapy of inflammatory skin disorders
Wan, T. Y., Pan, Q., & Yuan, P. (2021) · Science Advances · doi:10.1126/sciadv.abe2888 · Cited by 226

See all 230

Authors: Li, A., Tanner, M. R., Lee, C. M., Hurley, A. E., De Giorgi, M., Jarrett, K. E., Davis, T. H., Doerfler, A. M., Bao, G., Beeton, C., & Lagor, W. R.
Affiliations: Rice University (US)
Baylor College of Medicine (US)
Corresponding author: Bao, Gang
Beeton, Christine
Lagor, William R.
ORCID: Li, Ang
Tanner, Mark R.
Lee, Ciaran M.
Hurley, Ayrea E.
Jarrett, Kelsey E.
Doerfler, Alexandria M.
Bao, Gang
Beeton, Christine
Lagor, William R.
Venue: Molecular Therapy
Publisher: Elsevier BV
Published: 2020-06
Volume: 28
Issue: 6
Pages: 1432-1441
ISSN: 1525-0016 (ISSN-L)
DOI: 10.1016/j.ymthe.2020.04.017
PMC: PMC7264438
OpenAlex: W3016959969
Cited by: 230
References: 40
Citations by year: 2026: 26 · 2025: 33 · 2024: 33 · 2023: 36 · 2022: 38 · 2021: 43 · 2020: 19
FWCI: 11.02
Type: journal article
Language: en
Open access: hybrid
doi.org
Licence: cc-by-nc-nd
www.elsevier.com/tdm/userlicense/1.0/ (tdm)
www.elsevier.com/legal/tdmrep-license (tdm)
creativecommons.org/licenses/by-nc-nd/4.0/ (vor)
Locations: Molecular Therapy (publishedVersion, cc-by-nc-nd)
PubMed Central (publishedVersion, cc-by-nc-nd)
PubMed (publishedVersion)
Digital Commons-TMC (Texas Medical Center) (submittedVersion)
Figshare (submittedVersion, cc-by)
Health Research Alliance (cc-by)
Funders: American Heart Association (19POST34430092, 19PRE34380467, GM08231, HL007676, RR024574)
Cancer Prevention and Research Institute of Texas (RR140081, RP180672)
National Institutes of Health (RR024574, HL007676, GM08231, CA125123, UG3HL151545, HL132840, U42OD026645)
Topics: Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
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
Keywords: adeno-associated virus vectors, CRISPR/Cas9, SaCas9, pre-existing immunity, neutralizing antibodies, T cell response, hepatocyte apoptosis, compensatory liver regeneration, SpCas9
MeSH terms: Gene Editing, CRISPR-Associated Protein 9, RNA, Guide, CRISPR-Cas Systems, Dependovirus, Animals, Genetic Vectors, Humans, Immunization, Immunologic Memory, Biomarkers, Gene Expression, Immunophenotyping, T-Lymphocyte Subsets, Transgenes, Hepatocytes, Gene Order, Mice, Clustered Regularly Interspaced Short Palindromic Repeats

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.

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.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review

AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9

Abstract

Overview

Integrity signals

Figures

Verification

Claims the paper makes

Claims other papers make about this one

Self-check

Citations

Cites

Cited by

Related

Metadata

Record

About these checks