AAV-CRISPR Gene Editing Is Negated by Pre-existing Immunity to Cas9
Open accessAbstract
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.
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Preexisting T-cell populations may represent a greater risk to gene-edited systemic tissues than antibodies at any prevalence or concentration.
“preexisting T-cell populations may represent a greater risk to gene-edited systemic tissues than antibodies at any prevalence or concentration4–6.”
From Investigation of Cas9 Antibodies in the Human Eye (2022) · cites this paper as [5]
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.”
From Investigation of Cas9 Antibodies in the Human Eye (2022) · cites this paper as [5]
In mice, pre-existing SaCas9 immunity resulted in increased CTL response leading to hepatocyte apoptosis and loss of transgene.
“Studies showed pre-existing SaCas9 immunity in mice resulted in increased CTL response leading to hepatocyte apoptosis and loss of transgene (112).”
From Immunomodulation in Administration of RAAV: Preclinical and Clinical Adjuvant Pharmacotherapies (2021) · cites this paper as [112]
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- 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
- Venue
- Molecular Therapy
- Publisher
- Elsevier BV
- Published
- 2020-06
- Volume
- 28
- Issue
- 6
- Pages
- 1432-1441
- ISSN
- 1525-0016 (ISSN-L)
- 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 · 2019: 2
- FWCI
- 11.02
- Type
- journal article
- Language
- en
- Open access
- hybrid
- doi.org
- Licence
- 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
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