AAV2 delivery of the saCas9 gene results in presentation of an HLA-A∗02:01-restricted T cell epitope potent to induce T cell cytotoxicity

Susana S. Najera; Annalisa Nicastri; Sojin Bing; Abdul Mohin Sajib; Nicola Ternette; Ronit Mazor

doi:10.1016/j.omtm.2025.101506

AAV2 delivery of the saCas9 gene results in presentation of an HLA-A^∗02:01-restricted T cell epitope potent to induce T cell cytotoxicity

Susana S. Najera, Annalisa Nicastri, Sojin Bing, Abdul Mohin Sajib, Nicola Ternette (Lead / Corresponding author), Ronit Mazor (Lead / Corresponding author)

Cell Signalling and Immunology

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Abstract

In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A^∗02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.

Original language	English
Article number	101506
Number of pages	9
Journal	Molecular Therapy Methods and Clinical Development
Volume	33
Issue number	3
Early online date	9 Jun 2025
DOIs	https://doi.org/10.1016/j.omtm.2025.101506
Publication status	Published - 11 Sept 2025

Keywords

class I
CRISPR
gene editing
genome editing
immunogenicity
immunotoxicity
MHC I

ASJC Scopus subject areas

Molecular Medicine
Molecular Biology
Genetics

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10.1016/j.omtm.2025.101506Licence: CC BY-NC-ND

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Cite this

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title = "AAV2 delivery of the saCas9 gene results in presentation of an HLA-A∗02:01-restricted T cell epitope potent to induce T cell cytotoxicity",

abstract = "In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A∗02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.",

keywords = "class I, CRISPR, gene editing, genome editing, immunogenicity, immunotoxicity, MHC I",

author = "Najera, \{Susana S.\} and Annalisa Nicastri and Sojin Bing and Sajib, \{Abdul Mohin\} and Nicola Ternette and Ronit Mazor",

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T1 - AAV2 delivery of the saCas9 gene results in presentation of an HLA-A∗02:01-restricted T cell epitope potent to induce T cell cytotoxicity

AU - Najera, Susana S.

AU - Nicastri, Annalisa

AU - Bing, Sojin

AU - Sajib, Abdul Mohin

AU - Ternette, Nicola

AU - Mazor, Ronit

PY - 2025/9/11

Y1 - 2025/9/11

N2 - In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A∗02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.

AB - In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A∗02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.

KW - class I

KW - CRISPR

KW - gene editing

KW - genome editing

KW - immunogenicity

KW - immunotoxicity

KW - MHC I

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