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Updated: Oct 23, 2025
Use of Single Chain MHC Technology to Investigate Co-agonism in Human CD8+ T Cell Activation
Published on: February 28, 2019
Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells
Vijaya L Simhadri1, Louis Hopkins1, Joseph R McGill1
1Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.
Abstract:
CRISPR-Cas9 mediated genome editing offers unprecedented opportunities for treating human diseases. There are several reports that demonstrate pre-existing immune responses to Cas9 which may have implications for clinical development of CRISPR-Cas9 mediated gene therapy. Here we use 209 overlapping peptides that span the entire sequence of Staphylococcus aureus Cas9 (SaCas9) and human peripheral blood mononuclear cells (PBMCs) from a cohort of donors with a distribution of Major Histocompatibility Complex (MHC) alleles comparable to that in the North American (NA) population to identify the immunodominant regions of the SaCas9 protein. We also use an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC Class II (MHC-II) proteins on dendritic cells. Using these two data sets we identify 22 SaCas9 peptides that are both presented by MHC-II proteins and stimulate CD4+ T-cells.
Insights
Pre-existing immune responses to Cas9 may impact CRISPR gene therapy. This study identified specific Staphylococcus aureus Cas9 (SaCas9) peptides that trigger immune responses, crucial for safe clinical development.
Area of Science:
- Immunology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 gene editing holds promise for treating human diseases.
- Pre-existing immune responses to Cas9 proteins can hinder clinical applications of CRISPR-Cas9 gene therapy.
Purpose of the Study:
- To identify immunodominant regions of Staphylococcus aureus Cas9 (SaCas9).
- To determine SaCas9 peptides presented by MHC Class II (MHC-II) proteins and recognized by CD4+ T-cells.
Main Methods:
- Utilized 209 overlapping peptides spanning the SaCas9 sequence.
- Employed human peripheral blood mononuclear cells (PBMCs) from diverse Major Histocompatibility Complex (MHC) allele donors.
- Applied an MHC Associated Peptide Proteomics (MAPPs) assay to identify SaCas9 peptides presented by MHC-II on dendritic cells.
Main Results:
- Identified immunodominant regions of the SaCas9 protein.
- Detected 22 SaCas9 peptides presented by MHC-II proteins.
- Confirmed these peptides stimulate CD4+ T-cells, indicating immune recognition.
Conclusions:
- Characterized specific SaCas9 T-cell epitopes.
- Provided critical data for assessing and mitigating immune responses in SaCas9-based gene therapies.
- Informed the clinical development of CRISPR-Cas9 therapies using SaCas9.
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