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.
Highlights
CRISPR–Cas[9] mediated genome editing offers unprecedented opportunities for treating human diseases
The mass spectrometry (MS)-based Major Histocompatibility Complex (MHC) associated peptide proteomics (MAPPs) assay is an analytical tool that identifies the peptides from the protein that are naturally processed and presented on the MHC molecules expressed by antigen-presenting cells[15,16]
Part 1: a set of 209 overlapping peptides spanning the primary sequence of SaCas[9] (Supplementary Fig. 1) were used to identify peptides that elicited CD4+ T-cell proliferation in peripheral blood mononuclear cells (PBMCs) sourced from 21 healthy donors
Summary
CRISPR–Cas[9] mediated genome editing offers unprecedented opportunities for treating human diseases. Consistent with these findings, in silico assessment of the Cas[9] protein indicates that several peptides derived from SpCas[9] bind to MHC proteins with high affinity[9] These and other studies present evidence of memory T-cells to Cas[9] in the human population[3,4,5,7,9] due to exposure to Staphylococcus[5] the identification of biologically/ clinically meaningful T-cell epitopes has not been accomplished, i.e., Cas9-derived peptides that are generated by the proteolytic machinery of antigen-presenting cells (APCs), presented by MHC molecules and elicit T-cell proliferation. Similar strategies can be designed where SaCas[9] proteins are expressed in antigen-presenting cells, and peptides expressed on MHC Class I (MHC I) proteins that stimulate CD8+ T-cells are identified but are not part of this study
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