Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange

Abstract

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation. Here we show a proof-of-concept for reprogramming MHC-specificity by performing CRISPR-Cas9-assisted cassette exchange. Using murine antigen presenting cell lines (RAW264.7 macrophages), we demonstrate that the generation of Cas9-induced double-stranded breaks flanking the native MHC-I H2-Kd locus led to exchange of an orthogonal H2-Kb allele. MHC surface expression allowed for easy selection of reprogrammed cells by flow cytometry, thus obviating the need for additional selection markers. MHC-reprogrammed cells were fully functional as they could present H2-Kd-restricted peptide and activate cognate T cells. Finally, we investigated the role of various donor template formats on exchange efficiency, discovering that templates that underwent in situ linearization resulted in the highest MHC-reprogramming efficiency. These findings highlight a potential new approach for the correcting of MHC mismatches in cellular transplantation.

Multiple Knockout of Classical HLA Class II β-Chains by CRISPR/Cas9 Genome Editing Driven by a Single Guide RNA

“…As discussed above, the possibility to expand BLCLs expressing functional single HLA-II molecules in the context of an intact processing machinery also opens new avenues to the molecular characterization of the peptidome displayed by individual HLA-II Ags, a burning question fueled by the galloping recent advances in cancer immunotherapy. Finally, in a longer term perspective, our method might also prove useful in clinical protocols of personalized cellular therapy, for instance, for the generation of HLA-II-compatible hematopoietic stem cells for transplantation (45) or for the reduction of organ graft rejection by HLA-II knockout endothelial cells as previously suggested (9, 46). Rescue of HLA-DP expression and recognition by alloreactive T cells in HLA-II knockout BLCLs.…”

Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering

“…Genetic engineering of HLA alleles was proposed recently as a means of developing immunocompatible donor stem cells 39,40 . However, whether HLA-engineered stem cells are suitable for allotransplantation has not been demonstrated.…”

Molecular Design, Optimization, and Genomic Integration of Chimeric B Cell Receptors in Murine B Cells

“…While many years of work have aimed to reprogram immune cells for therapeutic purposes, such as CAR T cell therapy, these have almost exclusively relied on viral-based gene transfer. Recently, genome editing platforms providing targeted integration, most notably CRISPR-Cas9, have become promising tools to further improve current immune cell therapies, by offering potential advantages related to safety, uniform expression levels and potency (54, 58–60). Establishing a preclinical genome editing platform based on primary murine B cells does not only show progress on cellular engineering of technically challenging target cell lines, but also allows the investigation of these cells as novel vehicle for adoptive immune cell therapies.…”