The discovery of the highly efficient site-specific nuclease system CRISPR–Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy1,2. The immunogenicity of Cas9 nuclease has been demonstrated in mice3,4. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues3,4,5,6. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease7,8. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell–mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.
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The underlying data are available from the corresponding author upon reasonable request. TRB sequencing data have been deposited in the immuneACCESS database at https://doi.org/10.21417/B7ZP86 (http://clients.adaptivebiotech.com/pub/wagner-2018-naturemedicine).
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The study was generously supported in parts by the Deutsche Forschungsgemeinschaft (German Research Foundation, SFB-TR36-project A3 grant to H.-D.V., P.R., M.S.H.), the German Federal Ministry of Education and Research (BCRT grant, all authors), a kick-box grant for young scientists by the Einstein Center for Regenerative Therapies (D.L.W.), and the Berlin Institute of Health medical doctoral research stipend (D.L.W.). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. We would like to acknowledge the assistance of the BCRT Flow & Mass Cytometry Lab, D. Kunkel and J. Hartwig; G. Grütz and the team of the Core Unit Biomarker—Immunological Study Lab, BCRT, Charité—Universitätsmedizin Berlin; M. Streitz for flow cytometry assistance and CMV lysates; K. Vogt for technical assistance with the TSDR analyses; A. Jurisch, K. Grzeschik, and R. Noster for technical assistance; and A. Floriane Hennig and U. Kornak (Institute for Medical Genetics and Human Genetics, Charité – Universitätsmedizin Berlin) for the PX458_T2mali DNA plasmid. We thank the groups of M. Seifert and A. Thiel (both BCRT, Charité—Universitätsmedizin Berlin) for supplying the reagents, and T. Roch and M. Frentsch for critical discussions.
D.L.W., L. Amini, D.J.W., M.S.-H., P.R., and H.-D.V. have a patent pending on CRISPR Associated Protein Reactive T Cell Immunity (European patent application EP18163491.6, 2018). L.-M.B. and L. Akyüz have no financial competing interests.
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Wagner, D.L., Amini, L., Wendering, D.J. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat Med 25, 242–248 (2019). https://doi.org/10.1038/s41591-018-0204-6
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