Other - Gene-Therapy / Immunity
Previous studies showed that there is a pre-existing adaptive immunity toward CRISPR-associated nucleases (Cas) in humans. Clinical studies of viral gene therapy suggest that immune responses toward vectors may decrease the efficacy and safety of these treatments. Thus, clinical translation of CRISPR-Cas-based therapies will require careful immune monitoring as well as strategies to suppress unwanted immune responses in patients. We characterized the T-cell responses toward the whole protein of the CRISPR-associated nuclease derived from Streptococcus pyogenes (SpCas9) in the peripheral blood of healthy human donors. SpCas9-reactive T-cells were found in 95% of all the human donors comprising both effector and regulatory subsets with distinct functional properties. SpCas9-reactive regulatory T-cells suppressed the proliferation and cytokine production of their effector counterpart in vitro. Through depletion of regulatory T-cells, we established SpCas9-reactive effector T-cell lines that lysed SpCas9-expressing target cells in a dose-dependent manner. Further, we established an efficient and rapid method to identify the entire SpCas9-reactive T-cell repertoire using a customized peptide library covering the complete amino-acid sequence of SpCas9. Our method allows immune monitoring for risk assessment before and during clinical trials employing Cas-derived therapeutic approaches. Moreover, it provides the basis for an unsupervised characterization of the antigenic structures that mount the pre-existing Cas-reactive T-cell response. In addition, SpCas9-reactive effector T-cell lines could be used to evaluate the immunogenicity of autologous Cas-modified cell products before infusion into the patient. Regulatory T-cells with specificity for Cas epitopes may be an attractive option for a targeted immunosuppression strategy during gene therapy.