High prevalence of S. pyogenes Cas9-specific T cell sensitization within the adult human population – A balanced effector/regulatory T cell response

Summary Paragraph

The field of gene therapy has been galvanized by the discovery of the highly efficient and adaptable site-specific nuclease system CRISPR/Cas9 from bacteria.^1,2 Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues.^3-5 Immunocompetent mice treated with CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the Cas9 protein, that impact the efficacy of treatment and can cause tissue damage.^5,6 Most applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into the target cell. Thus, a putative humoral antibody response may be negligible.⁵ However, intracellular protein degradation processes lead to peptide presentation of Cas9 fragments on the cellular surface of gene-edited cells that may be recognized by T cells. While a primary T cell response could be prevented or delayed, a pre-existing memory would have major impact. Here, we show the presence of a ubiquitous memory/effector T cell response directed towards the most popular Cas9 homolog from Streptococcus pyogenes (SpCas9) within healthy human subjects. We have characterized SpCas9-reactive memory/effector T cells (T_EFF) within the CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, SpCas9-specific regulatory T cells (T_REG) profoundly contribute to the pre-existing SpCas9-directed T cell immunity. The frequency of SpCas9-reactive T_REG cells inversely correlates with the magnitude of the respective T_EFF response. SpCas9-specific T_REG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired T_EFF response in vivo. Furthermore, the equilibrium of Cas9-specific T_EFF and T_REG cells may have greater importance in Streptococcus pyogenes-associated diseases. Our results shed light on the T cell mediated immunity towards the much-praised gene scissor SpCas9 and offer a possible solution to overcome the problem of pre-existing immunity.