High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population

Abstract

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][4][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 (TEFF) within the CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with the magnitude of the respective TEFF response. SpCas9-specific TREG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response in vivo. Furthermore, the equilibrium of Cas9-specific TEFF and TREG 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. TextSpCas9 was the first Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated nuclease hijacked to introduce DNA double-strand breaks at specific DNA sequences. 1 Through the ease of target adaption and the remarkable efficacy, it advanced to the most popular tool for re-writing genes in research and potential clinical applications. The major concern for clinical translation of CRISPR/Cas9 technology is the risk for off-target activity causing potentially harmful mutations or chromosomal aberrations. 2,7 High-fidelity Cas9 enzymes were developed to reduce the probability of these events. 8 Furthermore, novel Cas9-based fusion proteins allow base editing or specific epigenetic reprogramming without inducing breaks in the DNA. 9,10 Most approaches are based on the original SpCas9 enzyme that originates in the facultatively

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…First, the source of Cas9 nucleases, that is, S. pyogenes and Staphylococcus aureus , are common human pathogens. Recent reports have highlighted pre-existing immunity towards both SpCas9 and SaCas9 in the human population, with a high prevalence of both Cas9-reactive T cells and antibodies [71]. Although it is still unclear whether adeno-associated virus delivery of Cas9 leads to the immune rejection of transduced cells in vivo , strategies to control the anti-Cas9 T cell responses, such as transient immunosuppression or engineering Cas9 proteins with mutated T cell epitopes, are being considered [72].…”

Evaluating the Probability of CRISPR-based Gene Drive Contaminating Another Species

“…pyogenes is a facultative pathogenic bacteria mostly restricted to humans, with about 10-20% of the population being asymptomatic carriers (Shaikh et al 2010;Roberts et al 2012) . It is thus no surprise that recent investigations have found anti-Cas9 antibodies and Cas9-reactive T cells in several human populations (Charlesworth et al 2019;Wagner et al 2019) . It is thus possible that certain humans are immune to gene drives, and future studies will undoubtedly shed light on this question.…”

Genschere Crispr‐Cas9 in den Schlagzeilen

“…Aber auch vor einer Anwendung in der somatischen Gentherapie ist noch viel Forschungsarbeit notwendig, wie Untersuchungen der Charité‐Universitätsmedizin Berlin zeigen . Die als erste entdeckte Nuklease, Sp‐Cas9 des CRISPR‐Cas9‐Systems, stammt aus dem Bakterium Streptococcus pyogenes , einem verbreiteten Erreger von Infektionskrankheiten beim Menschen.…”