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

Abstract

Paragraph 32The field of gene therapy has been galvanized by the discovery of the highly efficient and 33 adaptable site-specific nuclease system CRISPR/Cas9 from bacteria. 1,2 Immunity against 34 therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative 35 treatment and may pose significant safety issues. [3][4][5] Immunocompetent mice treated with 36 CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the 37 Cas9 protein, that impact the efficacy of treatment and can cause tissue damage. 5,6 Most 38 applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into 39 the target cell. Thus, a putative humoral antibody response may be negligible. 5 However, 40 intracellular protein degradation processes lead to peptide presentation of Cas9 fragments on 41 the cellular surface of gene-edited cells that may be recognized by T cells. While a primary T 42 cell response could be prevented or delayed, a pre-existing memory would have major impact. 43Here, we show the presence of a ubiquitous memory/effector T cell response directed towards 44 the most popular Cas9 homolog from Streptococcus pyogenes (SpCas9) within healthy human 45 subjects. We have characterized SpCas9-reactive memory/effector T cells (TEFF) within the 46 CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, 47SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9-48 directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with 49 the magnitude of the respective TEFF response. SpCas9-specific TREG may be harnessed to 50 ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response 51 in vivo. Furthermore, the equilibrium of Cas9-specific TEFF and TREG cells may have greater 52 importance in Streptococcus pyogenes-associated diseases. Our results shed light on the T 53 cell mediated immunity towards the much-praised gene scissor SpCas9 and offer a possible 54 solution to overcome the problem of pre-existing immunity. 55 Text 56SpCas9 was the first Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 57 associated nuclease hijacked to introduce DNA double-strand breaks at specific DNA sequences. 1 58Through the ease of target adaption and the remarkable efficacy, it advanced to the most popular 59 tool for re-writing genes in research and potential clinical applications. The major concern for 60 clinical translation of CRISPR/Cas9 technology is the risk for off-target activity causing potentially 61 harmful mutations or chromosomal aberrations. 2,7 High-fidelity Cas9 enzymes were developed to 62 reduce the probability of these events. 8 Furthermore, novel Cas9-based fusion proteins allow base 63 editing or specific epigenetic reprogramming without inducing breaks in the DNA. 9,10 Most 64 approaches are based on the original SpCas9 enzyme that originates in the facultatively 65 2 pathogenic bacterium Streptococcus ...

Where Will We Draw the Line? Public Opinions of Human Gene Editing

“…Although lacking in specifics, many participants seemed to share the concern that it is premature to introduce germline editing in the clinic, given the unknowns surrounding its safety and efficacy. The safety profile of CRISPR/Cas9 has yet to be fully characterized; safety concerns include the potential for off-target effects, embryo mosaicism, and human adaptive immunity to Cas9 proteins, which may trigger an inflammatory attack or prevent the genetic correction (Charlesworth et al, 2018; Wagner et al, 2018). Two recent reports also suggest that genetically edited cells may be at increased risk to become oncogenic (Haapaniemi, Botla, Persson, Schmierer, & Taipale, 2018; Ihry et al, 2018).…”

Multi-omic Analyses Reveal Minimal Impact of the CRISPR-Cas9 Nuclease on Cultured Human Cells

“…CRISPR-Cas9 is presently the most popular gene editing tool in basic research, biotechnology, and medicine. To interrogate the potential side effects of the system on the host, previous data have mainly addressed the off-targeting mutagenesis as well as the immunogenicity. ,− In the present study, we focus on the core component of CRISPR-Cas9 system, the Cas9 protein, and we ask whether this bacterial-derived endonuclease could influence the cellular homeostasis. Because the long-term expression of Cas9 might trigger nonspecific editing, we chose to analyze the cells transiently transfected with the Cas9 protein such that our data mainly reflect the cellular response to the protein but not to the effects associated with the extended expression of Cas9 or the immune activity.…”

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

“…The current size of the rAAV vector (v3) (Fig. 4b) is ~4.7kb and, if needed, there is sufficient room to incorporate target sequences for the hematopoietic-specific microRNA miR-142 into the St1Cas9 expression cassette to prevent its expression in antigen-presenting cells (APCs) and induce robust tolerance to Cas9 13,[68][69][70][71][72] .…”