Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans

Abstract

The CRISPR-Cas9 system has proven to be a powerful tool for genome editing, allowing for the precise modification of specific DNA sequences within a cell. Many efforts are currently underway to use the CRISPR-Cas9 system for the therapeutic correction of human genetic diseases. The most widely used homologs of the Cas9 protein are derived from the bacteria Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes). Based on the fact that these two bacterial species cause infections in the human population at high frequencies, we looked for the presence of pre-existing adaptive immune responses to their respective Cas9 homologs, SaCas9 (S. aureus homolog of Cas9) and SpCas9 (S. pyogenes homolog of Cas9). To determine the presence of anti-Cas9 antibodies, we probed for the two homologs using human serum and were able to detect antibodies against both, with 79% of donors staining against SaCas9 and 65% of donors staining against SpCas9. Upon investigating the presence of antigen-specific T-cells against the two homologs in human peripheral blood, we found anti-SaCas9 T-cells in 46% of donors. Upon isolating, expanding, and conducting antigen re-stimulation experiments on several of these donors' anti-SaCas9 T-cells, we observed an SaCas9-specific response confirming that these T-cells were antigen-specific. We were unable to detect antigenspecific T-cells against SpCas9, although the sensitivity of the assay precludes us from concluding that such T-cells do not exist. Together, this data demonstrates that there are pre-existing humoral and cell-mediated adaptive immune responses to Cas9 in humans, a factor which must be taken into account as the CRISPR-Cas9 system moves forward into clinical trials.

Lessons learned from lung and liver in-vivo gene therapy: implications for the future

“…A possible roadblock to effective clinical translation of the CRISPR/Cas9 research revolution is the recent finding that a large proportion of humans have pre-existing immunity to two of the widely-used Cas9 orthologues, saCas9 and spCas9 [99]. The antibody reaction found against both orthologues is troubling for the in-vivo use of ribonucleoprotein (RNP) mixtures of recombinant Cas9 protein and synthetic gRNAs without any form of cloaking.…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…As the CRISPR‐Cas systems are of prokaryotic origin, it remains to be seen whether innate or adaptive immune responses will hinder their clinical application. Both Streptococcus pyogenes and Staphylococcus aureus are human pathogens; preexisting antibodies against the Cas9 proteins from these bacterial species have been identified in humans . The presence of humoral and cell‐mediated immunity to Cas proteins is a challenge but could be mitigated by immunosuppression, limiting exposure to a single dose, and/or using Cas proteins from microbes that humans have not encountered and acquired immunity against .…”

“…While awaiting the first news of how CRISPR genome editing performs in the clinic, researchers are working to surmount some of the technique's known limitations. Namely, there is a dearth of sufficient methods for delivering genome‐editing molecules into disease‐affected patient tissues; desired edits must be performed without causing any genetic collateral damage; and the possibility of preexisting immunity or a post‐treatment immune reaction to bacterial CRISPR proteins must be assessed . As some scientists refine the technology and gear up for clinical use, others continue to find novel medical uses for CRISPR enzymes.…”

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

“…The authors targeted Cas9 to a binding site for BCL11A within the promoter of γ‐globin and demonstrated efficient switch from human β‐globin to γ‐globin expression in red blood cells. A potential obstacle for human translation is the broad pre‐existing immunity to Cas9 in humans (Charlesworth et al , ; Wagner et al , ), which may be avoided by administration of immunosuppressive drugs. In vivo gene correction or gene insertion would require additional delivery of the DNA repair template and low HR frequencies would be expected in HSCs that have not been stimulated with cytokines, which is standard in ex vivo HSC therapies.…”