Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes

Abstract

The CRISPR-Cas9 system has raised hopes for developing personalized gene therapies for complex diseases. Its application for genetic and epigenetic therapies in humans raises concerns over immunogenicity of the bacterially derived Cas9 protein. Here we detect antibodies to Streptococcus pyogenes Cas9 (SpCas9) in at least 5% of 143 healthy individuals. We also report pre-existing human CD8+T cell immunity in the majority of healthy individuals screened. We identify two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor PBMCs. In a proof-of-principle study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity. Our study highlights the problem of pre-existing immunity against CRISPR-associated nucleases and offers a potential solution to mitigate the T cell immune response.

Immunogenicity of Cas9 Protein

“…A potential solution to evade an immune response would be "epitope masking," wherein potentially immunogenic peptide sequences are identified and modified or removed to prevent their detection by the immune system, while still maintaining the function of the original protein. In a remarkable study published earlier this year, Ferdosi et al 19 identified 2 immunodominant SpCas9 T cell epitopes for the human leukocyte antigen (HLA)-A*02:01 ( Fig. 2) using an enhanced prediction algorithm that incorporates T cell receptor contact residue hydrophobicity and HLA binding and evaluated them by T cell assays using healthy donor peripheral blood mononuclear cells.…”

Clinical applications of CRISPR‐based genome editing and diagnostics

“…103 Furthermore, targeted mutation of Cas9 has allowed removal of specific T-cell epitopes, thereby engineering an "immunosilenced" version of Cas9 while preserving function. 104 Editing organs that are relatively immune privileged, such as the brain or eye, may present fewer complications therapeutically. 105 The gRNA is responsible for programming specificity based on complementarity with the DNA target sequence, but sometimes the Cas enzyme cuts nontargeted DNA sequences that pair with the gRNA in nearly all sequence positions.…”

Gene Editing by Extracellular Vesicles

“…Moreover, pre-existing antibodies against Cas proteins [ 17 ] and immune response to Cas and sgRNAs [ 18 ] can limit efficacy of CRISPR/Cas approaches. Reducing and evading immune recognition can be achieved by rationally designing Cas proteins (e.g., epitope masking or limiting presentation of Cas epitopes to the immune system) [ 77 ], using CRISPR/Cas systems from non-pathogenic organisms, inducing immune tolerance [ 78 ], or shielding Cas proteins in systemic circulation. Short-lived CRISPR/Cas complexes are sufficient for most clinical applications, especially in immune-privileged organs, and are less likely to induce a meaningful immune response.…”