Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes

Shayesteh R Ferdosi,Sri Krishna,Mo R Ebrahimkhani,Radwa Ewaisha,Karen S Anderson,Jin G Park,Farzaneh Moghadam,Samira Kiani

doi:10.1038/s41467-019-09693-x

Shayesteh R Ferdosi, Sri Krishna + Show 6 more

Open Access

https://doi.org/10.1038/s41467-019-09693-x

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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.

Highlights

The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas[9] system has raised hopes for developing personalized gene therapies for complex diseases
We identify two immunodominant SpCas[9] T cell epitopes for HLA-A*02:01 by an improved prediction algorithm and T cell assays using healthy donor peripheral blood mononuclear cells (PBMCs)
We first determined whether healthy individuals have detectable IgG Abs to SpCas[9]

Summary

Introduction

The CRISPR-Cas[9] system has raised hopes for developing personalized gene therapies for complex diseases. The expression of Streptococcus pyogenes Cas[9] protein (SpCas9) in mice has evoked both cellular and humoral immune responses[9,10], which raises concerns regarding its safety and efficacy as a gene or epi-gene therapy in humans These preclinical models and host immune reactions to other exogenous gene delivery systems[11,12,13] suggest that the pathogenic, non-self origin of Cas[9] may be immunogenic in humans. Unlike the use of Cas[9] for gene editing, which may only require Cas[9] presence in cells for a few hours, current techniques for CRISPR-based epigenetic therapies require longer term expression of Cas[9] in vivo, possibly for weeks and months[30,31], which poses the challenge of combating pre-existing immune response towards Cas[9]. We demonstrate that Cas[9] protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity

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