Rational Engineering of Minimally Immunogenic Nucleases for Gene Therapy

Abstract

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. On the horizon for FDA approval next year are several of the first treatment tools involving CRISPR based gene editing for sickle cell anemia and beta thalassemia (Frangoul et al. 2021). However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use(Charlesworth et al. 2019; Chew 2018; Mehta and Merkel 2020). Here we report reduced immunogenicity (Red)(i)-variants of two clinically-relevant nucleases, SaCas9 and AsCas12a. Through MHC-associated peptide proteomics (MAPPs) analysis, we identified putative immunogenic epitopes on each nuclease(Sekiguchi et al. 2018). Then, we used computational modeling to rationally design these proteins to evade the immune response(King et al. 2014). SaCas9 and AsCas12a Redi variants were substantially less recognized by adaptive immune components, including reduced binding affinity to MHC molecules and attenuated generation of cytotoxic T cell responses, while maintaining wild-type levels of activity and specificity. In vivo editing of PCSK9 with SaCas9.Redi variant was comparable in efficiency to wild-type SaCas9, but significantly reduced undesired immune responses. This demonstrates the utility of this approach in engineering proteins to evade immune detection and expand in vivo clinical use for a wide assortment of hematological disorders.