Rational engineering of minimally immunogenic nucleases for gene therapy

Genome editing using CRISPR-Cas systems is a promising avenue for the treatment of genetic diseases. However, cellular and humoral immunogenicity of genome editing tools, which originate from bacteria, complicates their clinical use. 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. Then, we used computational modeling to rationally design these proteins to evade the immune response. 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.1 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.

利用CRISPR-Cas系统开展基因组编辑，是治疗遗传疾病的极具前景的策略。然而，这类源自细菌的基因组编辑工具所具备的细胞免疫原性与体液免疫原性，使其临床应用面临诸多复杂挑战。本研究报道了两种临床相关核酸酶——SaCas9与AsCas12a的低免疫原性（Red(i)）变体。通过主要组织相容性复合体相关肽蛋白质组学（MHC-associated peptide proteomics, MAPPs）分析，我们在每种核酸酶上鉴定出了推定的免疫原性表位。随后，我们借助计算建模手段对这些蛋白质进行理性设计，使其能够逃避免疫应答。SaCas9与AsCas12a的Red(i)变体被适应性免疫组分识别的程度显著降低，具体表现为与MHC分子的结合亲和力下降、细胞毒性T细胞应答的生成被削弱，同时保留了野生型蛋白质的活性与特异性水平。利用SaCas9.Redi.1对PCSK9进行体内编辑的效率，与野生型SaCas9相当，但显著减少了非预期的免疫应答。这一结果证明了该方法在工程化蛋白质以逃避免疫识别方面的应用价值。