NaturemedicineCRISPR–Cas9核酸酶免疫原性研究

Cas9核酸酶及其免疫原性

S.pyogenes ca 9(Spcas 9)是第一个CRISPR相关核酸酶(Cas)，用于在特定DNA序列中引入双链断裂。由于靶点易适应性和显着效率，已经成为研究领域和潜在的临床治疗方法中最广泛使用的基因编辑工具。高保真度的Cas 9酶和Cas 9定向碱基编辑器被开发出来，以减少脱靶效应和染色体畸变的风险。但大多数酶变异体都是来源于兼性致病菌化脓性链球菌(S.pyogenes)的Spcas 9酶。化脓性咽炎和脓皮病是世界范围内与化脓性链球菌感染有关的最常见疾病之一。考虑到化脓性链球菌感染的高发率，我们假设Spcas 9可以在人体内诱发适应性记忆免疫反应。大多数治疗应用的目的是暂时表达Cas9核酸酶或直接将该蛋白导入靶细胞。因此，SpCas 9-特异性抗体可能是阴性的。然而，细胞内的蛋白质降解过程会导致基因编辑细胞表面出现Cas9片段，而SpCas 9-反应性T细胞可能会识别这些片段。

Cas9 反应性T细胞及效应因子研究

来自德国夏里特大学（Charité）医学免疫学研究中心的科学家，在最新一期Nature medicine发表研究结果：High prevalence of Streptococcus pyogenes Cas9-reactive T cellswithin the adult human population。作者为了检测SpCas 9诱导的T细胞应答，用重组Spcas 9刺激人外周血单个核细胞(PBMCs)，用流式细胞术检测CD3、CD4和CD8 T细胞，用一套T细胞活化标记物(CD137，CD 154)和效应细胞因子产生(IFN-γ，IFN-γ) 肿瘤坏死因子-α(TNF-α)、白细胞介素-2(IL-2)分析T细胞反应性

结果

Ubiquitous peripheral SpCas9-reactive T cell response in human donors

SpCas9-reactive T cell response contains a substantial proportion of Treg cells

SpCas9-reactive Tregcells suppress their SpCas9-reactive Teff counterpart

本研究TNF-α检测由ProteinSimple Ella 超敏微流控ELISA系统完成

TNF-α release assay. To test for the potential endotoxincontamination of the recombinant Cas proteins, detection of human TNF-α in supernatants of whole blood wasperformed with the Ella system assay (ProteinSimple), according tothe manufacturer’s instructions. TNF-α release was assessed in the whole blood assay by thegiven reagent alone and in presence of 10 μ g ml−1 BPI. Heparinized whole blood fromhealthy volunteers was diluted 1:10 with RPMI medium and stimulated with either500 ng ml−1 lipopolysaccharide, 25 ng ml−1 phorbol myristate acetate/2 μ g ml−1 ionomycin, 1 μ g ml−1 SEB, CMV pp65overlapping peptide pools at 1 μ g ml−1 or 5 μ g ml−1 SpCas9/SaCas9/Cpf1 for 24 h at 37 °C. Aftercentrifugation for 15 min at 1,000g at room temperature, the supernatantswere collected. Measurements were performed in the Immunological Study Lab ofthe BCRT. For each sample, the respective optical density values of TNF-α concentration calculated on the basisof a calibration curve were obtained by subtracting the blank provided by thevendor. The mean concentrations and standard deviations of the samples werecalculated.