Cas9核酸酶及其免疫原性

S.pyogenes ca 9(Spcas 9)是第一個CRISPR相關核酸酶(Cas)，用于在特定DNA序列中引入雙鏈斷裂。由于靶點易適應性和顯著效率，已經成為研究領域和潛在的臨床治療方法中最廣泛使用的基因編輯工具。高保真度的Cas 9酶和Cas 9定向堿基編輯器被開發出來，以減少脫靶效應和染色體畸變的風險。但大多數酶變異體都是來源于兼性致病菌化膿性鏈球菌(S.pyogenes)的Spcas 9酶。化膿性咽炎和膿皮病是世界范圍內與化膿性鏈球菌感染有關的最常見疾病之一。考慮到化膿性鏈球菌感染的高發率，我們假設Spcas 9可以在人體內誘發適應性記憶免疫反應。大多數治療應用的目的是暫時表達Cas9核酸酶或直接將該蛋白導入靶細胞。因此，SpCas 9-特異性抗體可能是陰性的。然而，細胞內的蛋白質降解過程會導致基因編輯細胞表面出現Cas9片段，而SpCas 9-反應性T細胞可能會識別這些片段。

Cas9 反應性T細胞及效應因子研究

來自德國夏里特大學（Charité）醫學免疫學研究中心的科學家，在最新一期Nature medicine發表研究結果：High prevalence of Streptococcus pyogenes Cas9-reactive T cellswithin the adult human population。作者為了檢測SpCas 9誘導的T細胞應答，用重組Spcas 9刺激人外周血單個核細胞(PBMCs)，用流式細胞術檢測CD3、CD4和CD8 T細胞，用一套T細胞活化標記物(CD137，CD 154)和效應細胞因子產生(IFN-γ，IFN-γ) 腫瘤壞死因子-α(TNF-α)、白細胞介素-2(IL-2)分析T細胞反應性
 

結果

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檢測由ProteinSimple Ella 超敏微流控ELISA系統完成

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.