Cas9核酸酶及其免疫原性

S.pyogenes ca 9(Spcas 9)是第一個(gè)CRISPR相關(guān)核酸酶(Cas)，用于在特定DNA序列中引入雙鏈斷裂。由于靶點(diǎn)易適應(yīng)性和顯著效率，已經(jīng)成為研究領(lǐng)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(kāi)發(fā)出來(lái)，以減少脫靶效應(yīng)和染色體畸變的風(fēng)險(xiǎn)。但大多數(shù)酶變異體都是來(lái)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(nèi)與化膿性鏈球菌感染有關(guān)的最常見(jiàn)疾病之一。考慮到化膿性鏈球菌感染的高發(fā)率，我們假設(shè)Spcas 9可以在人體內(nèi)誘發(fā)適應(yīng)性記憶免疫反應(yīng)。大多數(shù)治療應(yīng)用的目的是暫時(shí)表達(dá)Cas9核酸酶或直接將該蛋白導(dǎo)入靶細(xì)胞。因此，SpCas 9-特異性抗體可能是陰性的。然而，細(xì)胞內(nèi)的蛋白質(zhì)降解過(guò)程會(huì)導(dǎo)致基因編輯細(xì)胞表面出現(xiàn)Cas9片段，而SpCas 9-反應(yīng)性T細(xì)胞可能會(huì)識(shí)別這些片段。

Cas9 反應(yīng)性T細(xì)胞及效應(yīng)因子研究

來(lái)自德國(guó)夏里特大學(xué)（Charité）醫(yī)學(xué)免疫學(xué)研究中心的科學(xué)家，在最新一期Nature medicine發(fā)表研究結(jié)果：High prevalence of Streptococcus pyogenes Cas9-reactive T cellswithin the adult human population。作者為了檢測(cè)SpCas 9誘導(dǎo)的T細(xì)胞應(yīng)答，用重組Spcas 9刺激人外周血單個(gè)核細(xì)胞(PBMCs)，用流式細(xì)胞術(shù)檢測(cè)CD3、CD4和CD8 T細(xì)胞，用一套T細(xì)胞活化標(biāo)記物(CD137，CD 154)和效應(yīng)細(xì)胞因子產(chǎn)生(IFN-γ，IFN-γ) 腫瘤壞死因子-α(TNF-α)、白細(xì)胞介素-2(IL-2)分析T細(xì)胞反應(yīng)性
 

結(jié)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(cè)由ProteinSimple Ella 超敏微流控ELISA系統(tǒng)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.