Table1_Interferon alpha and beta receptor 1 knockout in human embryonic kidney 293 cells enhances the production efficiency of proteins or adenoviral vectors related to type I interferons.DOCX

Human embryonic kidney (HEK) 293 cells are widely used in protein and viral vector production owing to their high transfection efficiency, rapid growth, and suspension growth capability. Given their antiviral, anticancer, and immune-enhancing effects, type I interferons (IFNs) have been used to prevent and treat human and animal diseases. However, the binding of type I IFNs to the IFN-α and-β receptor (IFNAR) stimulates the expression of IFN-stimulated genes (ISGs). This phenomenon induces an antiviral state and promotes apoptosis in cells, thereby impeding protein or viral vector production. In this study, we generated an IFNAR subtype 1 knockout (KO) HEK 293 suspension (IFNAR-KO) cell line by using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) technology. Upon treatment with human IFN-α, the IFNAR-KO cells showed a constant expression of ISGs, including 2ʹ-5ʹ-oligoadenylate synthetase 1 (OAS1), myxovirus resistance 1 (Mx1), protein kinase RNA-activated (PKR), and IFN-induced transmembrane protein 1 (IFITM1), when compared with the wild-type HEK 293 (WT) cells, wherein the ISGs were significantly upregulated. As a result, the titer of recombinant adenovirus expressing porcine IFN-α was significantly higher in the IFNAR-KO cells than in the WT cells. Furthermore, the IFNAR-KO cells continuously produced higher amounts of IFN-α protein than the WT cells. Thus, the CRISPR-Cas9-mediated IFNAR1 KO cell line can improve the production efficiency of proteins or viral vectors related to IFNs. The novel cell line may be used for producing vaccines and elucidating the type I IFN signaling pathway in cells.人胚肾293细胞（Human embryonic kidney 293 cells，HEK 293）因转染效率高、生长速度快且具备悬浮培养能力，被广泛应用于蛋白质与病毒载体的生产。鉴于Ⅰ型干扰素（type I interferons, IFNs）具有抗病毒、抗肿瘤及免疫增强的生物学活性，其已被用于人类与动物疾病的预防与治疗。然而，Ⅰ型干扰素与干扰素α/β受体（IFN-α/β receptor, IFNAR）结合后，会刺激干扰素刺激基因（IFN-stimulated genes, ISGs）的表达，该现象可诱导细胞产生抗病毒状态并促进细胞凋亡，进而阻碍蛋白质或病毒载体的生产。 本研究借助成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats, CRISPR）/CRISPR相关蛋白9核酸酶（CRISPR-associated protein 9 nuclease, Cas9）技术，成功构建了干扰素α/β受体1型敲除（knockout, KO）的HEK293悬浮细胞系（命名为IFNAR-KO细胞系）。将IFNAR-KO细胞与野生型HEK293（wild-type HEK 293, WT）细胞分别用人干扰素α处理后，野生型细胞中的干扰素刺激基因显著上调，而IFNAR-KO细胞中的目标干扰素刺激基因——包括2'-5'-寡腺苷酸合成酶1（2'-5'-oligoadenylate synthetase 1, OAS1）、黏液病毒抗性蛋白1（myxovirus resistance 1, Mx1）、双链RNA依赖的蛋白激酶（protein kinase RNA-activated, PKR）及干扰素诱导跨膜蛋白1（IFN-induced transmembrane protein 1, IFITM1）——均维持稳定表达水平。 实验结果显示，表达猪干扰素α的重组腺病毒在IFNAR-KO细胞中的滴度显著高于野生型细胞；此外，IFNAR-KO细胞持续分泌的干扰素α蛋白量也显著高于野生型细胞。综上，经CRISPR-Cas9介导构建的IFNAR1型敲除细胞系，可提升与干扰素相关的蛋白质或病毒载体的生产效率，该新型细胞系可用于疫苗生产及细胞内Ⅰ型干扰素信号通路的机制研究。