Abstract:
Epstein-Barr virus (EBV) is a ubiquitous human tumor virus that establishes lifelong, persistent infections in B cells. The presence of EBV in cancer cells presents an opportunity to target these cells by reactivating the virus from latency. In this study, we developed a novel approach for EBV reactivation termed clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated EBV reactivation (CMER) strategy. Using modified CRISPR-associated protein 9 (dCas9) fused with VP64, we designed 10 single guide RNAs (sgRNAs) to target and activate the EBV immediate-early gene promoter. In Akata Burkitt lymphoma cells, 9 out of 10 CMER sgRNAs effectively reactivated EBV. Among these, CMER sgRNA-5 triggered robust reactivation across various cell types, including lymphoma, gastric cancer, and nasopharyngeal carcinoma cells. Importantly, the combination of CMER and ganciclovir selectively eliminated EBV-positive cells, regardless of their cell origin. These findings indicate that targeted virus reactivation by CMER, combined with nucleoside analog therapy, holds promise for EBV-associated cancer treatment.
OBJECTIVE: This study explores a novel strategy called clustered regularly interspaced short palindromic repeats (CRISPR)/dCas9-mediated Epstein-Barr virus (EBV) reactivation (CMER) to reactivate the Epstein-Barr virus in cancer cells. EBV is associated with various cancers, and reactivating EBV from latency offers a potential therapeutic strategy. We utilized an enzymatically inactive CRISPR-associated protein 9 (dCas9) fused with VP64 and designed 10 single guide RNAs to target the EBV immediate-early gene promoter. Nine of these sgRNAs effectively reactivated EBV in Burkitt lymphoma cells, with CMER sgRNA-5 demonstrating strong reactivation across different cancer cell types. Combining CMER with ganciclovir selectively eliminated EBV-positive cells, showing promise for EBV-associated cancer treatment.
摘要:
爱泼斯坦-巴尔病毒(EBV)是一种普遍存在的人类肿瘤病毒,B细胞持续感染。EBV在癌细胞中的存在提供了通过从潜伏期重新激活病毒来靶向这些细胞的机会。在这项研究中,我们开发了一种新的EBV再激活方法,称为成簇规则间隔短回文重复(CRISPR)/dCas9介导的EBV再激活(CMER)策略.使用与VP64融合的修饰的CRISPR相关蛋白9(dCas9),我们设计了10个单指导RNA(sgRNA)来靶向并激活EBV立即早期基因启动子。在AkataBurkitt淋巴瘤细胞中,10个CMERsgRNA中有9个有效地重新激活EBV。其中,CMERsgRNA-5在各种细胞类型中触发了强大的再激活,包括淋巴瘤,胃癌,和鼻咽癌细胞。重要的是,CMER和更昔洛韦的组合选择性消除EBV阳性细胞,不管他们的细胞来源。这些发现表明,CMER的靶向病毒再激活,联合核苷类似物治疗,对EBV相关的癌症治疗充满希望。
目的:本研究探索了一种新策略,称为成簇规则间隔短回文重复序列(CRISPR)/dCas9介导的EB病毒(EBV)再激活(CMER),以重新激活癌细胞中的EB病毒。EBV与各种癌症有关,从潜伏期重新激活EBV提供了一种潜在的治疗策略。我们利用与VP64融合的无酶活性的CRISPR相关蛋白9(dCas9),并设计了10个单指导RNA来靶向EBV立即早期基因启动子。这些sgRNA中的9个有效地重新激活了伯基特淋巴瘤细胞中的EBV,CMERsgRNA-5在不同的癌细胞类型中表现出强烈的再激活。结合CMER与更昔洛韦选择性消除EBV阳性细胞,显示EBV相关癌症治疗的希望。