Basic research on novel Epstein-Barr virus(EBV)-specific gene therapy against uncontrollable EBV- associated diseases

eb病毒（Epstein-Barr virus， EBV）与多种人类恶性疾病有关，这些疾病具有临床侵袭性，通常对常规癌症治疗反应较差，目前可用的抗疱疹病毒药物治疗无效。EBV核抗原1（EBNA1）是一种在所有受感染的增殖细胞中一致表达的潜伏病毒蛋白，是维持细胞中EBV发作所必需的。在本研究项目中，为了开发一种新的治疗策略，专门针对不受控制的EBV疾病，我们构建了一个突变体（mt） EBNA1，相对于野生型（wt） EBNA1，缺乏大部分的n端一半，并研究了mtEBNA1是否对维持病毒发作产生显性负作用，从而导致EBV感染的肿瘤细胞生长中断。得到的结果如下：1。以耐新霉素重组EBV（rebna1）转化的淋巴细胞和上皮细胞为模型，以腺病毒载体为媒介，研究了mtEBNA1的更多转导，在三种主要病毒潜伏期的转化细胞系中，引起了rEBV衍生的wtEBNA1表达水平和病毒基因组负荷的快速和显著降低。这一结果在单细胞水平上通过原位病毒基因组信号的细胞丢失得到进一步验证，从而表明mtEBNA1可以作为显性阴性（dn） EBNA1.2发挥作用。在体外和体内，dnEBNA1的转导显著地损害了天然ebv - berkitt淋巴瘤细胞和T/NK淋巴瘤细胞的生长，这与病毒发作的根除有关。dnEBNA1本身的表达在ebv未感染的细胞（包括正常的人成纤维细胞）中没有可检测到的细胞毒性。dnEBNA1的抑瘤作用是通过3种机制实现的：1)竞争性抑制wtEBNA1与oriP的结合，2)抑制Qp导致wtEBNA1的新生合成减少，3)直接诱导肿瘤细胞凋亡。这些结果表明，我们的dnEBNA1可以有效地阻止ebv依赖性恶性表型的细胞，而不受病毒潜伏期、组织来源或驻留病毒株的影响。因此，该突变体将提供一种额外的治疗方法，专门针对ebv相关的恶性肿瘤。此外，dnEBNA1可能有助于分析wtEBNA1可能的致癌作用。少

Epstein-Barr virus(EBV) is associated with a variety of human malignant disorders, which are clinically aggressive and often less responsive to the conventional cancer therapy, and currently available anti-herpesvirus drugs are ineffective treatments. EBV nuclear antigen 1(EBNA1), a latent viral protein consistently expressed in all infected proliferating cells, is essentially required in trans to maintain EBV episomes in cells. In this research project, to exploit a novel therapeutic strategy specifically acting against uncontrollable EBV diseases, we constructed a mutant(mt) EBNA1 lacking most of the N-terminal-half, relative to wild-type(wt) EBNA1, and examined whether the mtEBNA1 exerted dominant-negative effects on maintenance of the viral episome thereby leading to abrogation of EBV-infected tumor cell growth. The results obtained are as follows.1.Using lymphocyte and epithelial cell lines converted with neomycin-resistant recombinant EBV(rEBV) as models, adenovirus vector-mediat … More ed transduction of mtEBNA1 brought about rapid and striking reductions of rEBV-derived wtEBNA1 expression levels and viral genomic loads in converted cell lines of three major viral latencies. This outcome was further validated at the single cell level by cellular loss of viral genomic signals in situ, thereby indicating that mtEBNA1 can function as a dominant-negative(dn) EBNA1.2.The dnEBNA1 transduction significantly impaired growth of naturally EBV-harboring Burkitt's lymphoma cells and T/NK lymphoma cells in vitro and in vivo, in association with the eradication of viral episomes.3.Expression of dnEBNA1 per se caused no detectable cytotoxicity in EBV-uninfected cells including normal human fibroblasts.4.The tumor suppressive effects by dnEBNA1 was achieved by three mechanisms : 1) competitive inhibition of wtEBNA1 binding to oriP, 2) decrease of de novo wtEBNA1 synthesis due to suppression of Qp, and 3) direct induction of apoptosis in tumor cells.These results indicate that our dnEBNA1 can efficiently impedes the EBV-dependent malignant phenotypes in cells regardless of viral latency, tissue origin or resident viral strain. Therefore, the mutant will afford an additional therapeutic approach specifically targeting EBV-associated malignancies. Moreover, dnEBNA1 may facilitate analysis of possible oncogenic role(s) for wtEBNA1. Less