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

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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