Abstract
BackgroundNonviral vectors are attractively used for gene therapy owing to their distinctive advantages. Our previous study has demonstrated that transfer of human IFNγ gene into nasopharyngeal carcinoma (NPC) by using a novel nonviral vector, minicircle (mc), under the control of cytomegalovirus (CMV) promoter was effective to inhibit tumor growth. However, therapies based on CMV promoter cannot express the targeted genes in cancer tissues. Previous studies indicated that the development of human NPC was closely associated with Epstein-Barr virus (EBV) and demonstrated the transcriptional enhancer function of oriP when bound by EBV protein. Therefore, the present study is to explore the targeted gene expression and the anti-tumor effect of a novel tumor-specific gene therapeutic system (mc-oriP-IFNγ) in which the transgene expression was under the transcriptional regulation of oriP promoter.Methodology/Principal FindingsDual-luciferase reporter assay and ELISA were used to assess the expression of luciferase and IFNγ. WST assay was used to assess the cell proliferation. RT-PCR was used to detect the mRNA level of EBNA1. RNAi was used to knockdown the expression of EBNA1. NPC xenograft models in nude mice were used to investigate the targeted antitumor efficacy of mc-oriP-IFNγ. Immunohistochemistry was used to detect the expression and the activity of the IFNγ in tumor sections. Our results demonstrated that mc-oriP vectors mediated comparable gene expression and anti-proliferative effect in the EBV-positive NPC cell line C666-1 compared to mc-CMV vectors. Furthermore, mc-oriP vectors exhibited much lower killing effects on EBV-negative cell lines compared to mc-CMV vectors. The targeted expression of mc-oriP vectors was inhibited by EBNA1-siRNA in C666-1. This selective expression was corroborated in EBV-positive and -negative tumor models.Conclusions/SignificanceThis study demonstrates the feasibility of mc-oriP-IFNγ as a safe and highly effective targeted gene therapeutic system for the treatment of EBV positive NPC.
Highlights
The goal of cancer treatment is to selectively eliminate malignant cells while leaving normal tissues intact [1]
We have developed a novel minicircle vector in which transgene expression is under the transcriptional regulation of the oriP-CMV promoter
To determine the transgene expression provided by the novel Epstein-Barr Nuclear Antigen 1 (EBNA1)-regulated minicircle vectors in Epstein-Barr virus (EBV)-negative cells (293, NP69, CNE-1 and CNE-2 cells) and the only available EBVpositive nasopharyngeal carcinoma (NPC) cell line (C666-1), the minicircle-luci was compared with its parent plasmid p2WC31-luci and the intermediate plasmid from which p2WC31-luci was derived
Summary
The goal of cancer treatment is to selectively eliminate malignant cells while leaving normal tissues intact [1]. A unique feature of NPC is that nearly 100% of anaplastic or poorly differentiated nasopharyngeal carcinomas contain Epstein-Barr virus genomes and express EBV proteins [6], which are expressed exclusively in the malignant tissues but not in the surrounding normal tissues. This difference provides an exploitable opportunity for tumorspecific targeting. The FR element consists of 20 tandem 30-bp repeats and acts as a transcriptional enhancer for heterologous promoters when it is bound by EBNA1 Based on these features, the oriP-CMV promoter has been exploited for targeted gene therapy in EBV-positive NPC [12]. The present study is to explore the targeted gene expression and the anti-tumor effect of a novel tumor-specific gene therapeutic system (mc-oriP-IFNc) in which the transgene expression was under the transcriptional regulation of oriP promoter
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