Minicircle-oriP-IFNγ: A Novel Targeted Gene Therapeutic System for EBV Positive Human Nasopharyngeal Carcinoma

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.

In VivoElectroporation of Minicircle DNA as a Novel Method of Vaccine Delivery To Enhance HIV-1-Specific Immune Responses

“…It is produced in Escherichia coli by att site-specific recombination catalyzed by the phage ⌽31 integrase (15). Minicircle DNA has great advantages over conventional DNA vectors for biosafety and robust and persistent gene expression, which have been demonstrated in muscle, liver, heart, human carcinoma xenograft tumors, and iPS cells (16)(17)(18)(19)(20)(21). The unique feature of minicircle DNA to enhance levels and duration of protein expression allows us to investigate whether minicircle DNA functions as an innovative vaccine delivery platform.…”

Tumor suppressor BLU inhibits proliferation of nasopharyngeal carcinoma cells by regulation of cell cycle, c-Jun N-terminal kinase and the cyclin D1 promoter

“…The system could be improved by replacing the ubiquitous promoter derived from murine cytomegalovirus with a regulatory element that has high activity in the malignancies to control the expression of biotherapeutic target genes. Such elements include α-fetoprotein enhancer-promoter for hepatocellular carcinoma cells [21], and origin of plasmid replication (OriP) element [22,23]. …”

Minicircle DNA vectors for gene therapy: advances and applications

“…Nonetheless, further improvement of mcDNA constructs efficiency in terms of levels and duration of expression can certainly prompt their application as AMPs, provided that all the regulatory demands are met. In this context, recent investigations described straightforward strategies to improve gene transfer in eukaryotic cells by including scaffold matrix attachment regions (S/MAR) [51], tissue-specific promoters (TSPs) [52,53] or organelle-specific targeting signals [54], in the vectors backbone.…”

“…This remarkable expression efficiency also resulted in reduced relative cell growth rates when mcDNA-IFNg was administered to nasopharyngeal carcinoma cell lines (CNE-1, CNE-2 and C666-1), via Lipofectamine Ò 2000 liposomes, thus demonstrating its therapeutic potential. In a similar approach, Wu et al recently reported the administration of liposome-minicircles encoding IFN-g but under the control of different promoters (cytomegalovirus [CMV] and oriP [Epstein-Barr gene promoter] linked to CMV [oriP]), to evaluate a possible increase in the therapeutic effect [52]. The rationale underlying the use of oriP-CMV in this study is the concept that nasopharyngeal carcinoma cells contain Epstein--Barr virus (EBV) genome and a cell-specific expression of IFN can be obtained [52].…”

“…In a similar approach, Wu et al recently reported the administration of liposome-minicircles encoding IFN-g but under the control of different promoters (cytomegalovirus [CMV] and oriP [Epstein-Barr gene promoter] linked to CMV [oriP]), to evaluate a possible increase in the therapeutic effect [52]. The rationale underlying the use of oriP-CMV in this study is the concept that nasopharyngeal carcinoma cells contain Epstein--Barr virus (EBV) genome and a cell-specific expression of IFN can be obtained [52]. In vitro data revealed that mcDNA-oriP-IFN-g vectors achieved a selective transgene expression in EBV-positive cell line (C666-1) and are significantly less cytotoxic to normal, or EBV-negative cancer cells.…”