THE JOURNAL OF GENE MEDICINE RESEARCH ARTICLE J Gene Med 2006; 8: 1400–1406. Published online 19 October 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jgm.978 An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression Liang Shan Leyao Wang Juan Yin Peng Zhong Jiang Zhong* Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China *Correspondence to: Jiang Zhong, Department of Microbiology and Microbial Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China. E-mail: jzhong@fudan.edu.cn Received: 14 March 2006 Revised: 7 September 2006 Accepted: 18 September 2006 Abstract Background The baculovirus Autographa californica multiple nucleopolyhe- drovirus (AcMNPV) has been explored as a gene delivery vehicle for a variety of mammalian cell lines. However, the transient expression nature due to its incapability to replicate in mammalian cells and insufficient transduction efficiency limit its application. Methods Recombinant baculovirus vectors containing genetic elements from Epstein-Barr virus (EBV), OriP and EBNA-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, were constructed and tested for their ability to sustain and express transgene (enhanced green fluorescence protein (egfp)) in mammalian cells. Results The recombinant baculovirus containing OriP and EBNA-1 genes driven by the cytomegalovirus (CMV) promoter was capable of persisting in a significant proportion of infected mammalian cells, HEK293, Vero, Cos-7, and Hone-1, without any selective pressure. In HEK293, the expression of EGFP lasted for 60 days with markedly enhanced expression level. The persistence of baculovirus genome correlated with the expression of EBNA-1. Conclusions The improved baculovirus vector could mediate prolonged and enhanced foreign gene expression in some mammalian cells. Furthermore, an adequate level of the EBNA-1 protein was essential for the maintenance of the OriP-containing baculovirus genome. The new vector has potential for use in gene therapy. Copyright  2006 John Wiley & Sons, Ltd. Keywords baculovirus vector; gene therapy; transduction; mammalian cells; Epstein-Barr virus; oriP/EBNA-1 Introduction The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is widely used to express heterogeneous proteins in insect cells. Although AcMNPV only infects arthropods in nature, it can enter a variety of vertebrate cells efficiently in vitro and in vivo, without any visible cytopathic effect (CPE) [1–4]. The broad range of susceptible cell lines, the large capacity for foreign genes, and minimal cytotoxicity are features that make baculovirus an attractive gene delivery vector for mammalian cells. One of the major limitations of the baculoviral transduction vector is the short duration of transgene expression. Since the genome of baculovirus cannot replicate in mammalian cells, it is usually lost or diluted soon after infection. Although partial integration of the baculovirus genome into cellular chromosomes in the presence of selective pressure has been reported [5], Copyright  2006 John Wiley & Sons, Ltd.