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Journal of Virology, June 2004, p. 6556-6566, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6556-6566.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells

Oliver Dorigo,1,2 Jose S. Gil,1 Sean D. Gallaher,1 Brenton T. Tan,3 Maria G. Castro,4 Pedro R. Lowenstein,4 Michele P. Calos,5 and Arnold J. Berk1*

Molecular Biology Institute, University of California at Los Angeles,1 Department of Obstetrics and Gynecology, David Geffen UCLA School of Medicine, Los Angeles, California 90095,2 Department of Pathology,3 Department of Genetics, School of Medicine, Stanford University, Stanford, California 94305,5 Gene Therapeutics Research Institute, UCLA Cedars Sinai Medical Center, Los Angeles, California 900484

Received 9 December 2003/ Accepted 26 January 2004

Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.

* Corresponding author. Mailing address: Molecular Biology Institute, University of California at Los Angeles, 611 Charles E. Young Dr. East, Los Angeles, CA 90095-1570. Phone: (310) 825-9370. Fax: (310) 206-7286. E-mail: berk{at}mbi.ucla.edu.

Journal of Virology, June 2004, p. 6556-6566, Vol. 78, No. 12
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.12.6556-6566.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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