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Journal of Virology, May 2005, p. 6400-6409, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6400-6409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

An Adenovirus Type 5 (Ad5) Amplicon-Based Packaging Cell Line for Production of High-Capacity Helper-Independent {Delta}E1-E2-E3-E4 Ad5 Vectors

Daniele Catalucci, Elisabetta Sporeno, Agostino Cirillo, Gennaro Ciliberto, Alfredo Nicosia, and Stefano Colloca*

Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Roma, Italy

Received 19 July 2004/ Accepted 4 January 2004

Production of multiply deleted adenoviral (Ad) vectors with increased cloning capacity and reduced immunogenicity to adenovirus gene products requires the concomitant generation of efficient packaging cell lines. High expression levels of the complementing genes must be achieved in a coordinated fashion with viral replication. This is a particularly difficult task in light of the significant cytotoxicity displayed by adenoviral proteins. To this end, we developed a novel adenovirus-based amplicon with an Epstein-Barr virus origin of replication, Ad type 5 (Ad5) inverted terminal repeats, all Ad5 early region 2 (E2) genes, and the early region 4 (E4) open reading frame 6 (ORF6) under the control of a tetracycline-dependent promoter. The amplicon (pE2) was stably maintained in multiple copies in the nuclei of 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 (EBNA1) and allowed replication as a linear DNA upon induction of E2 and ORF6 gene expression. A stable cell line (2E2) was generated by introducing pE2 into 293EBNATet cells expressing the tetracycline-dependent transcriptional silencer and the reverse Tet transactivator (rtTA2). Upon induction with doxicycline, 2E2 cells produced higher levels of polymerase, precursor terminal protein (pTP), and DNA binding protein than noninduced 2E2 cells infected with first-generation Ad5 vector and supported efficient amplification of a multiply deleted Ad5 vector lacking E1, E2, E3, and E4 genes (Ad5{Delta}E1-4). The high cloning capacity of Ad5{Delta}E1-4 (up to 12.6 kb) was exploited to construct a vector encoding the entire hepatitis C virus (HCV) polyprotein. Infection of HeLa cells by the resulting vector showed high levels of correctly processed HCV proteins.

* Corresponding author. Mailing address: Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Roma, Italy. Phone: 39-06-91093231. Fax: 39-06-91093225. E-mail: Stefano_colloca{at}merck.com.

Journal of Virology, May 2005, p. 6400-6409, Vol. 79, No. 10
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.10.6400-6409.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.





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