title: An adenovirus type 5 (Ad5) amplicon-based packaging cell line for production of high-capacity helper-independent deltaE1-E2-E3-E4 Ad5 vectors.
abstract: 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 (Ad5DeltaE(1-4)). The high cloning capacity of Ad5DeltaE(1-4) (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.
authors.affiliation: Istituto di Ricerche di Biologia Molecolare P. Angeletti, Via Pontina Km 30.600, 00040 Pomezia, Roma, Italy. Stefano_colloca@merck.com
authors.forename: Daniele
authors.initials: D
authors.lastname: Catalucci
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Sporeno
authors.forename: Agostino
authors.initials: A
authors.lastname: Cirillo
authors.forename: Gennaro
authors.initials: G
authors.lastname: Ciliberto
authors.forename: Alfredo
authors.initials: A
authors.lastname: Nicosia
authors.forename: Stefano
authors.initials: S
authors.lastname: Colloca
mesh_terms: D000256:Adenoviridae / Q000235:genetics* / Q000378:metabolism; D017498:Adenovirus E1 Proteins / Q000235:genetics*; D017502:Adenovirus E2 Proteins / Q000235:genetics*; D017503:Adenovirus E3 Proteins / Q000235:genetics*; D017504:Adenovirus E4 Proteins / Q000235:genetics*; D002460:Cell Line / Q000378:metabolism; D002999:Clone Cells; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism; D017353:Gene Deletion; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D016174:Hepacivirus / Q000378:metabolism; D006801:Humans; D014162:Transfection; D014764:Viral Proteins / Q000378:metabolism
chemical_list: D017498:Adenovirus E1 Proteins; D017502:Adenovirus E2 Proteins; D017503:Adenovirus E3 Proteins; D017504:Adenovirus E4 Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D014764:Viral Proteins; C417029:EBV-encoded nuclear antigen 1