title: Generation of helper-dependent adenoviral vectors by homologous recombination.
abstract: Helper-dependent adenoviral vectors (HD-Ad) represent a potentially valuable tool for safe and prolonged gene expression in vivo. The current approach for generating these vectors is based on ligation of the expression cassette into large plasmids containing the viral inverted terminal repeats flanking "stuffer" DNA to maintain a final size above the lower limit for efficient packaging into the adenovirus capsid (approximately 28 kb). The ligation to produce the viral plasmid is generally very inefficient. Similar problems in producing first-generation adenoviral (FG-Ad) vectors were circumvented with the development of a system taking advantage of efficient homologous recombination between a shuttle plasmid containing the expression cassette and a FG-Ad vector backbone in the Escherichia coli strain BJ5183. Here we describe a method for fast and efficient generation of HD-Ad vector plasmids that can accommodate expression cassettes of any size up to 35 kb. To validate the system, we generated a HD-Ad vector expressing the fusion protein between beta-galactosidase and neomycin resistance genes under the control of the SR alpha promoter, and one expressing the enhanced green fluorescent protein under the control of the cytomegalovirus promoter. The viruses were rescued and tested in vitro and for in vivo expression in mice. The data collected indicate the possibility for achieving a high level of hepatocyte transduction using HD-Ad vectors derived from plasmids obtained by homologous recombination in E. coli, with no significant alteration of liver enzymes and a less severe, transient thrombocytopenia in comparison with previous reports with similar doses of a FG-Ad vector.
authors.affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Toietta
authors.forename: Lucio
authors.initials: L
authors.lastname: Pastore
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Cerullo
authors.forename: Milton
authors.initials: M
authors.lastname: Finegold
authors.forename: Arthur L
authors.initials: AL
authors.lastname: Beaudet
authors.forename: Brendan
authors.initials: B
authors.lastname: Lee
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D003434:Crossing Over, Genetic; D004274:DNA, Recombinant; D004926:Escherichia coli; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006378:Helper Viruses / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D009355:Neomycin / Q000494:pharmacology; D019076:Transgenes; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D004274:DNA, Recombinant; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase; D009355:Neomycin