title: Development of an AdEasy-based system to produce first- and second-generation adenoviral vectors with tropism for CAR- or CD46-positive cells.
abstract: BACKGROUND
  The AdEasy system has acquired preeminence amongst the various methods for producing first-generation, early region 1 (E1)-deleted human adenovirus (HAdV) vectors (AdVs) as a result of the fast and reproducible recovery of full-length AdV genomes via homologous recombination in Escherichia coli.
  
  
  METHODS
  From the classical AdEasy system, a new production platform was derived to assemble first- and second-generation [i.e. E1- plus early region 2A (E2A)-deleted] AdVs displaying on their surface HAdV serotype 5 (HAdV5) fibers (F5) or chimeric fibers (F5/50) comprising the tail of F5 and the fiber shaft and knob of HAdV serotype 50 (HAdV50). The CD46-interacting chimeric fibers allow for the high-level transduction of various human primary cell types of clinical interest with low or no surface expression of the Coxsackievirus and adenovirus receptor.
  
  
  RESULTS
  A new set of pAdEasy plasmid 'backbones' with or without E2A and encoding F5 or F5/50 was constructed and recombined in E. coli strain BJ5183 with a 'shuttle' plasmid coding for β-galactosidase. The resulting clones yielded AdV preparations with similar high titers following their rescue and propagation in producer cells. The AdVs with F5/50 were superior to those carrying F5 with respect to transducing human skeletal myocytes and mesenchymal stem cells.
  
  
  CONCLUSIONS
  In the present study, an AdEasy system tailored for the production of not only first-, but also second-generation AdVs equipped with the receptor-interacting fiber domains of the prototypic species C HAdV5 or of the species B member HAdV50 is presented. This system expands the range of applications for this robust and versatile AdV production platform.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands.
authors.forename: Josephine M
authors.initials: JM
authors.lastname: Janssen
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Skokan
authors.forename: Manuel A F V
authors.initials: MA
authors.lastname: Gonçalves
authors.forename: Antoine A F
authors.initials: AA
authors.lastname: de Vries
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics*; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004770:Enterovirus / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D020869:Gene Expression Profiling; D005822:Genetic Vectors / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D051916:Membrane Cofactor Protein / Q000235:genetics*; D059630:Mesenchymal Stem Cells / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D014161:Transduction, Genetic; D056189:Viral Tropism; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D051916:Membrane Cofactor Protein; D011994:Recombinant Proteins; D001616:beta-Galactosidase