title: AdEasy-based cloning system to generate tropism expanded replicating adenoviruses expressing transgenes late in the viral life cycle.
abstract: Replicating adenoviral vectors (RAds) hold great promise for the treatment of cancer. Significant therapeutic effects of these vectors do not only rely on tumor targeting but also on efficient release of viral progeny from host cells. Cytotoxic genes expressed late in the adenoviral life cycle can significantly enhance viral release and spreading. Therefore, an adenoviral cloning system that allows easy integration of established tumor targeting techniques together with late expression of transgenes can be a valuable tool for the development of RAds. We expanded the features of the widely used AdEasy adenoviral cloning system toward the production of tropism modified replicating adenoviral vectors that express transgenes late in the viral life cycle. Three vectors (pIRES, pFIBER and pAdEasy-Sce) that facilitate easy manipulation of the adenoviral fiber region were established. Unique BstBI and I-Sce-1 restriction sites facilitate the introduction of retargeting peptides in the fiber HI-loop and of genes of interest in the fiber transcription unit. We validated the system by constructing an E1-positive adenovirus with an RGD motif in the fiber HI-loop and green fluorescent protein (GFP) expressed from the fiber transcription unit (AdDelta24Fiber-rgd-GFP). Additionally, assessment of E1-negative replication-deficient vectors confirmed strict dependence upon E1 expression for the expression of transgenes inserted into the fiber transcription unit. This flexible cloning system allows for straightforward construction of tropism expanded replicating adenoviral vectors that express transgenes late in the adenoviral life cycle.
authors.affiliation: AMC Liver Center, Academic Medical Center/University of Amsterdam, the Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Lie-A-Ling
authors.forename: C T
authors.initials: CT
authors.lastname: Bakker
authors.forename: J G
authors.initials: JG
authors.lastname: Wesseling
authors.forename: P J
authors.initials: PJ
authors.lastname: Bosma
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000502:physiology*; D000257:Adenoviridae Infections; D017498:Adenovirus E1 Proteins / Q000235:genetics; D017503:Adenovirus E3 Proteins / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D009369:Neoplasms / Q000628:therapy*; D009842:Oligopeptides / Q000235:genetics; D019076:Transgenes; D018523:Tropism; D014779:Virus Replication
chemical_list: D017498:Adenovirus E1 Proteins; D017503:Adenovirus E3 Proteins; D036022:Capsid Proteins; D009842:Oligopeptides; C018315:hexon capsid protein, Adenovirus; D049452:Green Fluorescent Proteins; C047981:arginyl-glycyl-aspartic acid