title: Adenovirus hexon modifications influence in vitro properties of pseudotyped human adenovirus type 5 vectors.
abstract: Commonly used human adenovirus (HAdV)-5-based vectors are restricted by their tropism and pre-existing immunity. Here, we characterized novel HAdV-5 vectors pseudotyped with hypervariable regions (HVRs) and surface domains (SDs) of other HAdV types. Hexon-modified HAdV-5 vectors (HV-HVR5, HV-HVR12, HV-SD12 and HV-SD4) could be reconstituted and amplified in human embryonic kidney cells. After infection of various cell lines, we measured transgene expression levels by performing luciferase reporter assays or coagulation factor IX (FIX) ELISA. Dose-dependent studies revealed that luciferase expression levels were comparable for HV-HVR5, HV-SD12 and HV-SD4, whereas HV-HVR12 expression levels were significantly lower. Vector genome copy numbers (VCNs) from genomic DNA and nuclear extracts were then determined by quantitative real-time PCR. Surprisingly, determination of cell- and nuclear fraction-associated VCNs revealed increased VCNs for HV-HVR12 compared with HV-SD12 and HV-HVR5. Increased nuclear fraction-associated HV-HVR12 DNA molecules and decreased transgene expression levels were independent of the cell line used, and we observed the same effect for a hexon-modified high-capacity adenoviral vector encoding canine FIX. In conclusion, studying hexon-modified adenoviruses in vitro demonstrated that HVRs but also flanking hexon regions influence uptake and transgene expression of adenoviral vectors.
authors.affiliation: Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University of Witten/Herdecke, Witten-58453, Germany.
authors.forename: Manish
authors.initials: M
authors.lastname: Solanki
authors.affiliation: Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University of Witten/Herdecke, Witten-58453, Germany.
authors.forename: Wenli
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University of Witten/Herdecke, Witten-58453, Germany.
authors.forename: Liu
authors.initials: L
authors.lastname: Jing
authors.affiliation: Institute of Virology and Microbiology, Center for Biomedical Education and Research (ZBAF), Department of Human Medicine, Faculty of Health, University of Witten/Herdecke, Witten-58453, Germany.
authors.forename: Anja
authors.initials: A
authors.lastname: Ehrhardt
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D036022:Capsid Proteins / Q000276:immunology* / Q000378:metabolism*; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis; D005164:Factor IX / Q000032:analysis / Q000235:genetics; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D005822:Genetic Vectors; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D056189:Viral Tropism
chemical_list: D036022:Capsid Proteins; D004279:DNA, Viral; C018315:hexon capsid protein, Adenovirus; D005164:Factor IX; D008156:Luciferases