title: Differential integrity of TALE nuclease genes following adenoviral and lentiviral vector gene transfer into human cells.
abstract: The array of genome editing strategies based on targeted double-stranded DNA break formation have recently been enriched through the introduction of transcription activator-like type III effector (TALE) nucleases (TALENs). To advance the testing of TALE-based approaches, it will be crucial to deliver these custom-designed proteins not only into transformed cell types but also into more relevant, chromosomally stable, primary cells. Viral vectors are among the most effective gene transfer vehicles. Here, we investigated the capacity of human immunodeficiency virus type 1- and adenovirus-based vectors to package and deliver functional TALEN genes into various human cell types. To this end, we attempted to assemble particles of these two vector classes, each encoding a monomer of a TALEN pair targeted to a bipartite sequence within the AAVS1 'safe harbor' locus. Vector DNA analyses revealed that adenoviral vectors transferred intact TALEN genes, whereas lentiviral vectors failed to do so, as shown by their heterogeneously sized proviruses in target cells. Importantly, adenoviral vector-mediated TALEN gene delivery resulted in site-specific double-stranded DNA break formation at the intended AAVS1 target site at similarly high levels in both transformed and non-transformed cells. In conclusion, we demonstrate that adenoviral, but not lentiviral, vectors constitute a valuable TALEN gene delivery platform.
authors.affiliation: Department of Molecular Cell Biology, Leiden University Medical Center, Eithovenweg 20, 2333 ZC Leiden, The Netherlands.
authors.forename: Maarten
authors.initials: M
authors.lastname: Holkers
authors.forename: Ignazio
authors.initials: I
authors.lastname: Maggio
authors.forename: Jin
authors.initials: J
authors.lastname: Liu
authors.forename: Josephine M
authors.initials: JM
authors.lastname: Janssen
authors.forename: Francesca
authors.initials: F
authors.lastname: Miselli
authors.forename: Claudio
authors.initials: C
authors.lastname: Mussolino
authors.forename: Alessandra
authors.initials: A
authors.lastname: Recchia
authors.forename: Toni
authors.initials: T
authors.lastname: Cathomen
authors.forename: Manuel A F V
authors.initials: MA
authors.lastname: Gonçalves
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics*; D002877:Chromosomes, Human; D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000235:genetics*; D056426:Genetic Loci; D005822:Genetic Vectors; D057809:HEK293 Cells; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D014161:Transduction, Genetic
chemical_list: D001426:Bacterial Proteins; D003851:Deoxyribonucleases