title: Site-specific integration and tailoring of cassette design for sustainable gene transfer.
abstract: Integrative gene transfer methods are limited by variable transgene expression and by the consequences of random insertional mutagenesis that confound interpretation in gene-function studies and may cause adverse events in gene therapy. Site-specific integration may overcome these hurdles. Toward this goal, we studied the transcriptional and epigenetic impact of different transgene expression cassettes, targeted by engineered zinc-finger nucleases to the CCR5 and AAVS1 genomic loci of human cells. Analyses performed before and after integration defined features of the locus and cassette design that together allow robust transgene expression without detectable transcriptional perturbation of the targeted locus and its flanking genes in many cell types, including primary human lymphocytes. We thus provide a framework for sustainable gene transfer in AAVS1 that can be used for dependable genetic manipulation, neutral marking of the cell and improved safety of therapeutic applications, and demonstrate its feasibility by rapidly generating human lymphocytes and stem cells carrying targeted and benign transgene insertions.
authors.affiliation: San Raffaele Telethon Institute for Gene Therapy, Division of Regenerative Medicine, Gene Therapy and Stem Cells, San Raffaele Institute, Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Lombardo
authors.forename: Daniela
authors.initials: D
authors.lastname: Cesana
authors.forename: Pietro
authors.initials: P
authors.lastname: Genovese
authors.forename: Bruno
authors.initials: B
authors.lastname: Di Stefano
authors.forename: Elena
authors.initials: E
authors.lastname: Provasi
authors.forename: Daniele F
authors.initials: DF
authors.lastname: Colombo
authors.forename: Margherita
authors.initials: M
authors.lastname: Neri
authors.forename: Zulma
authors.initials: Z
authors.lastname: Magnani
authors.forename: Alessio
authors.initials: A
authors.lastname: Cantore
authors.forename: Pietro
authors.initials: P
authors.lastname: Lo Riso
authors.forename: Martina
authors.initials: M
authors.lastname: Damo
authors.forename: Oscar M
authors.initials: OM
authors.lastname: Pello
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.forename: Angela
authors.initials: A
authors.lastname: Gritti
authors.forename: Vania
authors.initials: V
authors.lastname: Broccoli
authors.forename: Chiara
authors.initials: C
authors.lastname: Bonini
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
mesh_terms: D000229:Dependovirus / Q000235:genetics; D018014:Gene Transfer Techniques; D006801:Humans; D016254:Mutagenesis, Insertional / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019713:Receptors, CCR5 / Q000235:genetics; D016662:Virus Integration / Q000235:genetics
chemical_list: D019713:Receptors, CCR5