title: Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity.
abstract: Techniques enabling precise genome modifications enhance the safety of gene-based therapy. DLC1 is a hot spot for phiC31 integrase-mediated transgene integration in vitro and in vivo. Here we show that integration of a coagulation factor VIII transgene into intron 7 of DLC1 supports durable expression of factor VIII in primary human umbilical cord-lining epithelial cells. Oligoclonal cells with factor VIII transgene integrated in DLC1 did not have altered expression of DLC1 or neighbouring genes within a 1-Mb interval. Only 1.9% of all expressed genes were transcriptionally altered; most were downregulated and mapped to cell cycle and DNA repair pathways. DLC1-integrated cells were not tumourigenic in vivo and were normal by high-resolution genomic DNA copy number analysis. Our data identify DLC1 as a locus for durable transgene expression that does not incur features of insertional oncogenesis, thus expanding options for developing ex vivo cell therapy mediated by site-specific integration methods.
authors.affiliation: 1] Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore [2] Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore.
authors.forename: J
authors.initials: J
authors.lastname: Sivalingam
authors.affiliation: 1] Department of Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore [2] CellResearch Corporation, Singapore, Singapore.
authors.forename: T T
authors.initials: TT
authors.lastname: Phan
authors.affiliation: 1] Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore [2] Division of Medical Sciences, Laboratory of Applied Human Genetics, Humphrey Oei Institute of Cancer Research, National Cancer Centre, Singapore, Singapore.
authors.forename: O L
authors.initials: OL
authors.lastname: Kon
mesh_terms: D001483:Base Sequence; D056915:DNA Copy Number Variations; D005169:Factor VIII / Q000096:biosynthesis*; D020690:GTPase-Activating Proteins / Q000235:genetics*; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D019426:Integrases / Q000235:genetics; D016254:Mutagenesis, Insertional; D014162:Transfection; D019076:Transgenes; D025521:Tumor Suppressor Proteins / Q000235:genetics*
chemical_list: C112352:DLC1 protein, human; D020690:GTPase-Activating Proteins; D025521:Tumor Suppressor Proteins; D005169:Factor VIII; D019426:Integrases