title: Correction of chromosomal mutation and random integration in embryonic stem cells with helper-dependent adenoviral vectors.
abstract: For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.
authors.affiliation: Gene Therapy Division, Research Center for Genomic Medicine, Saitama Medical School, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan.
authors.forename: Fumi
authors.initials: F
authors.lastname: Ohbayashi
authors.forename: Michael A
authors.initials: MA
authors.lastname: Balamotis
authors.forename: Atsuhiro
authors.initials: A
authors.lastname: Kishimoto
authors.forename: Emi
authors.initials: E
authors.lastname: Aizawa
authors.forename: Arturo
authors.initials: A
authors.lastname: Diaz
authors.forename: Paul
authors.initials: P
authors.lastname: Hasty
authors.forename: Frank L
authors.initials: FL
authors.lastname: Graham
authors.forename: C Thomas
authors.initials: CT
authors.lastname: Caskey
authors.forename: Kohnosuke
authors.initials: K
authors.lastname: Mitani
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002875:Chromosomes / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology / Q000502:physiology; D018390:Gene Targeting / Q000379:methods; D030342:Genetic Diseases, Inborn / Q000235:genetics / Q000628:therapy; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D040641:Quantitative Trait Loci; D013234:Stem Cells / Q000502:physiology; D014161:Transduction, Genetic
chemical_list: D007041:Hypoxanthine Phosphoribosyltransferase