title: Adenovirus-based targeting in myoblasts is hampered by nonhomologous vector integration.
abstract: Chromosomal correction of dystrophin gene mutations is a most desirable therapeutic solution for Duchenne muscular dystrophy, as it allows production of the full-length dystrophin under the control of locus-specific promoters. Here we explored gene targeting in conditionally immortal mouse dystrophin-deficient myoblasts. We constructed an adenoviral vector for the correction of the mdx mutation, containing 6.0 kb of sequence homologous to the target locus (partial intron 21 through to exon 24 with the normal sequence of exon 23) and a neomycin expression cassette inserted in intron 23. Adenovirus-based gene targeting was previously reported to be beneficial in mouse embryonic stem cells, resulting in one targeted integration per three integration events. However, we found no targeted integration events among 144 stably transduced G418-resistant myoblast clones, reflecting efficient random integration of the adenoviral vector in myogenic cells. We found that mouse myoblasts are capable of integrating recombinant adenoviral DNA with an efficiency approaching 1%. Interestingly, dermal fibroblasts integrate adenoviral DNA up to 100 times less efficiently than myoblasts from the same mice. We also show that the efficiency of recombinant adenoviral DNA integration is influenced by preinfection cell density, possibly indicating the importance of cellular DNA replication for adenoviral integration.
authors.affiliation: School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey TW20 0EX, United Kingdom.
authors.forename: Olga
authors.initials: O
authors.lastname: Isman
authors.forename: Michael L
authors.initials: ML
authors.lastname: Roberts
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Morgan
authors.forename: Ian R
authors.initials: IR
authors.lastname: Graham
authors.forename: Kirstin
authors.initials: K
authors.lastname: Goldring
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Lawrence-Watt
authors.forename: Qi Long
authors.initials: QL
authors.lastname: Lu
authors.forename: Matthew G
authors.initials: MG
authors.lastname: Dunckley
authors.forename: Andrew C G
authors.initials: AC
authors.lastname: Porter
authors.forename: Terence A
authors.initials: TA
authors.lastname: Partridge
authors.forename: George
authors.initials: G
authors.lastname: Dickson
mesh_terms: D000256:Adenoviridae; D000818:Animals; D004261:DNA Replication; D020405:Dermis; D016189:Dystrophin; D053595:Embryonic Stem Cells / Q000378:metabolism; D005347:Fibroblasts; D018390:Gene Targeting; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D018101:Mice, Inbred mdx; D020388:Muscular Dystrophy, Duchenne / Q000235:genetics / Q000378:metabolism / Q000628:therapy*; D009154:Mutation; D032446:Myoblasts; D040641:Quantitative Trait Loci / Q000235:genetics; D016662:Virus Integration
chemical_list: D016189:Dystrophin