Adenovirus-Based Targeting in Myoblasts Is Hampered by Nonhomologous Vector Integration
| Autor: | Jennifer E. Morgan, Michael L. Roberts, Andrew C.G. Porter, Ian R. Graham, Diana J. Lawrence-Watt, Qi Long Lu, Kirstin Goldring, Matthew G. Dunckley, Olga Isman, George Dickson, Terence A. Partridge |
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| Rok vydání: | 2008 |
| Předmět: |
DNA Replication
Virus Integration Genetic Vectors Quantitative Trait Loci Biology medicine.disease_cause Adenoviridae Viral vector law.invention Dystrophin Myoblasts Mice law Genetics medicine Animals Humans DNA Integration Molecular Biology Embryonic Stem Cells DNA replication Gene targeting Dermis Genetic Therapy Fibroblasts Molecular biology Muscular Dystrophy Duchenne Gene Targeting Mutation Mice Inbred mdx Recombinant DNA Molecular Medicine Expression cassette |
| Zdroj: | Human Gene Therapy. 19:1000-1008 |
| ISSN: | 1557-7422 1043-0342 |
| DOI: | 10.1089/hum.2008.063 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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