Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV AAV rep hybrid amplicon vector

• Published in: Gene therapy Vol. 10; no. 19; pp. 1691 - 1702
• Main Authors: BAKOWSKA, J. C, DI MARIA, M. V, CAMP, S. M, WANG, Y, ALLEN, P. D, BREAKEFIELD, X. O
• Format: Journal Article
• Language: English
• Published: Basingstoke Nature Publishing Group 01-09-2003
• Subjects: Adenoviruses; Animals; Biological and medical sciences; Cell Line, Transformed; Classical genetics, quantitative genetics, hybrids; Deoxyribonucleic acid; Dependovirus - genetics; DNA; Embryo fibroblasts; Fibroblasts; Fibroblasts - metabolism; Fundamental and applied biological sciences. Psychology; Gene therapy; Genetic aspects; Genetic Engineering; Genetic Therapy - methods; Genetic vectors; Genetic Vectors - administration & dosage; Genetic Vectors - genetics; Genetics of eukaryotes. Biological and molecular evolution; Genomes; Genomic analysis; Herpes simplex; Humans; Mice; Mice, Inbred Strains; Mice, Transgenic; Mutagenesis, Site-Directed; Physiological aspects; Rep gene; Simplexvirus - genetics; Transduction, Genetic - methods; Transgenes; Transgenic animals; Transgenic mice; Vectors; Vertebrata; United States; Human; Integration; Adenoviridae; AAVS1; Transgenic animal; Hybrid; Virus; Associated virus; Gene amplification; transgenic mice; Length; AAV; amplicon; Locus; Gene therapy; Fibroblast; Vector
• ISSN: 0969-7128; 1476-5462
• DOI: 10.1038/sj.gt.3302061

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.