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Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

Gene Therapy volume 14, pages 1078–1091 (2007)Cite this article

Abstract

Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal β-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of β-galactosidase (β-gal) could be detected in human β-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of β-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated β-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed β-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and β-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.

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Acknowledgements

We thank Suzanne McDavitt for skilled preparation of the manuscript. This work was supported by the German Research Foundation Grant OE259/1-1 (AO), NCI CA69246 (XOB and CF) and NINDS NS24279 (XOB).

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  1. A Oehmig

    Present address: 3Present address: Natural and Medical Sciences Institute, University of Tuebingen, Tuebingen, Germany.,

Authors and Affiliations

  1. Departments of Neurology and Radiology, Molecular Neurogenetics Unit, Massachusetts General Hospital, and Program in Neuroscience, Harvard Medical School, Boston, MA, USA

    A Oehmig, M L Cortés, K F Perry, M Sena-Esteves & X O Breakefield

  2. Institute of Virology, University of Zurich, Zurich, Switzerland

    Cornel Fraefel

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Correspondence to X O Breakefield.

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Oehmig, A., Cortés, M., Perry, K. et al. Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector. Gene Ther 14, 1078–1091 (2007). https://doi.org/10.1038/sj.gt.3302960

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