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


Oehmig, A; Cortés, M L; Perry, K F; Sena-Esteves, M; Fraefel, C; Breakefield, X O (2007). Integration of active human beta-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector. Gene Therapy, 14(14):1078-1091.

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 b-galactosidase
(BGAL) gene of 100 kb. Temporary expression and
functional activity of b-galactosidase (b-gal) could be
detected in human b-gal-deficient patient and glioblastoma
(Gli36) cells upon infection with the basic BGAL amplicon
vector. Sustained expression of b-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 b-gal activity or
site-specific integration. In contrast, 80% of the rep-plus
clones possessed b-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 b-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.

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 b-galactosidase
(BGAL) gene of 100 kb. Temporary expression and
functional activity of b-galactosidase (b-gal) could be
detected in human b-gal-deficient patient and glioblastoma
(Gli36) cells upon infection with the basic BGAL amplicon
vector. Sustained expression of b-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 b-gal activity or
site-specific integration. In contrast, 80% of the rep-plus
clones possessed b-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 b-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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14 citations in Web of Science®
15 citations in Scopus®
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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:05 Vetsuisse Faculty > Institute of Virology
Dewey Decimal Classification:570 Life sciences; biology
Language:English
Date:2007
Deposited On:25 Mar 2009 08:52
Last Modified:05 Apr 2016 12:38
Publisher:Nature Publishing Group
ISSN:0969-7128
Publisher DOI:https://doi.org/10.1038/sj.gt.3302960
PubMed ID:17460718

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