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Studies of the adenovirus 5 L1 gene aimed at developing L1 gene deficiencies for use in gene therapy vectors
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Arslanoglu, Alper (1999) Studies of the adenovirus 5 L1 gene aimed at developing L1 gene deficiencies for use in gene therapy vectors. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b1369247~S1
Abstract
Gene therapy is a novel approach to the treatment of human disease that is in its very early
stages of development. Its purpose is to add to and/or alter the pattern of gene expression in
cells so as to achieve a therapeutic benefit and is being developed for application to such
diverse conditions as simple inherited diseases, cancer, AIDS, and cardiovascular disease.
Poor uptake of DNA by cells in vivo is a significant technical barrier for gene therapy.
Viruses have evolved to carry their nucleic acid contents into cells very efficiently and are
thus considered as potential vectors for gene therapy, provided their pathogenicity and other
adverse features can be overcome.
Adenovirus is a virus type, which is a prime candidate for development as a vector for
efficient transfer of genes into human tissues. In recent years, there have been many studies
in order to develop replication deficient adenoviral vectors as gene therapy vectors. Current
disadvantages of these vectors include their limited capacity to accommodate exogenous
DNA and elucidation of a host immune response against these vectors. Currently they have
a capacity for about 9 kilobasepairs of DNA, which is not sufficient for some therapeutic
purposes.
The work described in this thesis aimed to provide a way to produce highly deleted
adenoviral vectors containing deletions in their L1 52/55-kD, L1 52/55-kD and IVa2, or L1
52/55-kD and IX coding regions. These genes encode proteins, which contribute to virus
particles, and their assembly and some of which have been reported to increase expression
of the viral late (structural protein) genes. Together with the deletions available in currently
used vectors, these new deletions would create vectors with an increased capacity to
accommodate exogenous DNA. Furthermore, these deletions were expected to make the
vectors more replication-deficient and less immunogenic by decreasing the expression of
residual viral genes.
Initially, cell lines expressing adenovirus L1 52/55-kD, L1 52/55-kD and IVa2, or L1 52/55-kD
and IX proteins were constructed for use as complementing cell lines. These were
designed to supply in trans the relevant proteins that would be missing during attempts to
construct recombinant viruses with deletion mutations in these gene(s). 293-L1 cells were
proven in their ability to complement missing L1 52/55-kD protein function using existing
adenovirus L1 52/55-kD point mutants ts369 and H5pmSOO1. However, attempts to isolate
an L1 52/55-kD coding region deleted recombinant adenovirus using this cell line were
unsuccessful, possibly due to the nature of sequences missing in the deleted L1 52/55-kD
coding region which might have currently undefined cis acting regulatory functions.
L1 52/55-kD protein is known to form a complex with IVa2 and the latter protein has been
reported to activate the major late promoter. The effects of L1 52/55-kD protein on the
adenovirus major late promoter transactivation were therefore investigated by transient
expression experiments carried out by transfection of COS cells with a major late promoter-dependant reporter gene (CAT) and expression vectors for L1 52/55-kD and/or IVa2. These
experiments did not reveal any role for the L1 52/55-kD protein in the activation of
adenovirus major late promoter.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology R Medicine > RB Pathology |
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Library of Congress Subject Headings (LCSH): | Gene therapy, Adenoviruses | ||||
Official Date: | September 1999 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | Department of Biological Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Leppard, Keith | ||||
Extent: | xv, 187 leaves | ||||
Language: | eng |
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