Brown, Jason Lee (2000) Expression systems for adenovirus late proteins. PhD thesis, University of Warwick.

Preview

PDF WRAP_THESIS_Brown_2000.pdf - Submitted Version - Requires a PDF viewer. Download (23Mb) | Preview

Request Changes to record.

Abstract

During the past few decades a new approach has emerged for the treatment of
human disease. In that short period, the concepts and techniques of gene therapy have
progressed from being entirely fanciful to experimental clinical application. A major
stumbling block for gene therapy is the inefficiency of gene transfer and the transient
nature of therapeutic gene expression. Attempts to deliver therapeutic genes using
replication-defective adenoviruses have been hampered by a strong host immune
response to the vector, leading to clearance of transduced cells and loss of transgene
expression. Current adenovirus vectors have an additional disadvantage of being able
to carry only approximately 10 kb of exogenous DNA. The work here describes
attempts to improve upon existing adenoviral vectors by addressing these limitations
In order to reduce the host immune response to the vector, additional deletions
in the residual viral coding regions are required to prevent expression of immunogenic
proteins. Deletion of the major late transcription unit (MLTU), which encodes
virtually all of the viral structural proteins, would achieve this and would also increase
the transgene carrying capacity of the vector. In order to create such a vector, a cell
line would be required to complement the growth of the deleted vector by providing
late gene functions in trans. The work presented here describes the successful cloning
and subsequent analysis of the Ad5 MLTU with expression driven by the major late
promoter (MLP). The expression plasmids constructed express one or more late
proteins from each late gene segment in a transient assay. The plasmids also carry the
EBNA-I and oriP sequences from Epstein-Barr virus which allow the plasmids to be
stably maintained in eukaryotic cells. Stable cell lines were constructed using these
plasmids but no late protein expression from the MLTU could be detected. Attempts
to activate expression from the major late promoter by providing viral transactivating
factors in trans also proved to be ineffective.
To address these problems, an alternative inducible promoter was chosen. The
sheep metallothionein Ia promoter was cloned upstream of the MLTU and this
construct was then cloned into an episomal expression vector. This plasmid was also
used successfully to regulate the expression of late proteins during transient
transfection studies. However, a stable cell line constructed using the same plasmid
did not show any expression of late proteins.
The reasons for the inability of any cell line constructed to express late proteins
are still undetermined. Possible reasons discussed are plasmid rearrangement,
promoter down-regulation and possible blocks to post-transcriptional processing and
translation of the complex MLTU transcript. Suggested future studies include testing
of these possibilities in order to gain further insight into the regulation of expression
from the MLTU construct, ultimately leading to the construction of a cell line capable
of complementing the growth of adenovirus vectors with late gene deficiencies.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QR Microbiology R Medicine > RB Pathology
Library of Congress Subject Headings (LCSH):	Gene therapy, Adenoviruses
Official Date:	September 2000
Dates:	Date Event September 2000 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Leppard, Keith
Extent:	152, [12] pages
Language:	eng

Request changes or add full text files to a record

Repository staff actions (login required)

View Item

Downloads