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.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:246818 |
| Date | January 2000 |
| Creators | Brown, Jason Lee |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/60074/ |
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