Telomerase Directed Gene Therapy

Bilsland, Alan (2002) Telomerase Directed Gene Therapy. PhD thesis, University of Glasgow.

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Abstract

Stabilisation of telomere length is considered to be an essential step in cellular immortalisation in vitro and in human cancers. The telomerase ribonucleoprotein reverse transcriptase catalyses the addition of new telomeric repeat sequence to the ends of linear eukaryotic chromosomes and counteracts the cell division associated telomeric attrition that leads to cellular senescence. Its expression has been detected in approximately 85% of all human malignancies but is not detectable in the majority of normal somatic tissues and, therefore, telomerase represents an attractive target for the development of novel molecular therapeutics. Although telomerase activity is modulated on a number of levels, a primary level of regulation is the transcription of the telomerase sub-unit genes. In the present study, I describe the development of a transcriptionally directed cytotoxic gene therapy approach targeted against telomerase positive cancer cells. Transfection experiments using fragments of the human telomerase RNA component (hTERC) and the human telomerase reverse transcriptase (hTERT) promoters revealed large differences in promoter activity between mortal cells and cancer cells. The promoter fragments were sub-cloned into plasmids containing the coding sequence of nitroreductase (NTR), a bacterial enzyme that catalyses the chemical reduction of the non-toxic pro-drug CB1954 resulting in the formation of a powerful bi-functional alkylating agent that kills both dividing and nondividing cells. Stable cell lines harbouring hTERC-NTR and hTERT-NTR expression vectors were sensitised to CB1954 to an extent that was dependent on hTERC and hTERT promoter activity, with cell lines that had high promoter activities showing significant sensitisation, while those with low promoter activities were not significantly sensitised. The hTERC-NTR and hTERT-NTR expression constructs were cloned into adenovirus (Ad) delivery vehicles and the efficiencies of infection and expression of NTR were characterised in infected cell lines. The major RNA species that was expressed in infected cells was a splice variant that encoded a truncated NTR protein, but the function of NTR was not significantly impaired. Infection with the Ad-hTERC-NTR and Ad-hTERT-NTR gene therapy vectors resulted in a sensitisation to CB1954 that was dually dependent on promoter activity and infection efficiency. Two cancer cell lines that had high hTERC and hTERT promoter activities were significantly sensitised to CB1954, while a mortal foetal lung fibroblast cell strain and a normal adult human mammary epithelial strain, in addition to a bladder cancer cell line with low promoter activity, were not sensitised despite efficient infection with adenovirus. Therefore, the data presented herein support the further development of telomerase-nitroreductase expression vectors for anti-cancer gene therapy.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: W Nicol Keith
Keywords: Genetics, Oncology
Date of Award: 2002
Depositing User: Enlighten Team
Unique ID: glathesis:2002-75725
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Dec 2019 09:15
Last Modified: 19 Dec 2019 09:15
URI: http://theses.gla.ac.uk/id/eprint/75725

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