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Published on: February 9, 2015
The activity of the feline thyroglobulin promoter is compromised by flanking adenoviral sequence
Laura Blackwood1, David John Argyle
1Division of Small Animal Clinical Studies, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK. l.blackwood@liv.ac.uk
Abstract:
The use of human adenovirus (Ad) vectors for transcriptionally targeted thyroid gene therapy was investigated, aiming to develop a novel therapy for feline hyperthyroidism. Ad5 (El, E3 deleted) vectors were constructed in which the feline thyroglobulin promoter controls expression of the reporter gene chloramphenicol acetyl transferase (CAT). In FRTL-5 cells, CAT expression from adenoviral constructs harbouring the expression cassette was much reduced compared to controls transfected with the same cassette in a plasmid backbone, despite higher transduction efficiencies when viral vectors were used. Transfections with the "shuttle" plasmids utilised to create the adenoviral vectors also resulted in reduced expression compared to controls. In both viruses and shuttle plasmids, consistently lower expression was noted when the cassette was in the left to right orientation than the right to left. These results suggest cis acting elements in the flanking adenoviral sequences may compromise the activity of the feline thyroglobulin promoter and thus make Ad5 an unsuitable vector for transcriptionally targeted gene therapy in feline hyperthyroidism.
Insights
Human adenovirus vectors were explored for feline hyperthyroidism gene therapy. Results indicate Ad5 vectors may be unsuitable due to reduced gene expression, hindering potential thyroid treatments.
Area of Science:
- Veterinary Medicine
- Molecular Biology
- Genetics
Background:
- Feline hyperthyroidism is a common endocrine disorder in cats.
- Gene therapy offers a potential novel treatment approach.
- Adenovirus (Ad) vectors are commonly used in gene delivery.
Purpose of the Study:
- To investigate the efficacy of human adenovirus type 5 (Ad5) vectors for transcriptionally targeted gene therapy in feline hyperthyroidism.
- To assess the expression of a reporter gene (chloramphenicol acetyl transferase - CAT) under the control of the feline thyroglobulin promoter within Ad5 vectors.
Main Methods:
- Construction of Ad5 vectors with the feline thyroglobulin promoter controlling CAT gene expression.
- Transduction of FRTL-5 cells with Ad5 vectors and control plasmid transfections.
- Comparison of CAT expression levels between adenoviral constructs and plasmid backbones.
- Evaluation of gene expression based on the orientation of the expression cassette within the vectors.
Main Results:
- Adenoviral constructs showed significantly reduced CAT expression compared to plasmid controls, despite higher transduction efficiencies.
- Shuttle plasmids used for vector construction also exhibited reduced expression.
- Gene expression was consistently lower when the expression cassette was oriented from left to right compared to right to left.
Conclusions:
- Cis-acting elements within the flanking adenoviral sequences may inhibit the feline thyroglobulin promoter's activity.
- Ad5 vectors appear unsuitable for transcriptionally targeted gene therapy for feline hyperthyroidism.
- Further research is needed to identify alternative vectors or strategies for feline thyroid gene therapy.
