Better understanding of canine telomerase and its potential applications in canine oncology

Abstract

Telomerase, discovered in 1985, is considered a near-universal marker of malignancy and therefore has a potential use in cancer therapeutics and diagnostics. In this study, I used several approaches to gain a better understanding of telomerase and its potential applications in the canine context, for both cancer therapeutics and diagnosis. Having already developed an effective siRNA viral vector in vitro, the challenge still remained to deliver it efficiently in vivo. Thus, I initially investigated two possible approaches for in vivo delivery. First, I investigated a cell-based system for direct delivery to the tumours. Specifically I optimised a system for efficient gene-transfer to endothelial cells using a green fluorescent protein plasmid vector, and monitored systemic delivery by ex vivo imaging of dye-labelled cells in a canine xenograft tumour mouse model. In parallel, in vitro I investigated the gene transfer mediated by a novel dendrimer vector that can form nanoparticles with DNA and accumulate in tumour sites in vivo after i.v. administration. In order to utilize these delivery systems, I developed a DNA plasmid-based siRNA vector and tested its efficacy on canine tumour cells. To investigate telomerase as a cancer biomarker, I conducted a study that aimed to detect circulating telomerase reverse transcriptase (TERT) mRNA in serum taken from canine cancer patients. For this I developed several systems for effective RNA isolation from serum and used both conventional and quantitative PCR assays to detect TERT expression. Although for the first time I can confirm the existence of mRNA in serum of canine cancer patients, in this clinical study, I could only detect telomerase transcripts in a very small proportion of canine cancer patients. In a final pilot study to investigate anti-ageing technologies, I looked at the potential for drug-dependant telomerase induction rather than inhibition. For this I investigated the ability of three candidate drugs to induce TERT mRNA activation in canine embryonic fibroblasts. In this study, telomerase induction was measured using the quantitative PCR method that I had developed for serum detection. In summary, I have demonstrated that a cell-based delivery vehicle has a potential application in cancer therapy, but that more development is required before it can be applied clinically. I have also reported here that PPIG3 dendrimer-based gene transfer in vitro is low in canine cancer cells and thus require more optimisation and development before it can be utilised as an efficient systemic delivery vehicle. For the siRNA experiment, unfortunately, I did not observe any telomerase genesilencing in canine cancer cells using the plasmid-based siRNA expression vector, and therefore the gene sequence of cTR that we were targeting as well as the siRNA plasmid-vector that we used needs further validation in canine cells. I also suggest that TERT mRNA may not be a good serum biomarker for canine cancer diagnostics as I did not find TERT transcript in most of our serum samples from canine cancer patients, although circulating mRNA of a housekeeping gene was detected. Finally, in a pilot study, I have demonstrated that telomerase can be induced in normal canine somatic cells using small molecules. However, the long-term effects of telomerase induction on ageing must be determined in future studies.