Gene Therapy for Cancer
D. J. Argyle, Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, USA

Gene Therapy is not a new idea, the concept evolving in parallel to the discovery of both DNA and the genetic basis of disease. In simple terms gene therapy is the introduction of nucleic acid into a cell to ameliorate a disease process. For this to be effective, the gene has to be delivered to sufficient number of target cells in the body requiring a vehicle or vector for delivery. In addition, the gene has to be expressed at a sufficient level and for a length of time appropriate for the disease. However, although the concept of delivering genes to patients to ameliorate disease is both simple and attractive, the technology to achieve this is still in development. Gene Therapy holds many promises but there are still technical hurdles to overcome before it can become accepted clinical practice.

Companion animal cancer is of major significance in terms of animal health and welfare. Among the domestic species the canine population has the highest rate of malignant tumors (166 cases/100,000 dogs per year) and is second only to the horse for the benign tumor rate (134 cases/100,000/year). Despite advances in surgical techniques and the use of radiotherapy and chemotherapy, cancer still remains a disease of high mortality and alternative treatments warrant further investigation. In conventional therapies such as surgery, radiotherapy, or chemotherapy, the therapeutic index (TI) is dictated by the need to preserve vital structures or protect them from toxicity related to the therapy. The development of gene therapy is seen as a means of supporting or replacing these rather crude therapies with a much more targeted approach based upon an understanding of the disease at a molecular level. Central to this approach is the expectation that toxicity will be reduced and the therapeutic index increased. However, the use of genes that are potentially harmful to normal cells means that for gene therapy to become accepted clinical practice, it will have to demonstrate that it can deliver good clinical responses without unacceptable clinical side effects. Consequently, the introduction of potentially toxic genes into cancer cells requires that expression of that gene be limited only to cancer cells, such that normal tissue is spared. Several control mechanisms of targeting have been described and include surface targeting of the vector (e.g. receptor based approaches) and transcriptional targeting using transcriptional machineries that are unique to cancer cells.

The Use of Transcriptional Machineries to Target Transgene Expression

The production of protein within a cell requires that the appropriate gene be transcribed into mRNA and then translated into protein. This process is complex and subject to multiple levels of control. The regulation of transcription is the key initiating event in this process and is mediated by the interaction between the enhancer/promoter region of the appropriate piece of DNA and the specific proteins or transcription factors that bind to this region. For transcriptional targeting in cancer gene therapy, a promoter that is either tissue specific or tumor specific is used to drive expression of a novel transgene.

Tissue Specific Promoters

Despite the fact that almost every cell in the body contains a complete copy of the genome, phenotypic heterogeneity is largely achieved through differences in the patterns of gene expression that are, in the main, controlled at the level of transcription. Activation or repression of promoters is achieved through interactions with specific transcription factors. Consequently, some tissues express proteins that are specific to that tissue because the promoter for the appropriate gene is only activated in that tissue. Thus, the success of transcriptional targeting is dependent on differential expression of genes in cancer cells compared to normal cells. Several studies have focused on using tissue specific promoters such as tyrosinase (melanoma) osteocalcin (osteosarcoma) and MUC1 (breast cancer) to drive expression of therapeutic genes in specific cancer types. Table 1 gives an overview of some of the promoters that are or have been evaluated in human clinical gene therapy trials.

In cancer gene therapy, any tissue specific promoter (TSP's) used should have activity that is restricted to the target tissue. In reality, many so-called TSP's have low level background activity in other tissues. Ideally, this should be minimal to increase the TI of the strategy. In terms of companion animal tumours, the author's laboratory has explored a number of systems for tissue specific expression. In the first, we exploited the tissue specific expression of tyrosinase, an essential enzyme involved in the melanin biosynthesis pathway, which is restricted to melanocytes. The use of the tyrosinase promoter elements to drive tissue specific expression in-vitro and in-vivo in melanoma cells and whole tumours has been reported. To examine the usefulness of this promoter in canine melanoma we conducted a series of transient transfection studies of canine cells using a reporter gene construct driven by the mouse tyrosinase promoter elements. The cloned murine tyrosinase promoter construct was kindly provided by Dr R Vile and is based on the chloroamphenicol acetyltransferase (CAT) reporter consisting of a 200bp fragment of the melanocyte specific enhancer region and a 270bp DNA fragment (-270bp) ending at the +9 of the tyrosinase sequence upstream of the CAT gene. The pBLCAT6 vector was used as an internal control. Tissue specific expression of the CAT construct containing the enhancer/promoter of the murine tyrosinase gene was examined in a number of canine cell lines including D17 (Osteosarcoma) and CML10 (Melanoma) and one mouse cell line (B16 Melanoma) (Fig. 1). Transfection of the tyrosinase promoter-containing vector into different canine cell lines demonstrated tissue specific expression in CML10 canine melanoma cells with no significant expression detected in other canine cell lines. In parallel we have explored the possibility of using thyroid specific gene expression to target toxic gene therapy to the feline thyroid gland to treat hyperthyroidism. In our own studies we have demonstrated that the feline thyroglobulin promoter exhibits tissue specific activity and may provide a tool for the transcriptional targeting of gene therapy to the feline thyroid gland as an alternative treatment modality in hyperthyroidism.

Cancer-Specific Targeting

The division between a tissue specific and a cancer-specific promoter is a very blurred one. However, as many of the TSP's are confined to cancers of specific tissues, the possibility of developing a transcriptionally targeted promoter with a broader spectrum of activity is an attractive one. The development of such a system relies upon the identification of a gene/protein that is unique to a broad range of cancer types.

In this laboratory we have focused on a number of systems that rely on the expression of genes in cancer cells that are silent in normal cells. Loss of the p53 tumour suppressor gene function has been implicated in the pathogenesis of many human malignancies and recent evidence suggests that p53 may play an important role in the development of domestic animal neoplasia. We have recently investigated p53 as a potential target for gene directed therapy for canine cancer. In brief, we demonstrated that the accumulation of p53 protein in cancer cells in vitro could bind to exogenously introduced gene products, resulting in the transcriptional activation of a reporter gene. The approach involved the construction of two vectors. In the first vector, a construct was engineered to produce a chimeric protein. Part of the protein is able to bind to the enhancer/promoter region of the second vector. The other part of the chimeric protein is the c-terminal domain of canine p53 that is able to bind to p53 and recruit the transcriptional activation domain (TAD) of p53. This leads to expression of a therapeutic or reporter gene in the second vector. This system represents a method of targeting therapeutic or toxic genes to cancer cells in-vivo.

Although mutations in the p53 gene are well recognized in human and veterinary oncology, there are still a large proportion of cancers that do not show aberrant p53 expression. From studies on cellular senescence, expression of the enzyme telomerase has now emerged as a central unifying mechanism underlying the immortal phenotype of cancer cells and has thus become a candidate for differentiating between normal and neoplastic cells. Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. In humans, telomerase expression is repressed in most somatic tissues and telomeres shorten with each progressive cell division. In contrast, telomerase activity is a common finding in many human malignancies resulting in stabilized telomere length. The level and frequency of telomerase activity and component gene expression in cancers reinforces this as a potential target for cancer therapies. It is now well documented that the level of telomerase in malignant tissue compared to normal tissue is much higher and this differential is greater than that for classical enzymatic targets such as thymidylate synthase, dihydrofolate reductase or topoisomerase II.

We have assessed the activity of human hTERT and hTR promoter sequences to drive expression of reporter genes in canine cell lines. Briefly, plasmid based constructs (either hTERT or hTR? driving expression of luciferase, kindly provided by Dr. Nicol Keith, Medical Oncology, University of Glasgow) were used in transient transfection studies of telomerase positive canine cell lines (MDCK, D17 and CML 10) and human 293T cells. All transfections were carried out in duplicate and luciferase was quantified using standard luminometry. Activity was measured as a percentage relative to an SV40 luciferase control plasmid and corrected for transfection efficiency. The results are shown in figure 2 and demonstrate activity of these promoters in canine cell lines. The activity of the hTERT promoter appears to be weaker than the hTR promoter in D17, CML10 and MDCK cells. Although the human promoters are clearly active in canine cells we have recently isolated the canine homologues and aim to utilize the canine specific promoters in future studies.


Although there are many problems associated with the technical aspects of gene therapy, one must be able to put this in to context with conventional drug development. It is only 12 years since the first human clinical trials in gene therapy, and this is a very short time when comparing the development of classical drug therapies. When considering the failures of gene therapy, it must be borne in mind that many of the described clinical trials have been marker or reporter studies or have been carried out in human patients who have already undergone rounds of conventional drug therapy and have little hope of survival. The holy grail of cancer gene therapy is systemic gene delivery and many of the hurdles facing gene therapy revolve around vector development and the design of new systems that can target metastatic disease in a controlled fashion. It is clear that the development of targeting systems will help to increase the therapeutic index, however, for the immediate future, it is apparent that gene therapy will sit alongside conventional drug therapy and act as adjunct rather than a sole treatment. Because of the hopes placed in cancer gene therapy and the ease at which DNA can be manipulated, it is very easy to rush into clinical trials in our veterinary patients. However, in the veterinary field, I hope that we can look at the mistakes made by our medical colleagues, and ensure that we have the science in place before we embark on clinical phases.

Biographical Profile
David Argyle graduated from the University of Glasgow and subsequently worked in small animal practice before returning to Glasgow to study companion animal immunology and oncology. He was awarded a PhD for his work on the feline interferon system in 1995 and subsequently joined the faculty staff. Up until recently, he was senior lecturer in clinical oncology and gene therapy within the department of veterinary clinical studies and is also head of clinical oncology referral service. He is now associate professor of oncology at the University of Wisconsin-Madison. His research interests include the basic biology of cancer and aging and the development of targeted molecular therapeutics for cancer. Dr. Argyle is a Royal College recognized specialist in veterinary oncology and is currently president of the European Society for Veterinary Oncologists.

In: Genes, Dogs and Cancer: 2nd Annual Canine Cancer Conference, 2002 - Aurora, OH, USA, Modiano J. F. (Ed.)
International Veterinary Information Service, Ithaca NY, 2002; P0408.0902


This article contains copyrighted material, the use of which has not always been specifically authorized by the copyright owner. I am making such material available in my efforts to provide background knowledge on areas related to canine cancer. I believe this constitutes a 'fair use' of any such copyrighted material as provided for in section 107 of the US Copyright Law. In accordance with Title 17 U.S.C. Section 107, the material in this article is distributed without profit for educational purposes.


Famous model Golden Rusty