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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.
Conclusions
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
FAIR USE NOTICE
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. |
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