![]() | Current Issues | No.52 January 2008 |
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Human DNA contains 20,000-25,000 genes. When one
of these genes is defective, missing or becomes mutated, this can lead
to adverse health consequences. On the other hand, the introduction of
a healthy gene to the system, in theory, can lead to a medical miracle.
To date, genetic therapy has shown to be effective in the fight against
cancer, and has been used to try and fix sight disorders. Showing its
own vision and looking ahead to the next potential reality, the World
Anti-Doping Agency (WADA) has been attempting to get ahead, instead
of playing catch-up to the cheaters.
Since 2001, WADA has funded research projects related to genetic therapy
and cellular manipulation, in order to determine if genetic doping has
occurred, and in 2003, WADA’s Prohibited List was amended to include
gene doping.
One of these research projects is headed by Dr. Jim Rupert of the University
of British Columbia (UBC). No stranger to sport science and genetic
research, Rupert focused his PhD research on altitude adaptation and
later worked with early mouse development in gene expression analysis.
Since 2005, his lab has been attempting to create a blood-based test
capable of detecting exogenous erythropoietin (EPO) activity. EPO is
a hormone that helps to increase red blood cell production. The more
EPO in the body, the greater capacity for the blood to carry oxygen.
With more oxygen in the blood, higher levels reach muscles, helping
aerobic activity, which can enhance activity in endurance athletes.
While altitude training is one method that helps to stimulate natural
production of EPO, the dawn of genetic therapy has led to scientific
theories on how to artificially create EPO through genetic engineering.
Rupert’s hypothesis for his detection method is that “gene
doping will result in a distinct pattern of changes in gene expression
that will be different from those that occur in response to natural
stimuli, and that these differences could form the basis of a test for
exogenous gene products.”
According to Rupert’s WADA application, the goals of this project
are to:
In other words, as Rupert explains, “Genes are the blueprints
for the proteins that make up much of the structure and “machinery”
of the body. The intermediate between genes and proteins are messenger
molecules called “mRNA” and the more active a gene is, the
more mRNA it produces (or “expresses”). Technically, measuring
RNA is easier than measuring protein, and, as the activity of genes
is tightly regulated and highly environmentally sensitive, measuring
specific mRNA molecules can reveal much about the state of the body
– including, we hope, detecting the effects of “doping”.”
While the current tests can identify whether athletes have injected
themselves with artificial or synthetic EPO (otherwise known as “rHuEPO”)
due to differences between the natural and artificial molecules, the
fear is the corresponding increase in EPO from putting the gene into
their cells would be indistinguishable from the body’s natural
production.
To date, Rupert and his team have identified thousands of genes, which
are differentially expressed between treatment groups. Rupert explains,
“We are currently taking the approach of examining both known
transcripts which map to the mouse genome and also unknown transcripts.
This is one advantage of SAGE over another technique such as microarrays
because it allows the identification of both known and unknown transcripts.
We are clustering our known transcripts into functional groups and some
interesting patterns are emerging. As one would expect, rHuEPO treatment
upregulates hundreds of genes involved in erythropoietic pathways as
well as iron regulation, heme production and other important cellular
and metabolic pathways. Interestingly, as we hypothesised, the number
of genes which change in response to rHuEPO treatment is significantly
smaller than those which change in response to hypoxia which indicates
that hundreds of pathways are involved in an adaptive response to hypoxia
(including erythropoietic pathways) versus relatively few involved in
RBC production.”
Right now, the team is validating “genes of interest” and
looking for variability due to different parameters such as gender,
exercise and time of day. Rupert anticipates entering into the final
stage sometime in 2008, which involves “confirming our results
by dosing healthy human athletes with rHuEPO in a double blind fashion
and looking for our predicted gene signatures in these individuals compared
to placebo and hypoxia-treated individuals. We are currently collaborating
with another UBC research group to obtain blood samples from trekkers
in Nepal which have been acclimatized to 5,300 metres for two weeks
to form our hypoxia-exposed pool.”
There are other types of tests that WADA is funding in order to come
up with a definitive method of detection. These include a test using
non-invasive molecular imaging technology by the Institution Municipal
Investigacio Medica (Barcelona), CEXS-UPS, the Center for Genomic Regulation,
and the PET Center – High Technology Institute. The gene for EPO
is not the only one on WADA’s radar.
Dr. Jane Roberts, the Head of Pharmaceutical Contract Research at the
HFL Laboratory in Cambridgeshire, UK, is looking at tests for detecting
the use of the gene IGF-1 (Insulin Growth Factor 1), which assists in
muscle growth. Previously, HFL has focused on doping detection in equine
and canine sport by identifying metabolites and detecting abuse using
mass spectrometry and immunochemistry.
In partnership with the Nottingham Trent University/University College
and the Royal Free Hospital London, her current test focuses on the
use of both proteomic as well as transcriptomic techniques to identify
changes in genetic expression.
Roberts explains, “We are carrying out an analysis of the entire
proteome of blood serum and comparing the data generated from treated
and untreated subjects using computer based artificial neural networks
(ANN's). ANN's work in a manner comparable to the way the human brain
works and are extremely powerful tools for detecting patterns within
complex data. By 'training' a neural network to identify pattern differences
between treated and untreated subjects it is then possible to assess
data from other samples and assign it as being from a treated or untreated
subject.”
At this stage, Roberts and her team have identified potential biomarkers
and are currently trying to more accurately quantify these markers,
followed by validation through large population samples. Ultimately,
the goal would be to use simple, rapid techniques like immunoassays
to identify these markers.
According to Dr. Olivier Rabin, the Director of Sciences at WADA, since
2001, the agency has dedicated US$31.4 Million of its budget towards
research matters. Of that, approximately US$6.5 Million, or just over
20% of that figure has been targeted toward gene doping practices. In
addition, the amount allocated towards genetic doping research has doubled
from US$1.2 million in 2006 to US$2.4 million in 2007.
Rupert feels that gene doping is not occurring at the moment as genetic
therapy is still highly experimental, but it is only a matter of time.
He notes, “Our project will provide a lot of basic data on blood
gene expression that all anti-doping researchers will be able to access.
Doping tends to trail medical developments by only a few months, and
there is a lot of research being done into gene therapy.”
Roberts adds that while it’s difficult to definitively state
if gene doping is currently occurring, “we do know that workers
in the area of gene therapy have been approached by athletes following
the publication of scientific results. Also, in a relatively recent
case involving a sports coach, evidence was gathered that he had sought
to obtain a gene therapy agent for the production of EPO called 'Repoxygen'.
It is also relatively straight-forward to manufacture some of the possible
agents such as plasmids and commercial devices for implantation, such
as electroporators, are available. It remains highly dubious if gene
doping using such untried techniques would be successful in the human,
however, the technologies are available and it is highly likely that
someone will try to abuse these.”
With less than 200 days until the Beijing Olympic and Paralympic Games,
and 750 until Vancouver 2010, the countdown clocks are not only racing
towards the start of the games, but also to mark the need to finish
ahead of the cheaters in the race for fair and equal sport.
Contact
Vincent Chew
International Council of Sport Science and Physical Education Berlin, Germany Email: vchew@icsspe.org ![]() http://www.icsspe.org/portal/index.php?w=1&z=5 |