Current IssuesNo.52
January 2008
 
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The Genetic Doping Race – Will the Testers Finish First?
Vincent Chew

 

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:
  1. to use SAGE (Serial Analysis of Gene Expression) to identify the transcriptional response in blood cells to exogenous EPO treatment in mice;
  2. to identify and characterise the homologous transcripts in human blood and establish which of these could be used as in a diagnostic test for EPO use; and
  3. to develop and evaluate a prototype qPCR-based test for exogenous EPO using these transcripts.
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





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