Figure 1. Gene doping timeline since it prohibition in 2003.

2003 WADA prohibits gene doping Since 2003 WADA considered gene doping a realistic threat to fair sports and put gene doping on the list of prohibited substances for professional athletes. 2004 Geneticists engineer marathon mice Engineered mice run twice as far as regular mice before becoming tired. 2006 First gene doping accusation for Thomas Springstein The one-time coach of the German Athletics Association was found guilty of giving athletes performance enhancing drugs without their knowledge. An email in which Springstein attempted to obtain Repoxygen, a type of gene therapy, was read by a prosecutor. 2008 Doctor offers gene doping to athletes at Beijing Olympics Summary: In a documentary by ARD television, a Chinese doctor offers stem-cell therapy to a reporter posing as an American swimming coach shortly before the Beijing Olympics. Anti-doping officials were appalled that the treatment was so readily available. 2010 Evaluation of detection of gene doping Gene doping detection windows were evaluated in murine models and a detection method based on Polymerase Chain Reactions (PCR) was proposed by Baoutina et. al. 2016 Rio Olympics spark discussion about gene doping The possible use of gene doping sparks debate and extensive media coverage leading up to and during the Rio Olympics. According to the International Olympic Committee’s medical and scientific director, Richard Budgett, samples collected during the Olympics will be tested at a later date after the games. 2017 Start of gene doping control analysis EPO cDNA amplification assays based on real-time PCR are performed on samples selected exclusively of research. Due to absence of interlaboratory tests among WADA accredited laboratories, the analysis was not performed on the Rio Olympic samples. 2018 iGEM TU Delft starts developing ADOPE Our team takes up the challenge of developing a high throughput, versatile and reliable detection method for gene doping.

Customer: Anti-doping agencies

The main customers for our gene doping detection method are anti-doping agencies. The national anti-doping agencies are all given directive by the World anti-doping agency (WADA), leader in stimulating research into anti-doping measures. Their jobs are to protect the spirit of sports, prevent unethical performance enhancement and protect athletes health. These three criteria are used to evaluate if a substance will be added to the list of banned substances, after which a detection method will have to be developed to enforce the ban.
Thus, they benefit from an implemented detection method for the substances which are on the list, in this case gene doping. In these tests, they profit from high data processing speed, making the analysis of doping tests faster so they might prevent doping users from starting a race as the results are in before then. In addition, it is advantageous for them to have high-throughput tests, allowing for the testing of many samples at the same time. Athlete privacy is also an important advantage, since this is in compliance with data protection laws. They also benefit from a test that is accessible to all WADA-accredited laboratories and that fits within the current testing framework. This means that tissue sampling cannot be considered for doping tests (Haisma, 2004) and that new doping tests can be implemented within the current testing practices and times. Finally, a method would be easily adaptable to new developments in the doping world, keeping up with the rat race of doping user versus doping detector.
In contrast to these benefits, there are a few outcomes that doping authorities strive to prevent. False positive test outcomes must be prevented at all costs. A single false positive result could ruin an athletes career unjustly, so everything possible must be done to prevent such an outcome. Finally, they strive to lower the costs of detection methods as much as possible.

Value: Advanced Detection of Performance enhancement

The product we present is ADOPE, which stands for advanced detection of performance enhancement. The result is not a single kit, rather, it is a comprehensive step-by step method, including products (adapted kits and newly developed proteins), as well as services for more efficient testing and data analysis. The result includes a model for selecting the proper detection time, a sample preparation method for isolating trace elements of gene doping DNA from the blood, a pre-screening method to quickly screen and rule out many samples, an unique test based on targeted sequencing using a newly developed fusion protein, and finally a sophisticated, self-learning algorithm for data analysis to group the sequencing output in a reliable manner.
These products and services answer the needs from the anti-doping agencies in several ways. Our efficient pre-screen reduces costs and increases throughput. The screen rules out most of the samples, leaving only a fraction to the relatively more expensive sequencing step.
Due to our highly specific fusion protein, our method sequences only a low amount of DNA. This reduces data-processing time greatly compared to methods based on whole genome sequencing.
The sequencing step reduces false positive test outcomes, delivering a DNA sequence from the athletes blood as definitive proof of doping use. Thanks to the versatility of the target sequences we use to find doping DNA as well as our self-learning algorithm, our test is easily adaptable to new developments in the doping world. In addition, our test does not require expensive or rare laboratory equipment apart from the consumables supplied with the test, making is accessible to all WADA-accredited laboratories.
The products and services also relieve pain for our user by fitting within the current testing framework. Thanks to our detection window analysis, testing can be done at the frequency of regular inter-competition testing as executed by local doping authorities. Our test also facilitates compliance with data laws. Due to targeting specific DNA, only the data that is needed for a reliable test outcome is collected.

We investigated the need for and interest in a gene doping detection method for equestrian sports with the Dutch Trotting and Flatracing Association (Nederlandse Draf- en Rensport, or NDR). We spoke with Mr. Camiel Mellegers of the NDR, who is in charge of doping testing for the Dutch matches in Trotting and Flatracing. He has explained to us that anti-doping policies differ in quite a few ways between human and equestrian sports. We will shortly discuss the differences that are relevant to the case of gene doping.

• The threat of gene doping is a worrying topic in equestrian anti-doping. It is one of the topics discussed on October 8th of 2018 in the 52nd International Conference of Horseracing Authorities in Paris, France (Chesser, 2018).
• The anti-doping criteria in horse racing are stricter than in humans. The human anti-doping list consist of substances that check two of the three criteria of increasing performance enhancement, endangering athlete or community health and being against the spirit of sports. equestrian sports are stricter; they do not allow any substance that is not naturally present in the body of the horse or that is present in elevated concentrations. Mellegers explained to us that this is because horses are never consenting parties when it comes to doping use, and so they must be protected more strictly.
• EPO is not a suitable target gene for equestrian gene doping, as it does not have a performance enhancing effect in horses. Other genes have been identified, that e.g. enhance muscle mass and strength, regulate oxygen, help overcoming pain, enhance injury repair or are involved with angiogenesis and the energy metabolism (Wilkin et al, 2017). Due to the versatility of our method, detection of other genes is easily achievable. Our model on the determination of the detection window will have to be adapted drastically, since it will have to take into account equestrian physiology as well as regulation cycles for the genes in question. All other components of our detection method will be easily convertible to horse racing.
• Doping testing in equestrian sports works via an A-B-C system. First, the A sample is tested for doping. If the result is positive, the horse owner or trainer can decide to appeal. As part of this appeal, the B sample is tested with a more thorough detection method. The C sample is stored for (anonymous) research purposes. “Your pre-screen and screen fits with this model well.” Mellegers assured us. Since the appealing party pays for the testing of the B sample him or herself, cost is also less of an issue.
• Testing speed is important in applying a detection method. “The time between placement matches and registration closing of the main match is often only one week,” Mellegers explains, “so a doping sample should be shipped to testing facilities abroad and analyzed in less than one week. Most of our tests take two to three days.” Herein lies the strength of our fast detection method compared to methods without pre-screen or efficient data-processing.
• Blood will probably always be collected as a sample in the future. Currently, a doping official has 20 minutes with a horse to collect a sample. They initially try to receive a urine sample, but if the horse does not comply they take a blood sample. “In the future, we are planning to collect both samples always so that we have enough sample material to do advanced testing in cases of appeal.” according to Mellegers. For ADOPE, this means that blood sampling will remain relevant in equestrian testing.
• Finally, we learned that transparency is incredibly important in equestrian sports. “Horse racing is built from dreams” Mellegers says. If you are not completely transparent, people will mistrust any outcome and this will damage the sport greatly. All doping test results must be published immediately. Since transparency and being open source are values ADOPE embodies, we believe that our test will be a good match to the equestrian anti-doping policies.

To conclude, we will shortly discuss the potential of our detection method in the equestrian sports, motivated by the topics above. The treat of gene doping is real in equestrian sports (Chesser, 2018) and no detection method is currently in place (Wilkin et al, 2017). A continued ban of gene doping is highly probable based on the anti-doping values in equestrian sports. Adapting ADOPE to fit within the equestrian anti-doping policies is fairly uncomplicated. The current model gene EPO will need to be changed to a gene more relevant in horse physiology, which is what our versatile detection method was developed to do. The detection window analysis will need to be adapted to the new target genes. Our method is a good match to the equestrian anti-doping policies due to our fast result, stemming from our targeted sequencing approach. The distinction we make in an inexpensive and fast pre-screen and more expensive, highly precise screen fits well within the A-B sample method in equestrian testing. Our approach of complete transparency fits with the core values of the sport, ensuring that the dream of horseracing remains intact.

We initially visited Dutch National Bloodbank Saunquin to learn more about our sample preparation method, and there met Ms Aïcha Ait Soussan, who’s main research interest is fetal DNA in maternal plasma. When discussing our detection method with her and her collegues Onno Verhagen and Professor Ellen (C. E.) van der Schoot from the experimental immunohematology department, it became apparent that they are struggling with many of the same problems in isolating fetal DNA from the mother blood as were are with isolating gene doping DNA from an athletes blood. They are both present in only little amounts compared to an enormous background of genomic DNA. A targeted sequencing approach as we are developing would interest them as well.

The Dutch Research Department for Food Safety at Wageningen University and Research (RIKILT) reached out to our supervisory group because of their real interest in the application of our gene detection method, with a specific focus on our targeted or selective sequencing based on the fusion protein we have developed. They see many applications for this technology, and have invited us to discuss them with us and our supervisory group after the jamboree.