Cyclists during the race
CRISPR

The future of gene doping and how to test for it

1 September 2021

All local recommended safety guidelines followed at the time of interview.

Whenever the world's best athletes clutch their medals in victory, there is often a question mark hanging over their success. Was it pure natural ability, or did they accept a little extra help with performance enhancing medications? Athletic performance may even be altered on a cellular level. But can you still identify doping when it has become a part of your DNA?

“The history of doping among athletes is as old as competitive sport itself”, says Dr. Patrick Diel, an endocrinologist at the German Sports University of Cologne. It started with the original Olympic Games 2,500 years ago when runners imbibed certain plants or mushrooms to enhance their performance. Doping among cyclists began well over a century ago, while Japanese athletes at the 1936 Olympics were reportedly the first to use blood transfusions (blood doping) to boost their endurance before an event, says Diel. But it took the death of British cyclist Tom Simpson, who had overdosed on amphetamines during the 1967 Tour de France, to really initiate anti-doping laws.

As someone who has run 16 marathons, Diel understands what compels people to take up sport. He is also a keen amateur cyclist who racks up 4,500 kilometers a year when he's not at the German Sport University Cologne conducting research across multiple fields, including the latest developments in doping.

When the dope-fueled Simpson collapsed and died cycling up a tough slope on a brutally hot day, the sports world could no longer ignore the problem of performance-enhancing drugs, so the first doping controls were introduced the following year at the 1968 Olympics and Tour de France. And "Ever since," says Diel, "there has been a kind of competition between the people abusing and the people trying to detect it."

The sports world could no longer ignore the problem of performance-enhancing drugs after a cyclist lost his life due to an over-dose at the 1967 Tour de France. "Ever since," says Diel, "there has been a kind of competition between the people abusing and the people trying to detect it." It is speculated that doping enhances performance by no more than 10-15%, but amongst motivated athletes, that is the difference between Gold and Silver. Gene-doping is the modern method of enhancing performance, but how does one test for it?
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Ever since the birth of anti-doping in the late 1960s, there has been a kind of competition between the people abusing and the people trying to detect it.
Dr. Patrick Diel, Molecular and Cellular Sports Medicine, German Sport University, Cologne

The culture of doping

History would imply that it's unlikely no Olympics gold medalist won without the aid of performance-enhancing drugs in the past 40 years. “When it comes to winning, though, we are talking about a difference of 0.1 or 0.2 seconds." That's not much, but at the very top of the sport it's the kind of margin that doping can make. And that is enough of an incentive.

"The old method for gene doping," Diel says, "was to give the athlete a drug. There was an increase in performance, but the effect was transient and wore off within days. The definition of gene doping was that it was a kind of abuse of gene therapy."

Some newer doping methods are simply exploiting scientific tools that were developed for patients with disease. For example, Diel says “a lot of pharma and biotech companies are working on ways to treat people with type two diabetes that address the regeneration of their skeletal muscle mass. A worthy medical goal, everyone would agree. The downside”, says Dr. Diel, “is that such drugs can all be abused for doping purposes when taken by a healthy, powerful young athlete." For WADA, this means more new substances to test for and detect.

Dr Diel
Dr. Patrick Diel is a highly experience endocrinologist working at the German Sport University Cologne in the department for Molecular and Cellular Sport Medicine. When the World Anti-Doping Agency (WADA) got involved in funding anti-doping research after the turn of the century, Diel started researching the use of myostatin, and several follow-up projects at the university's center of preventative doping research.
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We have many different techniques used to increase skeletal muscle mass, for example, and now all these techniques can be abused for doping purposes.
Dr. Patrick Diel, Molecular and Cellular Sports Medicine, German Sport University, Cologne

The start of gene doping

Diel continues to explain that “WADA now defines gene doping as the introduction and the use of DNA and RNA for enhancing performance, and also the use of cells which have been modified by gene techniques."

He cites targeting myostatin, which regulates muscle growth, as a good example of how complicated it is to detect drug cheats. Suppressing myostatin stimulates muscle growth, and there are several ways to do this.

Aside from using antibodies to block the myosatin receptor which isn’t classified as gene-doping, "you can also use gene editing - manipulating DNA and RNA - to suppress myostatin," says Diel, "which is gene doping, but it doesn't matter whether you use gene doping or a conventional technique. In the end, there's muscle growth resulting in increased strength and improved velocity and no active myostatin, and that's an issue of manipulation."

runners starting from the starting line
The readiness of athletes - both top-level and amateur - to embrace new cheating techniques was illustrated when Diel and his team published their research on the effects of suppressing myostatin in mice in a scientific journal. Shortly afterwards, he was astonished to find that the contents of the obscure paper were being discussed on internet chat forums by amateur runners and body-builders. "These people are asking each other, 'Have you read the article from the Cologne Sports University? Amazing!' That's how fast word gets around about possible new doping techniques. This is dangerous gene manipulation, and all they want to know is how well it works."
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We have some very sensitive PCR techniques. I think, detecting gene editing is not more difficult than detecting any form of doping. It's just different.
Dr. Patrick Diel, Molecular and Cellular Sports Medicine, German Sport University, Cologne

PCR and sequencing to detect gene doping

"Whether you define it as gene doping or regular doping is irrelevant," he continues, "because there are no clear borders." The old method of giving the athlete a drug to manipulate their DNA, say, had a transient effect and wore off within days. It's much more complicated to detect the use of growth hormones, for example, because they include peptides and the life span of these proteins is very short," Diel says.

This 'transient gene editing' means that "after a few hours it's gone and no longer detectable. But when you manipulate your genome, the information is there in your body for a very long period, maybe forever. Using sensitive PCR and sequencing techniques, you just need a few samples to check for any remaining traces of manipulated DNA."

"When you have a gene manipulation that is permanent and stable, it's very easy to detect. So any manipulation can be detected quite easily by PCR techniques - you can see the difference in the genome. And today, it's not a problem to sequence huge amounts of DNA in a very short time."

To summarize, it's not easy, but I think that detecting gene editing is not more difficult than detecting any other form of doping. It's just different."

Gene Expression, life science, female scientist working in a lab
Transient gene editing, using a drug to temporality affect DNA, means that "after a few hours it's gone and no longer detectable. But when you manipulate your genome, the information is there in your body for a very long period, maybe forever,” explains Diel. Using sensitive PCR and sequencing techniques, you just need a few samples to check for any remaining traces of manipulated DNA." Once a gene has been edited using the CRISPR/Cas-9 technique, the DNA can be extracted, amplified and checked using, for example, QIAGEN's QIAprep&amp CRISPR Kit. QIAGEN's CRISPR-Q Custom PCR Assays and CRISPR Q-Sanger Primers.
Looking for a faster, easier way to characterize gene edits?
Check out our new CRISPR screening workflow and see how you can verify the success of your editing experiment in 4 simple steps.
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