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Gene doping: What is it and how is it being combated?

Nick Busca
2 Oct 2018

Harder to detect than EPO, gene doping is a less reported front in the fight for clean cycling

The history of doping and anti-doping is something like Wile E. Coyote chasing the Road Runner: no matter how close Wile E. gets to the Road Runner, the latter is always one step ahead. This seems even more the case for a new, shadowy corner of doping that may sound like a science-fiction script, but actually has been around for at least two decades: gene (or genetic) doping.

But despite the rapid development of gene doping, a new testing methodology for gene doping may represent an important turning point against the use of genes for performance enhancement purposes.

ADOPE (Advanced Detection of Performance Enhancement) was presented at the University of Stirling, Scotland, in early September and is one of the very few known tests against gene doping.

The method was developed by a group of scientists of the Technical University of Delft, the Netherlands, and it will compete against more than 300 other teams at the 2018’s Genetically Engineered Machine competition; the award ceremony will be held in Boston, MA, on 28th October.

First things first: what is gene doping?

Gene doping is the 'misuse' of gene therapy for performance enhancement purposes. Gene therapy, on the other hand, is a technique that uses genes rather than drugs or surgeries to treat or prevent diseases.

The therapy consists in the delivery of external genetic material into a patient’s cells. The genetic material – which contains a specific expression that activates the proteins used to treat the disease – is inserted into the cells using an external vector (normally a virus).

Let’s take EPO, for example. The Erythropoietin – the protein that stimulates red blood cells production in the bone marrow, and consequently increases the levels of haemoglobin in the body and the oxygen delivery to the tissues – is normally secreted by the kidneys.

EPO injections have been the notorious performance enhancement that cyclists abused for several years, particularly in the 90s.

Today, even though cases of EPO positivity are still reported, it’s become harder to get away with this practice as anti-doping controls can detect external EPO pretty efficiently nowadays.

However, the gene doping alternative, which enhances EPO production through the insertion of new genetic material into an athlete, would eventually look like a natural product of the athlete’s own physiology and not like a banned substance.

Although gene therapy is still only used for rare diseases that have no cure (like severe combined immunodeficiency, blindness, cancer and neurodegenerative diseases) scientists have confessed that people from the world of sports have approached them and asked them to use these therapies as a way to enhance their sport performances.

WADA and gene doping

The World Anti Doping Agency (WADA) organised the first workshop to discuss gene doping and its threats in 2002, while the practice was listed on WADA’s list of illegal substances and methods the year after.

Since then WADA has been devoting part of its resources to enable the detection of gene doping (including the creation of several groups and panels of gene doping experts), and in 2016 a routine test for EPO gene-doping was implemented in the WADA-accredited laboratory in Australia, the Australian Sports Drug Testing Laboratory.

However, the testing methodologies for gene doping can be laborious and require a wide knowledge of a specific DNA sequence for the actual testing practice.

The method proposed by ADOPE, on the other hand, focuses on targeted sequencing and combines the beneficial principles of the other methods in a potentially more efficient and targeted way.

The ADOPE testing methodology

ADOPE testing methodology has been developed through tests conducted on bovine blood and it’s structured in two phases: the first one is a pre-screening phase that targets a potential gene-doped blood, while the second targets specific genetic sequences to verify whether the DNA has truly been gene-doped or not.

'In the pre-screen,' explains Jard Mattens, Human Practices Manager of the TU Delft team that developed ADOPE, 'we further develop the use of so-called dextrin-capped gold nanoparticles for gene doping detection.

'The principle is based on the fact that gold nanoparticles induce a gradual quantifiable colour change of the sample when it contains the "doping" DNA.'

In order to work on and test a 'gene-doped DNA' – but without the need to actually gene-dope athletes or animals – the TU Delft team artificially 'spiked' bovine blood with several complementary DNA sequences.

The aim of their tests was to target and find the 'gene-doped' sequences they added into the blood.

'We use bovine blood as a good substitute for human blood since the principle works in the same way,' explains Mattens.

'For our test, we add several DNA types to this bovine blood in different concentrations to mimick the concentration development over time according to what we modelled previously for humans.

'From that point on our detection method will be the same and the DNA we added to the bovine blood should be detected by our method.'

Once the potential gene-doped blood has been identified due to change of its colour, the second phase of the test follows, targeting the specific sequences that have been added to the blood.

'To verify this initial screening,' continues Mattens, 'we use a technically unique and innovative CRISPR-Cas – Transposase fusion protein.

'This can be seen as a nanomachine that is able to specifically detect the specific differences present in gene doping DNA.'

The CRISPR, or CRISPR-Cas9 (or gene editing), is a different and more advanced technique that allows geneticists that uses two molecules – an enzyme called Cas9 and a piece of RNA – in order to produce a change (mutation) into the DNA.

This technique was also banned by WADA from the beginning of 2018 as a more advanced gene-doping technique, but in the case of ADOPE the CRISPR-CAS technique is used to find the modified DNA instead of modifying it.

The specificity of ADOPE

The model of testing developed by ADOPE has been specifically conceived and developed to detect the gene that enables the production of EPO in the human body, but as the methodology is highly versatile, the researchers of TU Delft claim it can be 'extended to detect any kind of gene doping.'

Based on the cycle during which EPO is effective in the body, the most likely time when athletes would dope using this specific gene would be well before competition – but at the same time, other genes, targeting different proteins and physiological enhancements, may have a much quicker effect.

That is why ADOPE aims to implement the regular anti-doping tests throughout the whole training and racing calendar.

However, as the so-called 'cell free DNA' targeted by the tests is expected to be very low in urine (although present here as well), for the time being ADOPE only works on blood samples and its detection window is still limited.

'Based on an experimental test with non-human primates by Ni et al in 2011,' says Mattens, 'we expect the detection window to be just a few weeks.

'Further development of the method might make the same method work for urine as well in the future.'

The difference between ADOPE and other approaches

'Most [of the other gene doping testing] approaches rely on PCR based reactions [Polymerase chain reaction: a technique that make copies of a specific DNA region in vitro], which have many drawbacks,' add Mattens.

'These reactions are relatively laborious and require extensive previous knowledge of the DNA sequence. Furthermore, using these anti-doping testing technologies, making the probability of evading detection significantly higher.'

Alternatively, some other testing practices focus on the whole genome sequence; that is, the whole genetic material present in a cell or organism.

But the downside of this approach is that the whole genome sequence must be taken into account, which is time consuming, inefficient and could also be seen as an invasion of athletes’ privacy.

'Our approach,' says Mattens, 'focuses on targeted sequencing, which combines beneficial principles from both approaches in a complementary manner.

'It utilises the specificity principle of PCR, however it requires only one target site on the transgene (but requires multiple sites for searching), making the probability of evading detection significantly lower.

'[ADOPE] utilises the sequencing principle of whole genome sequencing, however in a more efficient and targeted manner, dramatically reducing the amount of data.

'As a result we believe targeted sequencing is a much better approach and the future of gene doping detection.'

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