Gene Doping in Sports

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GENE DOPING IN SPORTS

Karin Kõrgesaar 12277207

MSc Forensic Science

Supervisor: dr. Wim Best

Examiner: prof. dr. Ate Kloosterman

Number of words: 7167

08/03/2020

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Abstract

Continuous advances in gene therapy have arisen a potential threat of gene doping – the transfer of genes and/or the use of normal or genetically modified cells to enhance athletic performance. The major objective of this literature thesis is to analyse the possibility of using gene doping and to identify and critically evaluate current detection methods.

The results suggest that gene doping is a rather risky method to increase athlete’s muscle mass, strength or endurance. The severe health risks and an insufficient number of studies outweigh its’ advantages. Moreover, scientists all over the world have put a lot of effort into the development of highly sensitive and specific detection methods, which include various types of PCR, LAMP and NGS. All these assays show good resolution and accuracy, however, the World Anti-Doping Agency (WADA) still has not implemented a standard detection method to be used. These results suggest that more research is needed to implement a cost-effective, fast, portable and highly specific method. As long as there has not been any conclusive evidence of gene doping being used in sports, there is still time to continue with the research and improvements.

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1. Introduction

Doping in sport is not a new phenomenon and almost every year we hear about several doping scandals from all over the world. Usually what is understood as doping is the use of different substances (pharmaceuticals, narcotics, hormones, anabolic agents etc.) or the manipulation of blood and blood components that could improve athletic performance, thus, help athletes to cheat their way towards unfair victory (World Anti-Doping Agency (WADA), 2020). In 2001 the first discussion on gene therapy and its’ future impact as the new potential doping method in sport was initiated by the Medical Commission of the International Olympic Committee (IOC). During this encounter it was concluded that the misuse of genetic modification methods is very likely to take place, and it is important to establish specific testing measures to identify athletes misusing this technology (Cummiskey, 2002). Since 2004 the World Anti-Doping Agency (WADA) has taken the responsibility to publish an international doping list of prohibited substances and methods, which is looked over and renewed every year. The method of gene and cell doping is included in this list (WADA, 2020, p.6) and the following is prohibited by WADA:

1. “The use of nucleic acids or nucleic acid analogues that may alter genome sequences and/or alter gene expression by any mechanism. This includes but is not limited to gene editing, gene silencing and gene transfer.”

2. “The use of polymers of nucleic acids or nucleic acid analogues.” 3. “The use of normal or genetically modified cells” (WADA, 2020, p.6).

In this literature thesis, the potential threat of gene doping for enhancing athletic performance in today’s world is described and analysed. First, I will give some relevant background information on gene therapy, followed by the possibilities for using gene doping, including the main target genes and potential health risks for athletes. Then I will discuss the detection of gene doping, including sampling and different detection methods and their reliability.

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2. Genetics and Sports

2.1. Gene Therapy

Medicines cannot always treat the malfunctioning of the human body. Over the past decade there has been a noticeable progress in science. All these technological advances have shown that an important tool to cure serious diseases caused by genetic disorders might be using gene-editing techniques (Sheridan, 2011). Gene therapy uses either the transfer of genetically modified foreign genes into malfunctioning human cells, gene correction/alteration or gene knockdown to try to repair defects in a person’s DNA sequence that are disease related (Friedmann, 1992; Kay, 2011). Genome editing can be either ex vivo or the editing machinery can be delivered in vivo using viral and non-viral vectors, such as plasmids, for gene delivery. In an ex vivo approach, cells are isolated from a patient or a normal donor, cultured, transfected in vitro with a therapeutic gene and then delivered back to the patient by electroporation or gene gun, without the exposure of the patient to the gene transfer vector (Naldini, 2011; Sheridan, 2011). For many genetic immunodeficiencies, cancers and blood cell diseases (e.g. adenosine deaminase deficiency, sickle cell anemia, acute lymphocytic leukemia, etc.) ex vivo gene delivery is used targeting T-cells and hematopoietic stem and progenitor cells (Dunbar et al., 2018).

In vivo gene transfer uses viral vectors (e.g. retroviruses, adeno-associated virus (AAV),

lentivirus, herpesvirus) to introduce nucleic acids into cells to prevent, halt or reverse a pathological process (Kay, 2011; Mingozzi and High, 2011; Sheridan, 2011). Methods used to insert these viral and non-viral vectors are, for example, direct injection into the target tissue, intravenous infusion (IV), aerosol inhalation for lung delivery. Besides, some aiding technologies can also be used, such as electroporation, gene guns, or plasmids bound together with liposome nanoparticles (Haisma and Hon, 2006; Kay, 2011; Sheridan, 2011). Some viral vectors, for example, retrovirus, integrate their genetic material into the host DNA. Other viruses, such as adenovirus, do not integrate and remain as an extrachromosomal episome in the cell nucleus. Integrating vectors are mostly used for ex vivo and nonintegrating vectors for in vivo gene transfer (Brzeziańska, Domańska and Jegier, 2014b; Neuberger and Simon, 2017). Viral vectors can only be used for one type of gene modification – gene addition (Dunbar et al., 2018). Viruses are highly evolved biological constructs that can gain access to

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6 host cells and deliver their genetic material with high efficiency into the host cell. As a result, in gene therapy all viral vectors used are replication-defective and all viral coding sequences are replaced with an expression cassette of the interested gene, hence, viral vectors are evolved into harmless transport units (Thomas, Ehrhardt and Kay, 2003; Dunbar et al., 2018).

Today the gene therapy market is categorized by various target areas, i.e. rare genetic diseases, cancers, infectious diseases, neurological diseases and cardiovascular illnesses. According to Shahryari, et al. (2019) there are twenty gene therapy products that have been confirmed for use worldwide, eight different gene therapies in Europe and thirteen in the USA. Furthermore, lots of research for a varied types of applications and clinical trials are still ongoing and a further growth in the field of gene therapy is expected (Shahryari et al., 2019).

2.2. Gene Doping

The genetic makeup of humans is diverse and that leads to different athletic features and capabilities which can make some people more successful in sports than others. The advances in gene therapy have led athletes to try to alter normal human features and to improve their athletic performance by genetic modifications. Gene doping is the transfer of the functional gene or recombinant protein into the body to enable its expression or to adjust the expression of an existing gene in order to enhance an athlete’s physiological performance (Haisma and Hon, 2006; Fischetto and Bermon, 2013). The misuse of gene technology has become a new threat in the world of sport next to other doping agents. The World Anti-doping Agency has already added gene doping to the prohibited list of substances and methods and has initiated anti-doping research in laboratories all around the world to attempt to find new detection methods and improve the accuracy of the already existing ones (WADA, 2020). These genetic modifications can cause an increase in body weight, enhancement in muscle strength and mass, and boost an athlete’s endurance (Haisma and Hon, 2006). So far there have been successful test trials of gene doping on animal models (Lee et al., 2004a; Wang et

al., 2004; Hakimi et al., 2007), however, no athlete has been caught yet in using this method

(Van Der Gronde et al., 2013a; de Boer et al., 2019). On the other hand, with the low testing and detection possibilities, the threat is still there.

Nevertheless, there are still some difficulties in gene doping. First, the introduction of modified genes does not have a long-lasting effect, therefore, repeated (multiple)

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7 integrations of the transgene have to be performed for desired results (Brzeziańska, Domańska and Jegier, 2014a). This makes this process inconvenient and more expensive. Moreover, there is always the risk of undesirable side effects when using viral vectors, especially when gene therapy methods in use are designed for people with certain disease and not for healthy athletes to improve their performance (Haisma and Hon, 2006).

2.3. Potential target genes

Up to now there have been various genes discovered that could have the potential to enhance athletic performance. Table 1 summarizes different potential genes for gene doping in sports. In this paper three of the genes are discussed in more detail.

Gene/Product Full name Function

ACTN2/3 α Actinin ACTN2 expression enhances endurance and ACTN3 expression enhances fast-type muscles and strength.

Endorphin or Enkephalin

Endorphin or Enkephalin

Naturally occurring peptide that can relieve pain and delay fatigue.

EPO Erythropoietin Enhancing endurance by increasing oxygen supply to muscles.

FGF2 Fibroblast growth

factor

In combination with other proteins could increase muscle repair due to its angiogenic effects.

GH Growth hormone Increases muscle strength, promotes the use of lipids for energy to conserve protein storage.

HIF-1 Hypoxia-inducible

factor-1

Regulates transcription at hypoxia response element.

IGF-1 Insulin-like growth

factor

Enhances muscle growth.

MSTN Myostatin The inhibition of it increases muscle growth.

PEPCK-C

Cytosolic phosphoenolpyruvate

carboxykinase

Regulates glyconeogenesis. Overexpression increases the rate of endurance.

PPARδ

Peroxisome proliferator-activated

receptor-δ

Decreases the aggregation of triglycerides in muscle cells and increases the oxidative capacity. As a result, enhancing endurance.

VEGF Vascular endothelial

growth factor

Increases endurance by increasing blood perfusion in muscles, heart, liver and lungs.

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2.3.1. Erythropoietin (EPO)

The EPO gene encodes the production of glycoprotein hormone that increases the oxygen supply to the muscle by producing more red blood cells (erythropoiesis). The glycoprotein hormone is normally synthesized in the kidney, secreted into the blood plasma, where it binds to the erythropoietin receptor to promote red blood cell production, or erythropoiesis, in the bone marrow. EPO gene expression is activated in hypoxic conditions, i.e. the decrease of the O2-pressure in tissue (Haisma and Hon, 2006; Brzeziańska, Domańska and Jegier, 2014a). As a result, an increased number of circulating red blood cells improves body’s energy production by aerobic mechanisms, which for athletes means better endurance and is the main reason to use EPO in gene doping.

In gene doping an additional EPO gene might be introduced intramuscularly (IM) into the body via viral or non-viral vectors. This will lead to the overexpression of the

EPO gene, thus, boosting athlete’s hematocrit (i.e. the proportion of blood volume

made up of red blood cells) (Haisma and Hon, 2006). On the other hand, the EPO gene can also have a legitimate therapeutic use in healthcare. A large number of EPO stimulating agents (ESAs) including recombinant EPO (rEPO), rEPO biosimilars, and EPO-mimetics have been developed after a successful cloning of the human EPO gene to treat anemia in patients with chronic kidney disease or in cancer patients with chemotherapy-induced anemia (Elliott, 2008).

2.3.2. Growth Hormone (GH)

Growth hormone is a human protein hormone that regulates many physiological processes in our bodies, including the stimulation of body growth and body weight, development of muscle mass, carbohydrate metabolism, the utilization of fat and protein metabolism. It is secreted from the anterior pituitary gland (Moøller and Joørgensen, 2009). It stimulates glycogenolysis (i.e. glycogen breakdown to glucose) in muscle cells and increases the release of glucose from liver. This raises the blood glucose levels which is an important fuel for endurance exercise. Furthermore, in endurance sports when energy reserves are limited GH increases the use of lipids

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9 (lipolysis) instead of carbohydrates and reduces lipogenesis to conserve protein storage which, at the same time, works on muscles and promotes muscle-specific growth factors resulting in overall leaner body mass (Van Der Gronde et al., 2013a; Brzeziańska, Domańska and Jegier, 2014a). Therefore, a GH gene therapy in athletes could mean that sprinters get bigger thighs and calves and swimmers, broader shoulders. On the other hand, there have not been any studies of gene therapy with

GH in humans. However, there have been few studies in animal models with varying

results (Rodriguez, Gaunt and Day, 2007; Oliveira et al., 2010; Higuti et al., 2016).

2.3.3. Insulin-like growth factor 1 (IGF1)

Besides longer endurance some disciplines in sport require lots of power, thus, athletes seek opportunities to gain more muscle mass and force. IGF1 is an insulin-like peptide encoded by the IGF1 gene and mainly produced in the liver and supervised by the growth hormone (GH). The hypothalamus produces growth-hormone-releasing hormone (GHRH), which stimulates the pituitary gland to release GH, thus, stimulating the liver to generate IGF1. IGF1 plays an important role in regulating the rise of the skeletal muscle mass, in stimulating hypertrophy and in repair following damage (Harridge and Velloso, 2009; Fischetto and Bermon, 2013). An increase in its’ expression by intramuscular injection improves muscle mass and force and facilitates recovery (Van Der Gronde et al., 2013a). A study in 2004 showed that a combination of viral administration of IGF1 and increased resistance training lead to an improvement in muscular mass by 31.8% and strength by 28.3% (Lee et al., 2004b). Moreover, another study showed that IGF1 gene transfer can fasten the regeneration of skeletal muscle after an injury (Schertzer and Lynch, 2006). This makes IGF1 a great potential target for gene doping.

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10 2.4. Health risks with gene doping

Gene doping is a relatively new method and certain side-effects and long-term effects of these gene manipulations are still unclear. The biggest worry is related to the techniques that are used to insert a gene of interest into an athlete. Gene doping uses transgenes, which could accidentally affect our other genes, (e.g. germ cells), producing permanent alterations which will be carried on to the next generations (Oliveira et al., 2011; Van Der Gronde et al., 2013a).

The expression of the new genes introduced into athletes’ bodies could become impossible to control, causing overexpression and the toxic accumulation of the protein, causing unpredictable health issues. Moreover, the inserted gene might not express at all, which could lead to gene silencing (Wells, 2008; Van Der Gronde et al., 2013a). Another risk is an immune reaction and the integration of the virus. Gene doping uses viral vectors as the carriers and because gene therapy requires a relatively high concentration of recombinant virus, it could trigger aggressive host immune response or the integration of the virus. Last-mentioned could lead to cancer (Sharp, 2008; Van Der Gronde et al., 2013a). This is especially alarming because as we know gene doping is something prohibited and must be done undercover. Hence, doping is produced in laboratories that have not been controlled and authorized. The methods and chemical supplies used in these laboratories might be impure, thus contaminating the samples, which could also lead to unpredictable side effects (Mazzeo and Volpe, 2016).

The main risks of using EPO in doping is a higher chance of having a stroke, heart attack or thrombosis (Oravițan, 2019). The increased level of red blood cells in healthy humans makes their blood thicker, therefore, it would be harder for the body to pump blood successfully to all the tissues around the body, causing blockage of the microcirculation. If an extra EPO gene is introduced into healthy humans then the level and duration of EPO production is less controllable and this could lead to pathological EPO levels (Wells, 2008; Santamaria et al., 2013).

When using GH there are risks of insulin resistance, impaired glucose tolerance and limited efficiency of the cardiovascular and respiratory systems. In addition, the

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11 overexpression of GH is often associated with intracranial hypertension, headache, peripheral edema, carpal tunnel syndrome, joint and muscle pain, or cardiomegaly in trained persons (Harridge and Velloso, 2009; Brzeziańska, Domańska and Jegier, 2014b).

The significant health risks of IGF-overexpression in the case of IGF gene doping are cancer and cardiac hypertrophy (Harridge and Velloso, 2009; Oliveira et al., 2011). Besides, if overexpressed it would not be possible to control the increase of muscle mass, which could be more than an athlete expected. Huge amounts of IGF in circulation cause uncontrollable formation of connective tissue in heart, liver, lungs, potentially leading to a heart failure, or sleep apnoea (Fischetto and Bermon, 2013).

3. Detection

What makes gene doping so attractive is the difficulty of detecting the use of it. It is believed to be so complex because the introduced gene and the protein it expresses would be almost identical to their endogenous counterparts. As a result, it is difficult to identify a potential detection target for testing (Baoutina et al., 2010). So far there are no approved tests for detecting gene doping in athletes, however, the research done so far has been significant and is showing promising results.

There are two main groups of detection methods: direct and indirect. Direct methods target illegal substance or the genetic material or virus that delivered it. Indirect, differently, use the effect, immune response, differences in expression or metabolic changes for detection (McKanna and Toriello, 2010; Van Der Gronde et al., 2013b). The Indirect method could be, for example, ELISA test or Western blot detecting T-cells and antibodies against viral particles, or proteomic profiling using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF) (Neuberger et al., 2012; Brzeziańska, Domańska and Jegier, 2014b). The most reliable test would be to take a muscle biopsy, as skeletal muscle is a very probable injection site for delivery of the performance-enhancing genes, but it is an invasive method and probably not every athlete is expected to give consent to this technique (Perez et al., 2013). Hence, the detection of gene doping should take place at the DNA level. Blood is often the most suitable testing fluid. It is collected using a minimally invasive procedure and unlike other bodily fluids, it is likely to contain

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12 transgenic DNA at levels high enough for detection (Baoutina et al., 2013). When designing a detection assay it is important to have a deep understanding of the biology of gene transfer vector systems, i.e. biodistribution and clearance of vectors, and the structure of the target DNA molecule (linear, integrated, circular). This way the most sensitive assay will be established that is compatible with the target molecule (Perez et al., 2013).

3.1. PCR

Polymerase chain reaction (PCR) methods are the most common direct methods for detecting gene doping. In gene therapy vectors are usually analyzed by a PCR assay that targets unique sequences of the vector, for example, a promoter or another regulatory sequence – vector specific assay. In gene doping this is useless because the vectors that are used are unknown to a testing laboratory. An option is to target copyDNA sequences for the introduced gene (Baoutina

et al., 2013). The idea behind this is that transgenic gene constructs (i.e. plasmid- and

virus-derived and intact constructs) will leak into the bloodstream and their copyDNA can be detected and isolated from genomic DNA through their differences in the sequence. Gene doping uses copyDNA rather than genomic DNA due to its’ smaller size. Furthermore, most human genes consist of noncoding sequences (introns) and coding sequences (exons). In gene therapy and in gene doping, the transgenes do not consist of introns because they are derived from the complementary DNA. Therefore, this method will be able to identify the existence of exon-exon junctions. Furthermore, Bogani, et al. (2011) found that with PCR it is possible to trace the transgene in various tissues and body fluids offering more non-invasive methods for sampling.

A number of different PCR based assays have been suggested. These are, nested PCR, real-time or quantitative PCR (rt-PCR), nested qPCR and digital droplet PCR (ddPCR).

3.1.1. Nested PCR and nested qPCR

Nested PCR includes two rounds of PCR. In the first round, samples are pre-amplified and then diluted. After that the PCR product undergoes a second PCR. The second PCR product is visualized using gel electrophoresis. Although this PCR assay has a high level of sensitivity, it requires two different rounds of PCR, hence, causing an increased chance for laboratory

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13 contamination. Moreover, gel-electrophoresis is a rather subjective analysis and not automated. However, it does not enable the quantification of transgene molecules (Beiter et al., 2008; Perez

et al., 2013). Interestingly, there is another nested PCR method that uses qPCR in the

second-round testing. In this assay 5 replicates are amplified for 25 cycles, pooled and diluted and in the second round processed for 40 cycles in qPCR (Figure 1). As a result, quantification of the samples has made possible by creating a standard curve in the qPCR amplification round (Neuberger et al., 2016).

Figure 1. The setup of the nested qPCR (Neuberger et al., 2016).

3.1.2. Quantitative or real-time PCR (RT-PCR)

RT-PCR includes a fluorescent reporter molecule that binds to the DNA in the amplification reaction. These probes increase specificity and sensitivity. In 2010 Baoutina, et al. successfully introduced a one-step RT-PCR that targeted the exon/exon junctions in cDNA. The advantages of this method are that it did not use electrophoresis detection nor multiple amplification steps like previous PCR methods. (Baoutina, et al., 2010). In 2011 Ni et al. conducted an animal study with non-human primates using the same method and showed a high potential of the test to detect viral vector sequences in blood in the long term after IM injection (Ni et al., 2011). Few years later Baoutina et al. improved their RT-PCR method’s sensitivity by adding plasmid linearization to the PCR protocol (Baoutina, et al., 2013).

Dilution of the sample

qPCR round:

3 replicates, 40 cycles

Pre-amplification round:

5 replicates, 25 cycles

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3.1.3. Digital droplet PCR (ddPCR)

This assay is based on the partition of a PCR reaction into thousands of individual droplets prior to amplification. For each separation, the reaction endpoint is measured. Furthermore, this method can work with huge amounts of background DNA and still distinguish the transgenic DNA. Compared to traditional qPCR, quantification by ddPCR is absolute without the need for standard curves, thus, it is more tolerant to inhibitors and variation and it gives more precise and reproducible results. (Moser, et al., 2014).

3.1.4. The issues

The problem with PCR methods is that even the slightest change of the gene sequence can easily interrupt the primers, consequently, modifications of the doping gene with DNA sequence mutations can be used to evade detection and to give back false-negative results (de Boer et al., 2019). Moreover, alterations can also be done by introducing regulatory and signaling sequences. This might mean that only one exon/exon junction remains intact, making it more complicated for the PCR methods to detect the transgenes (Baoutina et al., 2013). For gene doping detection PCR methods need at least one standard wild-type cDNA junction (Baoutina et al., 2013; Neuberger et al., 2016).

3.2. Loop-mediated isothermal amplification (LAMP)

LAMP is a highly specific amplification method that uses four different primers aiming at six distinct gene regions on the target gene. The process is performed at a constant temperature using a strand displacement reaction. There is no need for heat denaturation of the double-stranded DNA into single-stranded. Thus, the reaction can be carried out using a simple water bath. Moreover, the results can be viewed directly with the naked eye, eliminating the need for gel electrophoresis, and testing can be done outside the laboratory, for example on the site of the sports competition (Notomi et al., 2015; Salamin et al., 2017).

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15 3.3. Next-generation sequencing (NGS) of the copyDNA

This is one of the latest assays that has been developed for gene doping detection. In this method, De Boer, et al. (2019) isolated from the human blood samples some gDNA with plasmids containing cDNA of potential doping genes. The isolated DNA was then fragmented, and cDNA fragments were hybridized to biotin-labelled lockdown probes that target all exon-exon junctions of all known gene doping transcripts. Captured fragments were then magnetically pulled down with streptavidin beads, PCR-amplified and sequenced. This detection panel is DNA specific, eliminating RNA molecules right at the beginning, as they also contain exon-exon junctions that might interrupt the detection. NGS allows the detection of multiple gene doping genes in one sample. De Boer, et.al (2019) detected the presence of

EPO, IGF1, IGF2, GH1 and GH2 with this method, nonetheless, the number of target genes can

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Table 2. Advantages and disadvantages of the detection methods.

Method Advantages Disadvantages

High sensitivity Laborious workflow

Nested PCR Increased specificity Subjective visualization using gel electrophoresis

Reliable quantification not possible Uses 5 replicates of a single test to

minimize variability More laborious compared with qPCR

Nested qPCR Quantification included Uses chemicals

High specificity More manual handling steps

Direct quantification Needs a standard curve for quantification

Probes increase specificity Possibly low signal-to-noise ratio for low copy

number detection

Quantitative real-time PCR Superior reproducibility Reduced risk of contamination Processing many samples Works with huge amount of

background DNA. Higher cost

No need for external standard curves

for quantification Some false positives

Digital droplet PCR More tolerant to inhibitors Enables absolute quantitation More tolerant to variations in amplification efficiencies

Simple Complex primer design

Fast results Necessity of wild-type cDNA junction

LAMP On-site availability Subjective interpretation of results

Results seeable with naked eye Sensitive to tampering

No expensive equipment

Less sensitive for tampering Higher cost

NGS High sensitivity Needs future adjustments

Detection of multiple genes at the same time

4. Discussion

The rapid advances in genetic engineering in today’s world have alerted the world of the likelihood of the abuse of even more effective and less detectable doping practice - gene doping. Since 2004 WADA has taken action against gene doping, to ban, detect and prevent it (WADA, 2020). So far there is no evidence that gene doping is being used to improve athletic achievements (Neuberger and Simon, 2017; de Boer et al., 2019). Before the 2016 Olympic Games in Rio de

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17 Janeiro, a novel method to detect gene doping was considered for testing during the games, unfortunately, it was not implemented due to lack of time to ensure proper analyst training and method validation (WADA, 2016). One of the main advantages of gene doping is the low detection rates. Even though so far there has not been any reported cases of its detection and use.

There are several target genes in gene doping (Table 1). However, not all of them have been studied thoroughly in humans and can be considered as relevant candidates. The best-studied and most likely to be used for gene doping are EPO, vascular endothelial growth factor (VEGF), peroxisome proliferator-activated receptor δ (PPARδ), cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) (Van Der Gronde et al., 2013b). Myostatin inhibition and fibroblast growth factor (FGF) show also higher likelihood to be used, yet, the latter is most effective in combination with other proteins and not solely on its own (Yun et al., 2010) and myostatin inhibition is still poorly controlled, thus, more experience will be required (Van Der Gronde et al., 2013b). Most other candidate genes (Table 1) have not had any gene therapy studies done on humans or in vivo. Hence, we do not know what the potential effects and health risks on humans might be. For example, cancer or cardiac hypertrophy are possible risks of the overexpression of

IGF (Harridge and Velloso, 2009; Van Der Gronde et al., 2013a). These are severe health risks to

consider. On the other hand, van der Gronde, et al. (2013) and Neuberger and Simon (2017) give examples that show how the need for victory makes athletes more prone to take risks and put their health in danger.

Currently one of the strongest arguments against using gene transfer in athletes for performance enhancement is the fact that current gene therapies are designed to treat serious diseases and not healthy humans (Neuberger and Simon, 2017; Dunbar et al., 2018). In addition, there are still serious side effects that go with this procedure and instead of enhancement, these could diminish athlete’s performance. For example, the use of viral vectors is brought out as one of the biggest health issues. There is still insufficient information in this field. It is unclear which vector doses should be used and in how many injection repetitions are needed for an effect, how an athlete’s immune system will react to the vector administration and its components. As mentioned above, in gene therapy the viral vectors are modified to not to be pathogenic. The production of gene doping viral constructs, on the other hand, is not carried out in a regular pharmaceutically accredited laboratory, thus, impurities might be present and cause contamination, which in turn could increase genotoxicity and immune response (Dunbar et al., 2018). Furthermore, by reading different research articles it becomes clear that we still do not

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18 know exactly how to control the transgene after its introduction to the body. Overexpression of a gene could cause serious health issues as mentioned above. We cannot deny the fact that every human is different and the interindividual differences between us humans prove that there cannot be a single and certain outcome for everyone when it comes to genetic modifications.

As mentioned before, gene doping is so appealing for athletes because so far there is no standard test or method approved by WADA or any of the doping laboratories to detect gene doping (Sugasawa et al., 2019). One of the most researched methods is PCR. The results of the various studies show high sensitivity and reliable accuracy for several PCR assays (Table 2). However, some PCR assays require more manual handling of the samples and subjective visualization, which could lead to contamination and false positives. In recent years PCR assays have been developed from single transgene detection to the identification of multiple different transgenes simultaneously in one test. Nevertheless, most of the models used in these papers are based on in vitro or animal studies and not a true representative of gene doping in athletes. Therefore, the conclusions they draw should be interpreted with caution. The way the transgene and its product are accepted in the body varies between species. Even though PCR assays show high sensitivity and specificity, these could be different when analysing human samples. On the other hand, the validation of detection methods in humans would be unethical. From a positive perspective, all the papers of PCR methods studied in this literature thesis show good results in spotting the viral genomes days, weeks or even months after vector transfer. Hence, the detection window is wide.

Another method is LAMP. This method does show quite a few advantages over PCR methods, for example, it does not require so much equipment as PCR and could be transported and used more easily on the sport competition sites, however, the amplification process is a lot more complicated and the visual detection would demand some expert skills because it is rather subjective (Table 2).

Lastly, the NGS method was described. This assay gives a higher copy number of detected cDNA in gDNA compared to PCR methods (de Boer et al., 2019). Moreover, it is less sensitive to the manipulations of cDNA than LAMP or PCR, which both depend on at least one cDNA junction with a fixed sequence for detection. In the future, it seems to be an idea to unite NGS with PCR. While NGS can give us a cDNA sequence, we could compare this sequence to databases and identify any tampering in the sequence. PCR methods could then be designed according to the sequence for confirmation tests of positive gene doping.

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5. Conclusion

This literature thesis investigated the possibilities of gene doping and its detection. Cases of gene doping have not yet been reported, however, with the significant advances in genetic engineering and low testing and detection rates, the possibility remains, and the anti-doping community has to stay alert. The research and development of potential detection methods have given promising results. There are several different methods tested (PCR, LAMP, NGS) that have succeeded in identifying transgenes. On the other hand, no single standard method has been accredited by WADA. This research paper demonstrates that current methods do have good results but do not completely fulfil all preconditions. An ideal gene doping detection method should be able to process a large number of samples and multiple genes simultaneously with high sensitivity and reliable accuracy. Furthermore, the method should be reproducible, validated, cost-effective and automated, so it would be easy to transport to the competition sites. Hence, the research still needs to go on and current methods need some modifications to comply with all the requirements.

Even though athletes usually crave for greatness and becoming a champion, it does not mean that they are all ready to risk their lives for a title. In an ideal world, there would not exist any doping in sports, nevertheless, it is still an existing and regularly appearing problem. Up to now, if the illegal enhancement of athletic performance is desired, it would rather happen by using the standard doping agents instead of gene modifications. It seems unreasonable for an athlete to pick the one method that could cause severe health issues or death instead of some minor side effects that would not affect the training and competing of an athlete. On the other hand, I would assume that the potentially severe health risks that accompany doping, especially gene doping, would be enough warning to not to use any of these methods. Therefore, I think because there is still so much uncertainty and danger for healthy humans, not many people would dare to use gene doping.

All in all, research in gene doping is ongoing and published papers indicate that scientists are still working on developing new detection methods and improving existing ones. Whether it is the lack of validation that prevents the use of these methods or the assumption that gene doping is still hypothetical, occasional gene doping testing would be advised. I agree with Mattsson, et al. (2016) that altering only few genes in our bodies will not secure us a notable transformation in our athletic skills, strength and endurance because sport performance is a multi-dimensional

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20 phenotypic trait. However, I believe that with the continuous decrease in the cost of the human genome sequencing, more new potential genes linked to athletic performance will be discovered and probably the number will continue to grow every year. Therefore, I think the current methods could already be implemented into WADA’s testing protocols at various sports competitions.

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6. References

Baoutina, A. et al. (2010) ‘Gene doping detection: Evaluation of approach for direct detection of gene transfer using erythropoietin as a model system’, Gene Therapy, 17, pp.1022-1032. doi: 10.1038/gt.2010.49.

Baoutina, A. et al. (2013) ‘Improved detection of transgene and nonviral vectors in blood’, Human Gene

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APPENDIX

Search strategy

The search for research papers that contain data about gene doping was done via accessing the PubMed database and Science Direct database through the search topic “gene doping” for all the documents published in the last ten years. Due to the low number of newer articles I expanded the timeframe and added in some articles since 2006. I also accessed the official webpage of World Anti-Doping Agency (WADA) for relevant information. To be included in my research I applied some criteria for the papers. For example, the papers had to be written in English, they had to contain the phrase “gene doping” and they had to be full-text accessible.

Gene Doping in Sports