Genetic engineering in sport

Imagine this: at the Olympic games in London in 2012, a little known athlete surprises everyone in the men’s 100 metres, thrashing the established favourite by almost a whole second. “Is he on drugs?” everyone asks. Tests find nothing, and he is labelled a freak of nature. But are his achievements thanks to natural ability or are they down to genetic modification?

In our darkest memories, we can all recall having lied, cheated, or stretched the rules, often to gain a personal advantage. For elite athletes the pressures that drive cheating are vastly increased. Sport requires that participants train hard over long periods, often from a young age, linking achievement to self esteem, emotional state, and lifestyle. The temptations of money and fame and pressure from parents, coaches, and fans drive athletes to accept extreme risk to gain even small advantages.

Perhaps it is unsurprising that some athletes turn to science and drugs to improve their chances. In 1967 the British cyclist Tom Simpson collapsed and died on the slopes of Mont Ventoux, in France, largely because of misuse of amphetamines. As tests were developed a number of high profile cases brought doping into the public eye: Ben Johnson was stripped of his gold medal in the 1988 Olympic games, and the whole Festina team was ejected from the Tour de France in 1998 after doping equipment was found in the car of a team official.

An independent international body was established to fight doping on a global scale—the World Anti-Doping Agency. It updates the world antidoping code, which contains a list of prohibited substances and practices; coordinates the testing and education of athletes and their coaches; and funds research into testing to remain one step ahead of potential cheats.

A new threat

In 2002 the agency recognised a new threat—“gene doping”—by including it in the list of prohibited practices, defined as “the non-therapeutic use of cells, genes, genetic elements, or of the modulation of gene expression, having the capacity to enhance athletic performance.”^w1

The techniques involved in gene doping are spawned from the techniques of gene therapy, in particular gene transfer with viral vectors (fig 1). The aim of gene manipulation in these two settings can be clearly distinguished. In gene therapy the goal is treatment and requires the replacement of a defective gene or expression of a therapeutic gene. Gene doping, however, aims to improve athletic performance by increasing or decreasing production of endogenous molecules.

Fig 1 Principles of gene transfer by viral vector

Does gene therapy work?

Cystic fibrosis

Recently, good progress has been made in achieving clinically relevant results from gene therapy. Patients with cystic fibrosis have shown improved lung function, few side effects, and good tolerance in phase I trials of transfer of a functional cystic fibrosis transmembrane conductance regulator gene, mediated by an adeno associated virus (AAV).^w2

Most recently, using a fluorescently marked AAV as vector delivered by nasal spray, a team from Johns Hopkins University has shown good levels of vector specific expression of a functional cystic fibrosis transmembrane conductance regulator gene in the lungs of rhesus monkeys.^w3 This is promising because expression levels have previously been disappointingly low in vivo, limiting the possible effectiveness of the treatment.

Factor IX deficiency

Factor IX deficiency (haemophilia B) is a rare but serious clotting disorder that is currently treated by regular injections of recombinant factor IX. Phase I trials of gene therapy that targeted the liver, where factor IX is usually produced, have had some success.

In monkeys, concentrations as great as 121% of normal have been achieved using a liver restricted, double stranded AAV as vector.^w4 Using a delivery system to the hepatic portal vein, a US group achieved concentrations of 110% of normal for one month in one patient.^w5 Unfortunately, this concentration fell because of an immune response against the AAV’s capsid protein, but the trial is being modified to suppress immune response at the time of gene transfer.^w6

X linked severe combined immunodeficiency

Possibly the most promising result for gene therapy has been in X linked severe combined immune deficiency, a disorder caused by a deficiency in γc cytokine receptors that prevents T lymphocyte and natural killer cell differentiation at an early stage.

The ex vivo transduction of bone marrow haematopoietic stem cells with a retroviral vector that encoded a functional γc gene and their transplantation back into the patients produced remarkable results in 10 young children.^w7 Nine of the 10 patients had good integration and expression of the gene, and substantial clinical improvement meant the patients could lead a near normal life for some time.

Melanoma

Gene therapy is not only useful in the correction of single gene disorders but also can be used to treat malignancy. Morgan et al have engineered tumour reactive lymphocytes by retroviral transduction of a T cell receptor gene specific to tumour associated antigen.^w8 In two out of 17 patients with refractory metastatic melanoma, good circulating concentrations of these genetically engineered lymphocytes were sustained and caused a regression in tumour size such that both patients were clinically disease free at 20 months after treatment. Although this response rate is low, remember that the sample included only patients with disease refractory to all other treatment and that the gene therapy could be further optimised.

Expression of transgenes is crucial

These recent successes in gene therapy are dependent upon the long term expression of transgenes either through integration of the vector into the genome (retrovirus) or through the episomal persistence of the vector. However, gene therapy remains in the early stages of development because of the known risks and the relatively unknown long term effects. This is not much of a hindrance to gene doping because sport lacks the regulation and ethical framework of medicine.^w9

Candidates for gene doping

The recent successes for gene therapy indicate that transfer of a functional gene is possible and biologically active protein concentrations are achieved. Most likely it is through manipulation of the most successful gene therapy techniques that athletes will achieve gene doping. In some cases preclinical research has direct application to improving sporting performance.

Generating red blood cells

Erythropoietin is a small glycoprotein that acts to increase the number of red blood cells (haematocrit) in response to a lack of oxygen. Recombinant erythropoietin has been widely abused in sport to improve aerobic performance. Erythropoietin has a short half life and requires frequent injection thereby lending itself to continuous production by gene delivery.

As early as 1998 Zhou et al successfully inserted the erythropoietin gene into mice and baboons by intramuscular injection of an AAV vector.^w10 Albeit in very few animals, they showed high levels of expression of erythropoietin and a significant increase in haematocrit (from 49% to 81% in mice and from 40% to 70% in baboons) that lasted for more than 12 weeks. This mirrors results from other laboratories that show lasting expression and raised haematocrit.^{w11 w12} These researchers expressed a caveat to their work: the risk of thrombosis significantly increases with raised haematocrit, requiring prophylactic phlebotomy.

Muscle growth

Insulin-like growth factor (IGF-1) is the downstream effector of growth hormone and induces muscle growth (hypertrophy) when locally infused.^w13 This finding and others informed trials that led to recombinant IGF-1 being used to treat muscle wasting diseases, such as muscular dystrophy, and to help patients recover from injury. Goldspink and his team inserted the mechanogrowth factor (a splice variant of IGF-1) gene into muscle tissue in vitro and reported a 25% increase in mean muscle cross sectional area after only three weeks.^w14

A US team injected an AAV vector that encoded the IGF-1 gene into one leg of a mouse, which caused hypertrophy. After a period of resistance training, the injected leg showed greater hypertrophy and muscle power compared with the other.^w15 Also, the “Schwarzenegger mice” showed less loss of muscle mass and power after a 12 week “detraining” period. In other words, they show that by increasing expression of IGF-1 mice gain a training advantage over those that were not “doped.”

Unlimited targets?

Other targets for doping include genes involved in muscle formation and regulation (peroxisome proliferator activated receptor delta, peroxisome proliferator activated receptor gamma coactivator 1, myostatin), response to hypoxia (hypoxia inducible factors), blood vessel formation (vascular endothelial growth factor), lung function, pain control (endorphins), and immune response (interleukin 1). The list of candidate genes is only likely to grow, especially after studies have linked particular alleles of α-actinin and angiotensin converting enzyme with aerobic performance,^{w16 w17} and a population study has identified several exercise related genes in the human genome.^w18

Not all good news

The X linked severe combined immune deficiency trial has since been overshadowed by the development of leukaemia in the two youngest participants.^w19 Genetic analysis showed that the engineered gene was inserted close to a proto-oncogene, LMO2, resulting in overexpression and malignant transformation—insertional mutagenesis. Most recently, another participant developed leukaemia, and one of the first patients died from complications.^w20

Gene transfer has enormous potential for abuse by athletes, as reflected in several animal trials and some human trials of gene therapy, as discussed above. For this reason alone, the World Anti-Doping Agency considers it a risk to fair competition. Another consideration is the potential health risks to athletes^w21: insertional mutagenesis, serious immune response, toxicity, and physiological effects of overexpression—for example, thrombogenesis with erythropoietin and tendon overload with IGF-1.

Is gene doping detectable?

The World Anti-Doping Agency is particularly concerned about gene doping in the immediate future because it is hard to detect. Strategies are being researched, but at the moment no practical tests are in use. A biopsy of the tissue where the gene had been inserted would allow the use of a genetic assay to identify insertion motifs, but taking multiple muscle biopsies is neither practical nor acceptable to athletes.

A physiological approach could be taken—for example, high concentrations of the gene product in the blood and downstream physiological parameters (such as haematocrit) would raise suspicion of gene doping. However, this approach would probably only provide a screening test. Experience with recombinant doping shows that determined athletes can avoid suspicion by keeping physiological parameters within the allowed levels through close monitoring. A more specific diagnostic test is needed.

Detection of the vector itself and the host’s immune response to infection may give a route to detection. One approach is the use of the reverse transcription polymerase chain reaction to amplify regions of integrated or extranuclear vectors. Detection of the immune response could be informative, but infection with viral vectors is common in the normal population, so serological testing may be inconclusive.

It has been suggested that the transgene product will be identical to its endogenous counterpart, but recent research shows otherwise. Using a method of isoelectric focusing adapted from the urine test for recombinant erythropoietin, the serum of macaques was analysed before and after transfer of the erythropoietin gene into skeletal muscle mediated by AAV.^w22 There was a detectable difference between endogenous erythropoietin and that produced by the transgene. This difference is thought to result from post-translational modifications specific to cell type—skeletal muscle compared with renal fibroblasts.

A similar approach may be possible to detect IGF-1: a change in the relative ratios of isoforms.^w14 This provides hope that gene doping will be detectable, at least when the target tissue is different from that where the endogenous gene product is produced.

Knowledge of the human genome gives another approach to testing for gene insertion. DNA microarrays allow analysis of the downstream genes switched on after gene transfer (fig 2).^w24 In this way, specific signatures associated with a particular gene transfer could be identified. For example, in the case of peroxisome proliferator activated receptor δ (PPARδ), downstream genomic switches involved in the conversion of muscle fibres could be traced back to the original gene transfer.

Fig 2 DNA microarray technology to identify changes in gene expression before and after gene transfer. Adpated with permission from Gibson and Muse^w23

A similar approach could be envisaged using proteomic arrays. However, the most promising use of proteomics would be serial profiling of athletes to allow detection of any changes in protein expression. Athletes could then be given a genomic or proteomic “passport,” without which they would not be allowed to compete. The logistics of any such programme may be prohibitive but with international cooperation through the World Anti-Doping Agency it may be possible.

Conclusion

Advances in gene therapy in the past few years have showed much promise, sparking concern about the possibility of creating genetically engineered Olympians. Despite this, the World Anti-Doping Agency says that “claims are overblown currently.” But others disagree: “[gene doping] is here or so close to here that it makes no difference.”^w25

Substantial improvements in athletic performance are probably not possible with current technologies,^w26 but illicit trials are a distinct possibility. Future developments need further monitoring, and the World Anti-Doping Agency is well placed to do this. International conferences that bring together researchers, ethicists, athletes, and policy makers seem a good way to shape future regulation, funding more research into testing and crucially providing education programmes for athletes, coaches, and the general public. It is unlikely that we will see the first genetically engineered Olympian in Beijing 2008, but do not be surprised if at London 2012 there is widespread testing and the first “gene doping” scandal.

Competing interests: None declared.

Provenance and peer review: Not commissioned; externally peer reviewed.

1. The World Anti-Doping Agency: The World Anti-Doping Code: The 2008 Prohibited List, International Standard. http://www.wada-ama.org/rtecontent/document/2008_List_En.pdf
2. Moss RB, Rodman D, Spencer LT, Aitken ML, Zeitlin PL, Waltz D, Milla C, Brody AS, Clancy JP, Ramsey B et al.: Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter, double-blind, placebo-controlled trial. Chest 2004, 125: 509-21.
3. Fischer AC, Smith CI, Cebotaru L, Zhang X, Askin FB, Wright J, Guggino SE, Adams RJ, Flotte T, Guggino WB: Expression of a truncated cystic fibrosis transmembrane conductance regulator with an AAV5-pseudotyped vector in primates. Mol Ther 2007, 15: 756-63.
4. Nathwani AC, Gray JT, Ng CY, Zhou J, Spence Y, Waddington SN, Tuddenham EG, Kemball-Cook G, McIntosh J, Boon-Spijker M et al.: Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 2006, 107: 2653-61.
5. Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K, Blatt P, Konkle B et al.: Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Hematol 2006, 13: 301-7.
6. Ponder KP: Gene therapy for hemophilia. Curr Opin 1. The World Anti-Doping Agency: The World Anti-Doping Code: The 2008 Prohibited List, International Standard. http://www.wada-ama.org/rtecontent/document/2008_List_En.pdf
7. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, Selz F, Hue C, Certain S, Casanova JL et al.: Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000, 288: 669-72.
8. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM, Royal RE, Topalian SL, Kammula US, Restifo NP et al.: Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006, 314: 126-9.
9. Haisma HJ, de Hon O: Gene doping. Int J Sports Med 2006, 27: 257-66.
10. Zhou S, Murphy JE, Escobedo JA, Dwarki VJ: Adeno-associated virus-mediated delivery of erythropoietin leads to sustained elevation of hematocrit in nonhuman primates. Gene Ther 1998, 5: 665-70.
11. Svensson EC, Black HB, Dugger DL, Tripathy SK, Goldwasser E, Hao Z, Chu L, Leiden JM: Long-term erythropoietin expression in rodents and non-human primates following intramuscular injection of a replication-defective adenoviral vector. Hum Gene Ther 1997, 8: 1797-806.
12. Kessler PD, Podsakoff GM, Chen X, McQuiston SA, Colosi PC, Matelis LA, Kurtzman GJ, Byrne BJ: Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein. Proc Natl Acad Sci U S A 1996, 93: 14082-7.
13. Adams GR, McCue SA: Localized infusion of IGF-I results in skeletal muscle hypertrophy in rats. J Appl Physiol 1998, 84: 1716-22.
14. Goldspink G: Research on mechano growth factor: its potential for optimising physical training as well as misuse in doping. Br J Sports Med 2005, 39: 787-8; discussion 787-8.
15. Lee S, Barton ER, Sweeney HL, Farrar RP: Viral expression of insulin-like growth factor-I enhances muscle hypertrophy in resistance-trained rats. J Appl Physiol 2004, 96: 1097-104.
16. Yang N, MacArthur DG, Gulbin JP, Hahn AG, Beggs AH, Easteal S, North K: ACTN3 genotype is associated with human elite athletic performance. Am J Hum Genet 2003, 73: 627-31.
17. Myerson S, Hemingway H, Budget R, Martin J, Humphries S, Montgomery H: Human angiotensin I-converting enzyme gene and endurance performance. J Appl Physiol 1999, 87: 1313-6.
18. DeFrancesco L: The faking of champions. Nat Biotechnol 2004, 22: 1069-71.
19. Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, Lim A, Osborne CS, Pawliuk R, Morillon E et al.: LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 2003, 302: 415-9.
20. Agence Française de Securite Sanitaire des Produits de Sante: 3rd complication case in DICS X gene therapy clinical trial. http://agmed.sante.gouv.fr/htm/10/filcoprs/050103en.htm. 2005.
21. Trent RJ, Alexander IE: Gene therapy in sport. Br J Sports Med 2006, 40: 4-5.
22. Lasne F, Martin L, de Ceaurriz J, Larcher T, Moullier P, Chenuaud P: “Genetic Doping” with erythropoietin cDNA in primate muscle is detectable. Mol Ther 2004, 10: 409-10.
23. Gibson G, Muse S, A primer of genome science. 2001, Sutherland MA: Sinauer Associates Inc.
24. Azzazy HM, Mansour MM, Christenson RH: Doping in the recombinant era: strategies and counterstrategies. Clin Biochem 2005, 38: 959-65.
25. Pincock S: Feature: Gene doping. Lancet 2005, 366 Suppl 1: S18-9.
26. Baoutina A, Alexander IE, Rasko JE, Emslie KR: Potential use of gene transfer in athletic performance enhancement. Mol Ther 2007, 15: 1751-66.

      

 Printable version    Download PDF   E-mail this to a friend   Respond to this article