Abstract
Introduction
As we approach the London 2012 Olympics, sport finds itself facing a highly specific threat to its ethos of fair play. Defined by the World Anti-Doping Agency (WADA) as ‘the non-therapeutic use of genes, genetic elements and/or cells that have the capacity to enhance athletic performance’, 1 gene doping presents a range of challenges to those involved in sport and exercise medicine and an urgent need for an understanding of its potential impact to healthcare. 2,3
Gene therapy vs. gene doping
Gene doping has a molecular basis similar to gene therapy, whereby selected genes are intentionally introduced into targeted human somatic cells 4 to facilitate the production of the ‘normal’ physiological variant of a protein. 5 Therapy is therefore not intended to be curative nor prevent transmission of the genetic mutation to any future children (since any modification to the human germline is universally illegal). 4
Although several variations in the technology exist, it is essentially dependent upon a therapeutic gene coupled with a regulatory element to ensure its appropriate expression; and a ‘vector’ (viral or synthetic) to enable its delivery to cells. 5
Early researchers came to see two basic principles emerging that could be applied when considering the probable efficacy of gene therapy in various conditions; with the highest probable efficacy occurring if:
The disease process is solely due to a lack of normal protein production by its associated target cells; The associated target somatic cells are readily accessible.
Furthermore, in order for a suitable therapeutic gene for a condition to be developed, the genetic basis of the disorder must be well understood in order to not only ensure the desired therapeutic outcome but also that the therapy itself does not cause unforeseen harm to the recipient.
In light of this, the majority of early research focused on single gene disorders such as cystic fibrosis (CF), as well as disease states in which affected cells can be readily identified as being ‘non-self’, for example, cancer cells and those infected with human immune deficiency virus (HIV). 4,6,7
Other initial proposed areas for the technology's application included to develop transplantable tissues for the treatment of trauma, degenerative conditions and even organ failure; as well as to enable the controlled delivery of therapeutic proteins such as haemopoietic growth factors and monoclonal antibodies. 4
However, despite such early optimism, by 2011 only a handful of conditions had been successfully treated by gene therapy (Table 1). 8–11
Successful and promising gene therapy clinical trials
Moreover, profound concerns have been voiced surrounding the safety of gene therapy following reports of severe adverse effects having occurred in trial subjects; including the development of leukaemias in a few children treated for SCID-X1 deficiency, 9,12,13 and the unexplained death of a participant in a gene therapy trial involving rheumatoid arthritis patients. 14
However, research within the field continues to evolve rapidly 15 with promising results being reported in clinical trials in conditions as wide-ranging as Parkinson's disease 16 and Duchenne muscular dystrophy (DMD) (Table 1). 11,17 Accordingly, it is widely held that with advancing methods, the use of gene therapy will become safer, more efficient and, as such, increasingly widespread. 11,18,19
Gene doping was first defined by the WADA in 2003 and coincided with the technology's inclusion on the agency's ‘Prohibited List’ of substances/methods. 20,21
Doping targets
Approximately 200 ‘fitness genes’ are now known. 22 Certain target genes have begun to emerge as ‘front runners’ in the development of doping agents, particularly within fields in which a competitive advantage has been demonstrated through the use of synthetic substances and the physiological basis of this advantage is well understood.
Red blood cell activity/delivery
Since its introduction as a therapeutic agent in the 1980s, synthetic erythropoietin (EPO) has been a drug of relevance across a number of sports, particularly those, such as cycling, in which the aerobic threshold of an athlete is known to be a major source of performance limitation. However, its inclusion on the WADA list of prohibited substances means that its use in competitive sports is strictly forbidden. 21
It is hardly surprising therefore, that as reports began to emerge of impressive results from early EPO-gene therapy trials (such as that by Svensson et al. in 1997 23 ), concern was soon raised with regards to the potential for the abuse of such products in professional sport.
In 1997, Svensson et al. reported the results of their trial in which the administration of a genetic-based EPO agent to mice and primate subjects with EPO-responsive anaemias caused a substantial increase in the subjects' haematocrit levels from 49% to 81% and 40% to 70%, respectively. Moreover, the rise was then sustained for over one year in the mice and 12 weeks in the primates; and, upon assessment of its safety profile (among the primate subjects), no adverse effects were found to have occurred in any of the monkeys to whom it was administered. 23
One of the most significant recent advances in genetic EPO agents has been the development of ‘Repoxygen’ by Oxford Biomedica in 2002. Comprised of a viral-vector with a human EPO gene, intra-muscular (IM) injection of the product has been trialled in the treatment of anaemia associated with renal diseases and chemotherapy. In addition, the EPO gene is under the control of a hypoxia response element and is, therefore, self-regulated, only being induced (when required) under hypoxic conditions. 24
However, while promising results from pre-clinical trials on mice subjects were widely publicized, Oxford Biomedica ultimately chose not to develop the product any further, as it was thought that it would not be financially viable in a market where exogenous EPO agents are already widely available. 25
As such, it remains unclear as to the present state of development/availability of the agent. 24,25
When interviewed by The Times newspaper, the President of Oxford Biomedica (Professor Alan Kingsman) stressed the corporation's tight security controls surrounding all of its products, adding that he would be ‘extremely surprised if anything we made got on to the black market’. 25 In addition, Professor Kingsman issued the stark reminder that Repoxygen had only been ever been trialled on mice subjects; and that should any person, therefore, attempt to use it in humans, they really would be ‘playing with fire’. 25
Of associated interest has been the potential for the use of vascular endothelial growth factor (VEGF) and/or other angiogenic factors in order to increase tissue blood supply, thus improving tissue oxygenation and nutrition as well as the efficient removal of local waste products. 11
However, while there have been extensive clinical trials involving the use of angiogenic factors, a review by Kontox and Annex in 2007 concluded that success within the field had been largely limited; likely due to the fact that physiological angiogenesis is a highly complex process involving a delicate interplay between numerous angiogenic factors. 26
The authors did, however, raise the possibility that the use of transcription factors such as hypoxia-inducible factor 1α (HIF1 α) may facilitate therapeutic progress. 26 Known to activate the production of angiogenic factors and, crucially, itself be activated by muscle hypoxia and endurance training, HIF1 α has been shown to increase the levels of VEGF and EPO mRNA after exercise. 27
As such, a gene doping analogue could potentially offer its would-be users a ‘2 for 1’ means of increasing oxygen delivery to tissues; with effects likely to be further amplified with concomitant ‘Repoxygen’ administration.
Skeletal muscle size, strength and endurance
Anabolic factors known to improve muscle mass such as human growth hormone (HGH) and insulin-like growth factor-1 (IGF-1) have already been cloned. 18
IGF-1 is known to be important in the regulation of skeletal muscle mass through stimulating hypertrophy and enabling repair after injury. 28 In a study by Musaro and colleagues, mice that had been injected as embryos with a transgene encoding an isofrom of IGF-1 demonstrated a 15% increase in muscle bulk even in the absence of any exercise programme. 29
Moreover, a separate study by Lee et al. involving the injection of an IGF-1 isoform (via an adenovirus vector) to rat subjects, 30 has indicated that such effects may be further amplified when combined with subsequent resistance training. In the study, while all injected rats demonstrated an increase in muscle mass, the effect was considerably amplified when combined with resistance training; with a 31.8% increase in muscle mass noted among exercised rats vs. the 14.8% increase in mass in non-exercised subjects. 30
It is also important to highlight that, in contrast to the aforementioned EPO study involving anaemic mice, subjects in both of the IGF-1 trials were essentially normal/healthy animals. As such, it seems hard to ignore the potential connotations of such trials in relation to the possible use of IGF-1 isoforms as performance-enhancing agents.
Perhaps of more concern is the potential development of mechano-growth factor (MGF) gene therapy. MGF is an isoform of IGF-1 that is released locally from skeletal muscle in response to mechanical factors, such as exercise, rendering it virtually undetectable in blood or urine samples. 2 Originally studied with regards to its possible therapeutic application in conditions relating to skeletal muscle atrophy such as Duchenne muscular dystrophy, it has been suggested that MGF may be particularly beneficial as a doping agent in light of its involvement in skeletal muscle repair following injury. 2
Furthermore, it has been predicted that even greater improvements in performance are likely should IGF-1 and MGF be used concomitantly, 18 and that any adverse effects are likely to be minimal due to them being localized to the muscle of administration. 2 As Harridge et al. highlight, athletes may only need to inject relatively small quantities of IGF-1 in order to gain the 1% increase in power output necessary for them to clinch that gold medal. 28
In conjunction with rising interest in IGF-1 transfer techniques, another target that has received tremendous attention in recent years with regards to its doping potential is the regulatory protein myostatin. Known to act as a source of negative feedback for muscle growth, myostatin first gained mass attention in 2004 when Schuelke et al. reported the case of a myostatin-deficient child. 2,31 The boy, whose mother was a professional athlete, was noted at birth to have hypertrophic limb muscle development, and at 4.5 years was already able to hold two 3-kg dumbbells in horizontal suspension with extended arms. 31 The report instantly prompted a wealth of speculation with regards to the possibility of developing a gene-based myostatin inhibitor and, in particular, the probability of such an agent's abuse in professional sport. 15
Cong et al. recently demonstrated that an adenovirus-mediated shRNA expression system could be used to down regulate myostatin expression via inhibition of the enzyme ‘muscle atrophy F-box’ (MAFbx); resulting in an increase in the muscle mass of injected mice subjects. 32
In common with the aforementioned IGF-1/MGF research, the authors highlight that such a therapeutic system could have particular benefits for conditions relating to muscular atrophy. 32 However, given fears of the potential abuse of gene based IGF-1/MGF therapies in sport, it would seem sensible to suggest that such concerns would also be pertinent to the development of Cong et al.'s MAFbx inhibitor (as well as any other gene therapy systems that enable the downregulation of mysotatin expression).
Detecting doping
While a definitive test for genetic doping is not presently available, the efficacy of potential methods is a subject of as much debate as the use of the technology itself.
Detecting pharmacological/non-genetic doping
The mainstay of doping detection has thus far relied upon random, periodic sampling of athlete's blood and urine by regulatory bodies. 3
In 2009 WADA reported that it had undertaken nearly 1900 ‘Out of Competition’ doping tests; including blood sampling for agents such as HGH and haemoglobin-based oxygen carriers (HBOCs), and urinary testing for EPO use. 33
While the sample collected (blood or urine) will then determine which agents may be tested for, the process itself is structured around the directly witnessed collection of the sample by a WADA-accredited supervisor. 34
In the case of urine collection, the sample is then divided and sealed in the presence of the athlete in to two collection vessels (A and B), before being directly transported for testing in one of WADA's 35 accredited laboratories. If sample A is found to be positive for a banned substance(s), sample B is then also tested. Should this second sample also be found to be positive, doping for the substance(s) is considered confirmed; and the case is referred to an appropriate regulatory body. 34
Furthermore, WADA may subsequently perform more specific analysis on collected samples depending on agents considered to be of high suspicion at that time and/or of particular relevance to an athlete's sport. For example, in 2006 it was found that 50% of urine samples collected tested positive for anabolic-androgenic steroids (AASs), three times the amount testing positive for the next most commonly detected agent. In response to this, WADA now undertakes mass spectrometry testing of all samples in order to enable the detection of even trace amounts of natural or synthetic steroids. 35
Detecting gene doping
Some have suggested that adaptation of the aforementioned detection methods will also be suitable for the detection of genetic doping, for example, via the detection of changes in gene expression profiles that may occur secondary to the bypassing of metabolic pathways. 18
However, others have highlighted several difficulties when attempting to apply pre-existing detection systems to genetic doping.
First, for a transgenic protein (a protein produced from a transferred gene) to be detected, it must be distinguishable from its physiological counterpart. As we have already seen, one of the fundamental aims of gene therapy is to enhance ‘normal’ physiological production of various proteins, thus meaning that any detection on this basis would be very challenging. Substances such as erythropoietin are known to have very short half-lives, therefore this would thus further reduce the probability of their detection via a random blood or urine sample. 36
The only present method that may enable site specific detection would be that of a muscle biopsy to test for injected viral particles. However, while deemed safe, any biopsy method is likely to be highly unpopular with professional athletes due to its invasive nature. As has been highlighted, 2 even if such sampling were to be enforced, engineered viral products may be indistinguishable from their endogenous counterparts and it is unclear how long such particles would persist following their injection.
Recent advances have suggested that a fine needle aspiration biopsy in combination with real-time polymerase chain reaction (PCR) techniques may offer a solution to this barrier, 11,37 and is one of the areas to have received both encouragement and funding from WADA. 33
Notably, Beiter et al. recently demonstrated that a direct PCR-based strategy could be utilized to detect the presence or absence of transgenic DNA from peripheral blood samples. 38 Utilizing a nested PCR assay, the authors were able to detect the presence of transgenic DNA (VEGF-A cDNA) in blood samples taken from all six of the mice subjects to whom it was administerd via intramuscular adeno-associated virus (AAV)-mediated gene transfer. Furthermore, the VEGF-A transgene remained detectable at 4 weeks in all six subjects; and, remarkably, was still detected in 4/6 of the mice at day 56 post administration. 38
This method would avoid the difficulties of ascertaining possible gene doping via muscle biopsy; and it only required minute amounts of blood (20 µl). While the authors highlight that transgenic efficiency is considerably greater in mice, they remain confident that the technique can be successfully used to detect gene doping in humans and have thus far developed target specific identification PCRs for several other key doping targets including EPO and IGF-1. 38
A final means of detecting gene doping may come from the immunological detection of antibodies produced in response to injected viral particles. If this were to be initiated as a detection method, repeated testing of athletes' immune responses to various viral agents would be necessary. 39 Moreover, since viral gene delivery requires the injection of a bolus dose of relatively high concentration (several million particles) of recombinant virus, it has been predicted that some very aggressive immune responses would be witnessed; and that these would be accompanied by significantly higher titres of antibodies than those witnessed as a result of typical viral infections. 40
Looking to the future, application of this detection method could be limited by the development of increasingly efficient vector systems that will not induce such immunological reactions. 36
Finally, it has been suggested that the detection of gene doping may be possible through the barcode-like labelling of agents. While proven to be successful in agricultural industries and stated to be a prerequisite for the development of any new therapeutic agent designed to induce genetic alteration, 2,11 it is questionable whether a commitment to such labelling would withstand the considerable financial gains likely to be rewarded to those willing to make a gene doping product available. 2
Conclusion
We live in a world where the performance of elite athletes has to be proven as the genuine article.
Campaigns such as ‘100% me’ by the United Kingdom Anti-Doping Authority (UKAD) 41 highlight the extent of the concern presently surrounding the issue of doping in sport. Backed by athletic ‘ambassadors’ such as Sir Chris Hoy, ‘100% me’ seeks to emphasize to athletes that ‘It's What's Inside That Counts’ through encouraging a commitment to the principle of fairness in sport and by stressing the values and characteristics which make a true champion. 41
However, as this article highlights, for some the allure of gold can make doping the forbidden fruit worth risking. Since gene doping may present impressionable athletes with the ultimate temptation of long-term performance enhancement with a low risk of ‘testing positive’ through present doping detection methods, it would appear that campaigns such as ‘100% me’ face a considerable challenge in order to ensure that the torch of fair play continues to shine over London 2012.
DECLARATIONS
Competing interests
None declared
Funding
None
Ethical approval
Not applicable
Guarantor
DG
Contributorship
DG initiated the overall concept and provided advice on the key areas of coverage; LB wrote the wrote; AS provided input from an endocrinologist's clinical perspective
Acknowledgements
None