Download Gene doping - Essays in Biochemistry

© The Authors Journal compilation © 2008 Biochemical Society
9
Gene doping
Stephen D.R. Harridge1 and Cristiana
P. Velloso
Division of Applied Biomedical Research, School of Biomedical and
Health Sciences, 4.14 Shepherd’s House Guy’s Campus, King’s College
London, London SE1 1UL, U.K.
Abstract
Gene doping is the misuse of gene therapy to enhance athletic performance.
It has recently been recognised as a potential threat and subsequently been
prohibited by the World Anti-Doping Agency. Despite concerns with safety
and efficacy of gene therapy, the technology is progressing steadily. Many
of the genes/proteins which are involved in determining key components of
athletic performance have been identified. Naturally occurring mutations in
humans as well as gene-transfer experiments in adult animals have shown that
altered expression of these genes does indeed affect physical performance. For
athletes, however, the gains in performance must be weighed against the health
risks associated with the gene-transfer process, whereas the detection of such
practices will provide new challenges for the anti-doping authorities.
Introduction
In the great construction that is the human body our genes could easily be
described as the architect’s plans. Encoded in the nuclei of all cells are the
genetic blueprints for the manufacture of the proteins that constitute all
the tissues and organs of the body. This blueprint takes the form of an array
of nucleotides that make up DNA. It is our ability to subtly redraw these
plans by manipulating DNA that has, in recent years, formed the basis of new
strategies for treating a number of diseases, a process known as gene therapy.
1To
whom correspondence should be addressed (e-mail [email protected]).
125
POIN004_C09_125-138_.indd
125
2/6/08
4:11:58 PM
126
Essays in Biochemistry volume 44 2008
In contrast with its use as a medicinal tool, gene ‘doping’ is where DNA is
manipulated in otherwise healthy individuals for the purposes of improving
certain physiological functions and ultimately athletic performance. The
principles underlying gene doping and gene therapy are essentially the same
and thus, before going on to address gene doping specifically, it is necessary
to outline the concepts involved in the development of gene therapy as a
legitimate therapeutic tool.
Gene therapy
As our understanding of molecular biology has increased, the possibility of
manipulating our genes to correct genetic disorders has begun to be realized.
Genetic disorders are those resulting from mutations, or errors in the sequence
of a gene. Some mutations are inconsequential to the function of a protein,
but others can result in production of a non-functional protein or one with
aberrant function. Cystic fibrosis is an example of a hereditary condition
which results from mutations in the CFTR (cystic fibrosis transmembrane
conductance regulator) gene. In other diseases, such as cancers, mutations can
accumulate during the life of the organism due to environmental mutagens
or simply errors made in duplicating DNA during the process of cell
proliferation.
The best candidate diseases for gene therapy are monogeneic diseases,
those caused by defects in a single gene. The cure is to insert a correct copy of
the gene thus restoring the ability to produce a functional protein. This strategy may not be suitable for diseases resulting from proteins with aberrant function or deregulated expression. In these cases, the strategy would be to deliver
a gene encoding a protein that can specifically inhibit gene expression, protein
expression or protein function.
The process of gene expression can be regulated at several levels: the
transcription of a gene from DNA to pre-mRNA; the processing of the premRNA to mature mRNA; the translation of mRNA to protein; and the stability of the mRNA or protein. In many cases, the amount of protein can be
regulated by inhibiting or stimulating the gene transcription to produce more
or less mRNA. In theory, this can be achieved easily by introducing extra
copies of the gene or by altering the levels of the factors that regulate the transcription of a particular gene. These are transcription factors; proteins that
bind to regulatory sequences in the DNA and interact with the enzymes of the
transcription machinery, thereby activating or inhibiting gene transcription.
Other regulatory factors are involved in the process of translation, whereas
post-translational modifications of a protein (proteolytic cleavage, glycosylation, phosphorylation) regulate its activity and stability.
Currently the major obstacle to gene therapy is inefficient delivery and
expression of the gene of interest into the target tissue. Therefore the focus of
much research is the optimization of gene-delivery methods. It is no surprise
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
126
2/6/08
4:11:59 PM
S.D.R. Harridge & C.P. Velloso
127
that the most efficient method of gene delivery to mammalian cells involves the
use of viruses, as their natural function is precisely to infect cells and utilize the
protein and DNA synthesis machinery of the host to replicate. Most viruses
used for gene therapy are modified to make them replication incompetent.
There are several categories of viral vectors that are currently being used, each
with distinct advantages and disadvantages [1].
Non-viral vectors include plasmid DNA (pure DNA containing the gene
of interest and sequences required for expression) and complexes formed by
DNA and synthetic vectors such as liposomes (lipid vesicles), cationic polymers
and cell-penetrating peptides. The disadvantage in their use is the much lower
efficiencies of transfection compared with viral vectors. A variety of methods
are being developed to improve delivery. These include application of electric
fields to the site of injection (electroporation), ultrasound, laser magnetic fields,
mechanical massage and pressurized vascular delivery where local occlusion of
blood vessels increases pressure in the vascular bed at the site of injection. The
cause of improved transfection efficiency using these techniques is unclear but
it is probably due to increased permeability of cell membranes [2].
Clinical trials are being conducted for diseases such as cystic fibrosis,
Duchenne muscular dystrophy, immunological blood disorders, cardiac
ischaemia and cancer. The results have on the whole been disappointing, as
the clinical responses expected from animal models have not been consistently
observed in humans. Gene therapy, however, has been used successfully to
treat immunodeficient children and adults by ex vivo treatment of haematopoietic stem cells [1,3], the precursors of mature lymphocytes and of other
blood cells that are found in the bone marrow. These studies also highlighted
one of the major risks of viral-based gene therapy, insertional mutagenesis.
In 2 out of 14 children suffering from X-linked severe combined immunodeficiency, insertion of the virus near a proto-oncogene locus led to development of T-cell proliferative disease, a type of leukaemia [3]. Another risk
of viral-based gene therapy is an immune response to the viral vector, and it
was a severe immune system reaction that caused the death of 19-year-old
Jesse Gelsinger in a 1999 clinical trial. When less severe, the immune response
can still limit the number of administrations and, ultimately, the efficacy of
treatment. Generation of autoimmune responses against self-antigens is also
a concern, particularly in trials that involve the use of immunostimulatory
molecules. Injection of vectors expressing T-cell stimulatory molecules into
melanoma lesions to boost response against tumour antigens resulted in development of autoimmune vitiligo in 12–14% of patients, probably as a result of a
reaction to antigens also present in normal melanocytes [4].
Recent developments suggest that it may be possible to take advantage of
a natural DNA repair process called homologous recombination to replace
sequences of DNA, and thus substitute existing genes with the desired variant [5]. This process has two advantages over viral and non-viral gene therapy:
gene expression is controlled by endogenous regulatory sequences and there
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
127
2/6/08
4:11:59 PM
128
Essays in Biochemistry volume 44 2008
is no risk of insertional mutagenesis. Homologous recombination occurs
when chromosomes align during mitosis and meiosis. Identical sequences of
DNA can crossover from one molecule to the next and the DNA segments
can be exchanged between chromosomes. Engineered gene constructs can be
introduced into the cell and recombination takes place within the homologous
sequences. The end result is that a single targeted locus has been replaced with
the engineered construct.
Despite the setbacks that have occurred in gene therapy trials (Table 1),
the fact that more than a thousand different clinical trials have taken place with
a relatively small number of reported complications suggests that risks, though
potentially severe, may be sufficiently infrequent to justify use of gene therapy
where no alternative is available. It is noteworthy, however, that there may be
an association between lack of observed complications and limited efficacy of
delivery. Safer and more efficient delivery vectors and methods are constantly
being developed, and some success has been obtained in large animals, prompting optimism in the field.
Gene doping
As with many medical advances in the past, gene therapy has not gone
unnoticed by sections of the athletic and body-building communities looking
to gain an unfair advantage. The WADA (World Anti-Doping Agency)
defines gene doping as the “non-therapeutic use of genes, genetic elements
and/or cells that have the capacity to enhance athletic performance”. In 2003
WADA amended its Prohibited List of Substances and Methods to include
gene doping. What genes might athletes be interested in manipulating? To
understand this we need to consider the key physiological mechanisms
limiting different athletic events and the key proteins involved. The demands
of marathon running differ markedly from those of sprinting, jumping,
throwing and weightlifting. The last four events require rapid development of
high muscle forces (i.e. generation of explosive power). Endurance events, on
the other hand, require the ability to sustain a sub-maximal level of power
output for a prolonged period of time. Although in theory every protein that
is known to affect physical performance and has been cloned can be a target
for genetic manipulation, this chapter will focus on a few of the key genes/
proteins that are probable candidates for gene doping, and in which there has
been considerable recent interest (Figure 1). Other reviews have recently been
published which may be of interest to the reader [6–8].
Gene doping for increased strength and power
All other things being equal, muscle force is determined by its size, specifically
its cross-sectional area. Athletes abusing drugs have traditionally used anabolic
steroids, analogues of the male hormone testosterone, to increase muscle
mass. More recently, GH (growth hormone) and IGF-1 (insulin-like growth
factor 1) have also been used. For the athlete, the use of gene doping provides
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
128
2/6/08
4:11:59 PM
POIN004_C09_125-138_.indd
129
no statistically significant
increases in urea synthetic
capacity post-administration.
human OTC cDNA. 18 subjects
randomized into six dose groups,
received a single dose of vector
response alive after 59 months).
rV-TRICOM: seven patients with
objective clinical responses (one
patient alive after 22 months).
ICAM-1 and LFA-3 (rv-TRICOM,
13 patients). Three vaccinations in
melanoma lesions at monthly
intervals. The genes above encode
T-cell costimulatory molecules [4].
rvB7.1: three patients with objective
clinical responses (patient with best
poietic stem cells.
(rv-B7.1, 12 patients) or B7.1,
in 2000 last reported normal
in 2005.
infection of CD34+ haematoVaccinia virus with B7.1
gene transfer. Patients treated
receptor subunit by ex vivo
immunodeficiency (X-SCID)
Malignant melanoma
In 13/14 patients, normal
T-cell counts achieved three after
Moloney retrovirus-derived
vector expressing c cytokine
X-linked severe combined
artery [20a].
infused into the right hepatic
observed in seven liver biopses.
(OTC) deficiency
Transgene-expressing cells
Human adenovirus type 5,
deleted in E1 and E4 containing
Ornithine transcarbamylase
Effectiveness
Study design
Disease
Table 1. Adverse events resulting from use of virus-based gene therapy
low grade fever, mild fatigue and myalgia.
patients. Low grade injection site reactions,
New onset autoimmune viligo in 3/25
near LMO-2 causing its overexpression.
leukaemia) due to retroviral insertion
disease (similar to acute lymphoblastic
Two patients developed T-cell proliferative
organ system failure.
intravascular coagulation, and multiple
biochemically detectable disseminated
inflammatory response syndrome,
98 h after gene transfer; systemic
One subject died (highest dose group)
Adverse events
S.D.R. Harridge & C.P. Velloso
129
© The Authors Journal compilation © 2008 Biochemical Society
2/6/08
4:11:59 PM
130
Essays in Biochemistry volume 44 2008
FORCE
POWER
Muscle
mass
Fibre
type
NFAT
IGF-I
Akt
myostatin
testosterone
expansion
SPEED
Ex vivo
In vivo
selection
Oxygen EPO
delivery VEGF
Metabolism
PGC1D
PPARG
transduction
ENDURANCE
Figure 1. A general summary of phenotypes and performance parameters that
can be manipulated by targeting selected genes (italics) for overexpression (p) or
inactivation (q)
Strategies for gene doping include injection of vector (♦) directly into the body to transduce/
transfect cells in vivo or removal of cells from the body, for ex vivo transduction/transfection,
selection and expansion before reintroduction to the athlete.
a considerable advantage over endogenous administration of synthetic drugs.
This is because of the simple fact that manipulation of a gene will result in the
production (or over-production) of a given hormone that is equal in sequence
to its endogenous counterpart, making detection extremely difficult. Skeletal
muscle is an attractive target for gene therapy because of the large size of
the muscle cells and the fact that muscle is a non-proliferating tissue, leading
to long-lived gene expression. In addition, skeletal muscle is particularly
amenable to gene delivery using plasmid DNA vectors which are low risk,
can be easily quality controlled, and can be produced in large amounts at a
relatively low cost.
IGF-1
IGF-1 is a 70-amino-acid polypeptide synthesized primarily in the liver under
the control of GH. The GH/IGF-1 axis is extremely important in regulating
postnatal growth and development. In addition to the liver, other tissues,
including skeletal muscle, can produce IGF-1. The IGF-1 gene comprises six
exons and a process of alternative splicing at the 5 and 3 ends can generate
different isoforms. Evidence from viral gene transfer studies in mice have
shown that the two murine 3 splice variants IGF-1Ea and IGF-1Eb (also
termed mechano-growth factor or MGF) can induce significant local muscle
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
130
2/6/08
4:11:59 PM
S.D.R. Harridge & C.P. Velloso
131
hypertrophy in young mice, but only IGF-1Ea works on adult mice [9].
Transgenic approaches have also confirmed the potency of IGF-1Ea as an
anabolic agent. Use of a myosin light chain promoter restricting overexpression
of IGF-1Ea to muscle tissue, leads to significant muscle hypertrophy in
the affected animals [10]. Interestingly, when viral IGF-1Ea gene transfer
is combined with high-resistance exercise in adult animals, significantly
greater increases in muscle size and strength occur than when gene transfer or
resistance exercise are considered separately [11] (Figure 2). Although such gene
studies have not been performed in human beings, the mRNA for IGF-1Ea and
MGF have been shown to be expressed in skeletal muscle and are increased
following muscle-building, high-resistance, strength-training exercise [12].
IGF-1 acts through the PKB (Akt; protein kinase B) signalling pathway.
In vivo transfection of myofibres with a Ras double mutant construct that
selectively activates PKB resulted in marked hypertrophy of these cells [13].
Thus hypertrophy can be achieved by overexpressing IGF-1 itself or activating
proteins in its signalling pathway.
Myostatin
In contrast with IGF-1 and anabolic steroids, which stimulate protein
synthesis, myostatin, a member of the TGF- (transforming growth factor )
family of growth factors, acts as a negative regulator of muscle growth. The
significance of this protein came to light following work on the Belgian Blue
and Piedmontese breeds of cattle which exhibit a ‘double-muscled’, highly
hypertrophied phenotype. These animals possess a mutation in their myostatin
gene which results in production of an inactive protein. Transgenic mice
specifically bred to knockout this gene have a highly hypertrophied phenotype
that is remarkably similar to that of the IGF-1-overexpressing mice, even
*
40
Mass
35
Isometric force
*
30
% Change
†
*
25
20
15
10
5
0
Control
IGF-1
RT
RT &
IGF-1
Figure 2. Changes in muscle mass and isometric force following resistance training,
IGF-1Ea gene transfer or a combination of both stimuli
*P<0.05, significantly different from control; †P<0.05, significantly different from IGF-1 and RT.
RT, resistance training. Based on data taken from [11].
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
131
2/6/08
4:12:00 PM
132
Essays in Biochemistry volume 44 2008
though the mechanisms of action of these two proteins are fundamentally
different [14]. Myostatin inhibits satellite cell proliferation, whereas IGF-1
does not. Gene doping strategies would thus aim to inhibit production of
myostatin or interfere with the function of the endogenous protein.
Manipulation of fibre type
In addition to force of contraction, the other determinant of power is the
speed at which a muscle shortens. It is the myosin heavy chain isoform, the
molecular motor contained in each fibre, that primarily determines speed of
shortening. Human muscle fibres may contain the slow-contracting MHC-I
(where MHC is myosin heavy chain), the fast-contracting MHC-IIA or even
faster contracting MHC-IIX isoforms. In sprinters it is the MHC-IIa isoform
which dominates, whereas in marathon runners MHC-I predominates. It
is possible to alter MHC expression through modifying the neural input
delivered to a muscle or its mechanical loading status. These physiological
manipulations ultimately affect the expression of specific transcription factors
which regulate expression of genes associated with fast or slow myosins.
Understanding of the regulation of myosin expression opens the possibility for
these transcription factors to be genetically altered potentially allowing athletes
to alter their natural endowment for a given population of fast and slow fibres.
For example, altering the activation of the transcription factor NFAT (nuclear
factor of activated T cells) in mice, by plasmid injection of either a specific
peptide inhibitor or a constitutively active isoform, can alter MHC mRNA
expression in fast and slow muscles, overriding neural input [15].
Gene doping for increased endurance
In contrast with sprinting, which is primarily determined and limited by the
mechanical properties and anaerobic metabolism, endurance performance
requires a high oxidative capacity within the muscle and good cardiovascular/
respiratory function. There is a constant demand for oxygen to be supplied to
the mitochondria so that it can act as the final electron acceptor in the electron
transport chain for the generation of ATP. Delivery of oxygen to working
muscles occurs through a complex interaction of the cardiovascular and
respiratory systems. Ultimately oxygen is transported in the blood primarily
bound to haemoglobin contained in red blood cells.
EPO (erythropoietin)
Athletes have long used training at altitude to stimulate the bone marrow
to produce more red blood cells and thus increase the oxygen-carrying
capacity of the blood. In times of hypoxia, EPO promotes red blood cell
production by stimulating CFU-E (erythroid colony-forming unit) progenitor
cells to proliferate and differentiate into mature erythrocytes. Recombinant
human EPO has been available for human use since 1989. Not surprisingly,
this drug was readily identified by endurance athletes for its ergogenic
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
132
2/6/08
4:12:01 PM
S.D.R. Harridge & C.P. Velloso
133
potential. Randomized controlled trials provided evidence of its efficacy
in improving endurance performance [16], and it has subsequently gained
particular notoriety among endurance athletes, most notably cyclists in the
Tour de France. As methods for the detection of EPO become more refined,
athletes may well be tempted to use gene doping techniques to increase its
endogenous expression. Following a single intramuscular injection of a vector
containing the EPO gene under control of a rapamycin-sensitive promoter,
regulated, inducible expression of EPO has been obtained in macaques for a
period of six years [17]. In this study, two problems previously identified in
EPO gene therapy were overcome: polycytaemia (life-threatening increases in
blood viscosity owing to high erythrocyte numbers) and anaemia (owing to
development of autoimmunity to endogenous EPO).
VEGF (vascular endothelial growth factor)
In addition to a better oxygen-carrying capability, an optimized vascular
system would also promote oxygen and nutrient delivery to working muscles.
VEGF is an important factor in stimulating the formation of new blood
vessels. Phase I and II clinical trials have already been undertaken using VEGF
viral gene therapy techniques to induce angiogenesis in patients with ischaemic
heart disease [18].
PGC1 [PPAR (peroxisome-proliferator-activated receptor) co-activator]
and PPAR
In addition to oxygen delivery, metabolic characteristics of muscle fibres
are important for endurance. Studies in mice show that transgenic animals
for either PGC1 or PPAR have an increase in type I fibres as assessed
by oxidative enzyme expression, muscle colour (which reflects myoglobin
content), sarcomeric protein expression and mitochondrial content [19,20].
Importantly, these mice outperform littermate controls in running endurance
and muscle fatigue resistance. The question remains whether elevated
expression of these factors in adult animals, as opposed to during embryonic
development, would have similar effects.
Endorphins, endurance and pain
The death of Tommy Simpson during the Tour de France in 1967 brought
to the fore the use of amphetamines as a tool to dull the sensations of pain
associated with fatigue. The sensation of pain is an adaptive mechanism that
helps us to avoid noxious stimuli, prevents us from pushing ourselves too
far and protects an injury while healing takes place. The nature of physical
activity required during high-level competitive sports often means that athletes
push their bodies through barriers of discomfort not usually tolerated. They
may also live with chronic pain. The endogenous opioid peptides, such as
-endorphins, affect both local neurons and the pain-modulating circuitry in
the central nervous system leading to pain relief.
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
133
2/6/08
4:12:01 PM
134
Essays in Biochemistry volume 44 2008
Strenuous exercise causes a release of -endorphin from the pituitary into
the bloodstream. Motor neurons are able to synthesize -endorphin and receptors for this substance are present on the membrane of skeletal muscle fibres.
In addition to pain relief, a direct effect of -endorphin on skeletal muscle has
been proposed since this substance has been shown to reduce fatiguability and
increase glucose consumption in isolated muscle preparations [20a]. Thus this
factor could have a double effect in terms of modulating physical performance:
the first in alleviating pain and helping an athlete push through sensations of
discomfort, the second in enhancing muscle endurance. The administration
of endogenous opioids using gene therapy approaches is being investigated to
provide relief for chronic pain, such as might occur after trauma or in advanced
stages of cancer. This has the advantage that the peptides could be synthesized
in the vicinity or inside target cells and thus avoid some of the complications
associated with long-term administration of opioids. In addition to their analgesic effect, endorphins may have roles in the response to stress, determining
mood, and regulating the release of hormones from the pituitary gland, notably GH and the gonadotropin hormones.
Can gene doping be detected?
The major attraction of gene doping for sport is the theoretical difficulty in
distinguishing the gene product of the exogenous gene from the product of
its endogenous counterpart. Nevertheless there is hope for the authorities
that detection methods will not lag behind the adoption of gene therapy
technologies for doping. Detection strategies have been reviewed recently
[18] and include detection of the vectors used for gene delivery, evidence
of an immune response to these vectors, alteration of various parameters
(level of mRNA and protein, relative mRNA or protein isoform expression,
post-translational modifications) which may differ between transcripts and
proteins produced from endogenous and exogenous genes. A critical factor
in gene delivery is the size of the DNA that can be introduced into a vector.
Some of the sequences that regulate gene expression can be very distant from
the protein-coding regions of the DNA molecule and may not even be known.
Thus the manipulation of a gene for insertion into a vector is likely to affect
some of the regulatory elements and its ‘natural’ expression may never be
accurately mimicked at all levels.
Among the most promising applications is the use of complex molecular
signatures to identify normal, exercise-regulated and aberrant levels of a given
biomarker. For example, external hormone administration or disease states
disturb the composition of blood and urine. Application of high-throughput
analyses of protein spectra in these body fluids will allow identification of
specific profiles indicative, at minimum, of disturbance in normal physiology. The challenge will be to relate these disturbances to gene doping. At
present, the application of this technique is mainly in cancer diagnosis, but
advances in this area will aid the fight against gene doping. In this regard, it
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
134
2/6/08
4:12:01 PM
S.D.R. Harridge & C.P. Velloso
135
is encouraging to note that the pattern of post-translational modifications of
proteins or production of specific protein isoforms may require physiological
signals that are bypassed by overexpression of the transgene. In addition, the
processing of the gene may differ in different tissues. Thus targeting the transgene to skeletal muscle might result in production of protein products distinct
from those occurring naturally if the major source of protein is another organ
(for example, the liver). Last but not least, it is important to note that it may
not be possible for gene-doped athletes to arrest gene expression, unless they
use inducible vectors. In this way, supraphysiological levels of protein or the
use of drugs required for inducible gene expression may be sufficient to indicate doping. From the point of view of the gene-doped athlete, it is worthwhile
to note that deregulated protein expression is a risk not only for detection but
also for continuing health. The body is a fine-tuned machine and the deregulation of gene expression in a healthy person may lead to long-term complications even if it is beneficial in the short-term. For example, IGF-1 may be an
anabolic substance, but it is also a potent mitogen thus increasing the risk of
tumour development. In transgenic mice, chronic EPO overexpression leads to
multiple organ degeneration and reduced life expectancy [21]. In people suffering from disease or poor health, the benefits outweigh the risks, but the same
risk/benefit calculation does not necessarily apply to a healthy individual.
A potential grey area is in the development of gene therapy for the treatment of sporting injuries. Here the aim is to increase the expression of important growth factors in muscle, bone, tendon and cartilage to facilitate the repair
processes. A doping issue might arise if the athlete were to carry a permanent
marker of treatment which might give a competitive advantage over their peers
as a result of the treatment.
Gene polymorphisms and ‘natural gene dopers’
Not all men are created equal, at least not genetically! We all have different
physiognomic traits that are genetically determined. It follows therefore that
those participating in competitive sport must possess genetically determined
traits that confer advantages in physical performance relative to the general
population. Indeed the HERITAGE family study provides evidence of a
significant genetic component to the ability of individuals to respond to a
programme of aerobic training [23].
There is also evidence of the existence of individuals with naturally occurring gene mutations that confer competitive advantages. Familial erythrocytosis is a condition associated with high haematocrit (red blood cell count) and
haemoglobin levels, as well as low serum EPO. The condition results from an
EPO receptor mutation that inactivates a negative regulatory region, thereby
conferring hypersensitivity of the receptor to EPO. The genetic mutation was
detected in a Finnish family which had several members involved in competitive endurance sports, notably Olympic cross-country skier and gold medallist
Eero Mantyranta [24].
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
135
2/6/08
4:12:01 PM
136
Essays in Biochemistry volume 44 2008
More recently a myostatin loss-of-function mutation was reported in a male
child who had supranormal muscle mass from birth [25]. At 4½ years of age,
he possessed significant skeletal muscle hypertrophy and strength relative to
age-matched controls. Other members of his family were exceptionally strong
according to clinical records, but they were not available for genetic testing.
Gene polymorphisms rather than mutations may also be associated with
increased performance. The difference between a mutation and a polymorphism is basically quantitative. A mutation is considered to be a variation in
sequence that is present in less than 1% of the population, whereas a polymorphism is a more common occurrence. ACE (angiotensin-converting enzyme)
and ACTN-3 (actinin 3) are genes with polymorphisms that have been correlated with distinct patterns of performance.
ACE catalyses the conversion of angiotensin I into angiotensin II.
Angiotensin II is involved in regulating blood pressure primarily by causing vasoconstriction. It is also a potent growth factor for cardiac and vascular
tissues. The ACE gene polymorphism is a 287 base pair sequence and the
gene copies (alleles) of the ACE gene are classified as insertion (I) or deletion
(D) depending on whether they contain this sequence. Humans have two alleles of each gene, one inherited from each parent. There are three possible allelic
combinations of the ACE gene: II, DD and ID. Several studies have suggested
a link between occurrence of the I allele with endurance and the D allele with
strength, but null correlations have also been found [26]. There are also two
alleles of ACTN-3: R and R577X or X for short. The R allele results in production of actinin whereas the X does not. Actinin is found only in fast fibres,
which are associated with the generation of explosive power. Again the studies
showed a correlation between the presence of the R allele and the modality of
exercise in elite athletes: 50% of sprinters are RR, 45% are RX and the remaining 5% are XX. Among endurance athletes 31% are RR, 40% are RX and 24%
are XX. All studies conducted so far on ACTN-3 and ACE have been correlational in nature, but there is no evidence showing an irrefutable cause/effect
relationship between the presence of an allele and performance gains [27].
Conclusion
As our knowledge of the molecular basis of performance and of the
technologies involved in legitimate gene therapy progresses, so do the chances
of these techniques being abused by athletes. Whether gene doping is currently
practised is not known. The challenge to the anti-doping authorities will be to
develop methods which are capable of detecting such practices. This will not
be straightforward.
Summary
•
Gene therapy technology has the potential to be abused for performance
gains by athletes.
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
136
2/6/08
4:12:02 PM
S.D.R. Harridge & C.P. Velloso
•
•
•
•
•
137
Objective clinical responses have rarely been observed in clinical trials.
There are significant risks associated with viral gene therapy and thus
gene doping
Genes have been identified that affect skeletal muscle size, metabolism
and contractile properties
The cardiovascular/respiratory systems can also be genetically
manipulated.
Development of gene doping detection methods will not be straightforward.
We would like to thank WADA.
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
Young, S.Y., Searle, P.F., Onion, D. & Mautner, V. (2006) Viral gene therapy strategies: from basic
science to clinical application. J. Pathol. 208, 299–318
Wells, D.J. (2004) Gene therapy progress and prospects: electroporation and other physical
methods. Gene Ther. 11, 1363–1369
Cavazzana-Calvo, M., Lagresle, C., Hacein-Bey-Abina, S. & Fischer, A. (2005) Gene therapy for
severe combined immunodeficiency. Annu. Rev. Med. 56, 585–602
Kaufman, H.L., Cohen, S., Cheung, K., DeRaffele, G., Mitcham, J., Moroziewicz, D., Schlom, J. &
Hesdorffer, C. (2006) Local delivery of vaccinia virus expressing multiple costimulatory molecules
for the treatment of established tumors. Hum. Gene Ther. 17, 239–244
Porteus, M.H. & Carroll, D. (2005) Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23,
967–973
Azzazy, H.M., Mansour, M.M. & Christenson, R.H. (2005) Doping in the recombinant era: strategies and counterstrategies. Clin. Biochem. 38, 959–965
Haisma, H.J. & de Hon, O. (2006) Gene doping. Int. J. Sports. Med. 27, 257–266
Schneider, A.J. & Friedmann, T. (2006). Gene doping in sports: the science and ethics of genetically modified athletes. Adv. Genet. 51, 1–110
Barton, E.R. (2006) Viral expression of insulin-like growth factor-I isoforms promotes different
responses in skeletal muscle. J. Appl. Physiol. 100, 1778–1784
Musaro, A., McCullagh, K., Paul, A., Houghton, L., Dobrowolny, G., Molinaro, M., Barton, E.R.,
Sweeney, H.L. & Rosenthal, N. (2001) Localized Igf-1 transgene expression sustains hypertrophy
and regeneration in senescent skeletal muscle. Nat. Genet. 27, 195–200
Lee, S., Barton, E.R., Sweeney, H.L. & Farrar, R.P. (2004) Viral expression of insulin-like growth
factor-I enhances muscle hypertrophy in resistance-trained rats. J. Appl. Physiol. 96, 1097–1104
Hameed, M., Lange, K.H., Andersen, J.L., Schjerling, P., Kjaer, M., Harridge, S.D. & Goldspink, G.
(2004) The effect of recombinant human growth hormone and resistance training on IGF-I mRNA
expression in the muscles of elderly men. J. Physiol. 555, 231–240
Pallafacchina, G., Calabria, E., Serrano, A.L., Kalhovde, J.M. & Schiaffino, S. (2002) A protein kinase
B-dependent and rapamycin-sensitive pathway controls skeletal muscle growth but not fiber type
specification. Proc. Natl. Acad. Sci. U.S.A. 99, 9213–9218
Walsh, F.S. & Celeste, A.J. (2005) Myostatin: a modulator of skeletal-muscle stem cells. Biochem.
Soc. Trans. 33, 1513–1517
McCullagh, K.J., Calabria, E., Pallafacchina, G., Ciciliot, S., Serrano, A.L., Argentini, C., Kalhovde,
J.M., Lomo, T. & Schiaffino, S. (2004) NFAT is a nerve activity sensor in skeletal muscle and controls activity-dependent myosin switching. Proc. Natl. Acad. Sci. U.S.A. 101, 10590–10595
Birkeland, K.I., Stray-Gundersen, J., Hemmersbach, P., Hallen, J., Haug, E. & Bahr, R. (2000) Effect
of rhEPO administration on serum levels of sTfR and cycling performance. Med. Sci. Sports Exercise
32, 1238–1243
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
137
2/6/08
4:12:02 PM
138
Essays in Biochemistry volume 44 2008
17.
Rivera, V.M., Gao, G.P., Grant, R.L., Schnell, M.A., Zoltick, P.W., Rozamus, L.W., Clackson, T. &
Wilson, J.M. (2005) Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer. Blood 105, 1424–1430
18. Stewart, D.J., Hilton, J.D., Arnold, J.M., Gregoire, J., Rivard, A., Archer, S.L., Charbonneau, F.,
Cohen, E., Curtis, M., Buller, C.E. et al. (2006) Angiogenic gene therapy in patients with nonrevascularizable ischemic heart disease: a phase 2 randomized, controlled trial of AdVEGF(121)
(AdVEGF121) versus maximum medical treatment. Gene Ther. 13, 1503–1511
19. Lin, J., Wu, H., Tarr, P.T., Zhang, C.Y., Wu, Z., Boss, O., Michael, L.F., Puigserver, P., Isotani, E.,
Olson, E.N. et al. (2002) Transcriptional co-activator PGC-1 drives the formation of slow-twitch
muscle fibres. Nature 418, 797–801
20. Wang, Y.X., Zhang, C.L., Yu, R.T., Cho, H.K., Nelson, M.C., Bayuga-Ocampo, C.R., Ham, J., Kang,
H. & Evans, R.M. (2004) Regulation of muscle fiber type and running endurance by PPAR. PLoS
Biol. 2, e294
20a. MacArthur, D.G. & North, K.N. (2004) A gene for speed? The evolution and function of
-actinin-3. Bioessays 26, 786–795
21. Khan, S., Evans, A.A., Hughes, S. & Smith, M.E. (2005) -Endorphin decreases fatigue and increases glucose uptake independently in normal and dystrophic mice. Muscle Nerve 31, 481–486
22. Heinicke, K., Baum, O., Ogunshola, O.O., Vogel, J., Stallmach, T., Wolfer, D.P., Keller, S., Weber,
K., Wagner, P.D., Gassmann, M. & Djonov, V. (2006) Excessive erythrocytosis in adult mice
overexpressing erythropoietin leads to hepatic, renal, neuronal, and muscular degeneration. Am. J.
Physiol. Regul. Integr. Comp. Physiol. 291, R947–R956
23. Bouchard, C., An, P., Rice, T., Skinner, J.S., Wilmore, J.H., Gagnon, J., Perusse, L., Leon, A.S. &
Rao, D.C. (1999) Familial aggregation of VO2max response to exercise training: results from the
HERITAGE Family Study. J. Appl. Physiol. 87, 1003–1008
24. de la Chapelle, A., Traskelin, A.L. & Juvonen, E. (1993) Truncated erythropoietin receptor causes
dominantly inherited benign human erythrocytosis. Proc. Natl. Acad. Sci. U.S.A. 90, 4495–4499
25. Schuelke, M., Wagner, K.R., Stolz, L.E., Hubner, C., Riebel, T., Komen, W., Braun, T., Tobin, J.F. &
Lee, S.J. (2004) Myostatin mutation associated with gross muscle hypertrophy in a child. N. Engl. J.
Med. 350, 2682–2688
26. Jones, A., Montgomery, H.E. & Woods, D.R. (2002). Human performance: a role for the ACE
genotype? Exercise Sport Sci. Rev. 30, 184–190
27. Raper, S.E., Yudkoff, M., Chirmule, N., Gao, G.P., Nunes, F., Haskal, Z.J., Furth, E.E., Propert,
K.J., Robinson, M.B., Magosin, S. et al. (2002) A pilot study of in vivo liver-directed gene transfer
with an adenoviral vector in partial ornithine transcarbamylase deficiency. Hum. Gene Ther. 13,
163–175
© The Authors Journal compilation © 2008 Biochemical Society
POIN004_C09_125-138_.indd
138
2/6/08
4:12:02 PM