Генотерапия – лечение с помощью высоких технологий генной инженерии. Методы генотерапии направлены на внедрение необходимых генетических конструкций, регулирование работы генов или удаление ненужного генетического материала. В спорте имеется риск получения различных механических повреждений опорно-мышечного аппарата. Поэтому, генотерапия в спортивной медицине занимается проблемами быстрого и полного восстановления
поврежденных структур.
Gene Therapy and Tissue Engineering in Sports Medicine
GENE
THERAPY OR DOPING OF THE FUTURE
Vladimir Martinek, MD;
Freddie H. Fu, MD; Johnny Huard, PhD
THE PHYSICIAN AND SPORTSMEDICINE - VOL 28 - NO. 2 - FEBRUARY
2000
In Brief: Treatment of sports injuries has
improved through sophisticated rehabilitation programs, novel operative
techniques, and advances in biomechanical research during the past two decades.
Despite considerable progress, treatments remain limited due to poor healing
capacity for anterior or posterior cruciate ligament rupture, central meniscal
tear, cartilage lesions, and delayed bone fracture. New biological approaches
seek to treat these injuries with growth factors to stimulate and hasten the
healing process. Gene therapy using the transfer of defined genes such as those
encoding growth factors represents a promising way to deliver therapeutic
proteins to the injured tissue. Tissue engineering, which may eventually be
combined with gene therapy, offers the potential to create tissues or scaffolds
for regeneration of defects occurring from trauma.
The treatment of sports-related
injuries has improved continuously during the last two decades. New, minimally
invasive operative techniques, especially arthroscopy, novel instruments,
modern rehabilitation and medications, as well as increasing knowledge of joint
biomechanics and trauma pathophysiology, have enhanced and accelerated therapy.
Despite this progress, deficits in injury treatment remain because of the
limited healing capacity of certain musculoskeletal system tissues. Ligaments,
tendons, menisci, and articular cartilage have a limited blood supply and a
slow cell turnover. For this reason, healing is prolonged and often results in
the formation of a dysfunctional scar or a tissue defect.
Various growth factors
effect musculoskeletal tissue healing (1). These growth factors are small
proteins that can be synthesized both by resident cells at the injury site (eg,
fibroblasts, endothelial cells, and mesenchymal stem cells) and infiltrating
repair or inflammatory cells (eg, platelets, macrophages, and monocytes). The
proteins are capable of stimulating cell proliferation, migration, and differentiation
as well as matrix synthesis (2,3), and their effects have been shown on
different tissues (2-12). Genes encoding most of the known growth factors have
been determined. Using recombinant deoxyribonucleic acid (DNA) technology,
researchers can produce large quantities of protein for use in treatment (1).
Although direct application
of recombinant human proteins has some beneficial effect on healing (4), their
relatively short half-lives in vivo often require high doses and repeated
injections. Another major limitation of using growth factors to promote healing
is the mode of delivery to the injury (13). Many strategies, including
polymers, pumps, and heparin, have been tested in attempts to achieve
consistent growth factor levels at the injured site (14,15). Despite the
improved local persistence of growth factor proteins with various approaches,
the results of these delivery techniques remain limited. Among the methods
developed for local administration of growth factors, gene transfer techniques
have proven the most promising (16).
Tissue defects are another
challenge for treatment of injuries. These occur as a direct consequence of
trauma (eg, cartilage lesion, osteochondral defect), treatment (eg, partial
meniscus resection), or poor or impaired healing capacity (eg, anterior
cruciate ligament [ACL] rupture, delayed bone union). Using autogenous or
allogeneic grafts is the most common therapeutic approach for treatment of
tissue defects such as these (17,18). Both treatments have disadvantages that
include additional trauma from graft harvesting (autografts) (19), transmission
of infectious diseases such as hepatitis B and HIV and immunorejection
(allografts) (20). Tissue engineering, a new technology, focuses on the
creation of tissues and scaffolds as a solution to these problems (21). The
technology combines the use of adequate scaffolds (shaped polymers that serve
as a matrix for tissue growth), selected growth factors, and responsive cells
to improve the healing of several musculoskeletal system tissues, especially
for treatment of cartilage and bone defects.
Gene therapy delivers
therapeutic genes into cells and tissues. Originally, it was conceived for the
manipulation of germ-line cells for treating heritable genetic disorders, but
that application has been greatly limited because of inefficient technology and
considerable ethical concern. Gene manipulation of somatic cells has been
widely accepted, however. Gene therapy applied to sports medicine includes
transfer of defined genes (such as those encoding growth factors or
antibiotics) into the target tissue. Thus, successful application of gene
therapy would promote production of therapeutic levels of desired proteins by
the transformed cells at the site of injury or inflammation.
Vectors. For gene therapy to allow
expression of an inserted gene, the DNA must be packaged into a vector and
enter the cell nucleus, where it is either integrated into the chromosomes of
the host cell (figure 1) or maintained in the nucleus as a separate episome.
The inserted gene is then transcribed into messenger RNA (mRNA), and mRNA is
transported into the cell cytoplasm, where it serves as the template for the
production of the therapeutic protein (eg, growth factors) in the ribosomes.
Consequently, the transduced cells become a reservoir of secreting growth
factors and cytokines capable of improving healing.
Viral and nonviral vectors
can be used to deliver genetic material into cells (table 1). Nonviral
gene-transfer systems such as liposomes are usually easier to produce and have
relatively low toxicity and immunogenicity, but their efficiency of gene
delivery is hindered by a low transfection rate. (Transduction refers to
introducing DNA into cells with a virus, while transfection refers to
introducing DNA into cells with a plasmid vector.) Despite new approaches in
enhancing cell transfection rates and development of new nonviral vectors (eg,
new liposomes made with diverse lipids that form different composition
aggregates containing the DNA), the efficiency of gene transfer remains low.
|
|
||
|
TABLE
1. Vectors Used for Gene Delivery Into Cells and Their Characteristics |
||
|
Gene Delivery |
Vector |
Characteristics |
|
|
||
|
Nonviral |
Liposome |
Low
efficiency of gene delivery |
|
|
||
|
Viral |
Adenovirus |
Infects
mitotic/postmitotic cells |
|
|
|
|
|
|
Retrovirus |
Low
toxicity/immunogenicity |
|
|
|
|
|
|
Adeno-associated virus |
Low
toxicity/immunogenicity |
|
|
|
|
|
|
Herpes simplex |
Infects mitotic/postmitotic
cells |
|
|
||
Currently, viral gene
vectors present a more efficient method for gene transfer (13). Before a virus can
be used as a vector in gene therapy, all genes for viral replication and genes
for pathogenic proteins must be removed and replaced by the desired gene(s).
Transfer relies on native viral ability to enter (infect) the cell (see figure
1). The virus attaches to the cell via a receptor, and the genetic material is
transported through the cytoplasm by viral and/or cellular proteins and enters
the nucleus. The most commonly used viruses include adenovirus, retrovirus,
adeno-associated virus, and herpes simplex virus (see table 1). New mutant
viral vectors with reduced cytotoxicity and immunogenicity are currently being
developed (12).
Delivery strategies. Various gene-transfer strategies,
including systemic and local delivery, can be used for gene transfer to
musculoskeletal tissues (figure 2) (13). Systemic delivery consists of
injecting the vector into the bloodstream, thus disseminating it to all organs
of the body; the technique is preferable when the target tissue cannot be
reached directly. This approach also has the advantage of better vector
distribution than that of direct, local injection of the vector. Major
limitations include low specificity of gene expression and large vector
concentration required for therapeutic effects. The lack of blood supply in
various tissues (eg, cartilage, meniscus), however, makes systemic delivery
inappropriate for most musculoskeletal system injuries.
Two basic strategies for local gene therapy in the musculoskeletal
system have been extensively investigated (see figure 2) (13). Vectors can be
directly injected in the host tissue (in vivo technique), or the cells from the
injured tissue can be removed, genetically altered (transduced [with a virus]
or transfected [with a plasmid]) in vitro, and reinjected in the injury site
(ex vivo technique). While the direct method is technically more simple,
indirect delivery poses less risk because gene manipulation takes place under
controlled conditions outside the body. With the ex vivo approach, growth
factors can be delivered with endogenous cells capable of responding to stimuli
and participating in the healing of the injured tissue. Tissue
engineering-based approaches to gene delivery that employ cells from various
tissues (eg, mesenchymal stem cells, muscle derived cell, dermal fibroblasts)
may offer additional ways to improve healing. Selecting the appropriate gene
delivery procedure depends on many factors that include the division rate of
the target cells, pathophysiology of the disorder, and accessibility of the
target tissues.
Limitations. For treatment of sports injuries,
the major concern for using gene therapy is safety. While gene therapy may
represent a "last chance" treatment option for severe disorders such
as cancer, Duchenne muscular dystrophy, Gaucher's disease, or cystic fibrosis,
the risk of side effects may be unacceptable in elective sports medicine. In
addition, integration of viral vectors into the host genome carries the risk of
insertional mutagenesis (22). Abnormal regulation of cell growth, toxicity from
chronic overexpression of the growth factor and cytokines, and malignancy are
all theoretically possible, but no cases have been reported. However, there is
no guarantee that integrated DNA sequences will not cause mutations or
malignancies years later. For this reason, long-term records of all human
trials in gene therapy need to be kept and exchanged among the research groups.
Most clinical trials of gene therapy are using the ex vivo approach, so the
virus is not directly introduced into patients and cells can be extensively
tested before implantation.
Loss of expression of the
transferred gene after a few weeks is a common and not fully understood
phenomenon. However, temporary and self-limiting gene expression could be
useful in the treatment of musculoskeletal injuries, in which only transient
high levels of growth factors are needed to promote healing response. Present
research is also focusing on the development of specific inducible promoters
that regulate the mRNA transcription. (Promoters are DNA sequences that are
adjacent to the functional genes and are required for expression and regulation
of gene transcription.) The inducible promoters could help control expression
of the transferred gene; they could modulate implanted genes as well as turn
them on and off. Although these systems are very attractive, they remain under
extensive investigation in many laboratories and are not yet ready for clinical
trial.
Although great strides in
gene therapy techniques have taken place, they still have not become
established treatments, partly because of the lack of appropriate gene vectors.
Many laboratories successfully focus on developing therapeutic viral and
nonviral vectors. Consequently, major advances in vector development can be
expected in the near future (12).
Tissue engineering is a
technology based on developing biological substitutes for the repair,
reconstruction, regeneration, or replacement of tissues (figure 3: not shown).
Its long-term goal is to construct biomaterials that are biocompatible,
biodegradable, and capable of integrating molecules (eg, growth factors) or
cells (23,24). Currently, many different ceramics, polymers of lactic and
glycolic acid, collagen gels, and other polymers have been tested in vitro and in
vivo (24). More recently, genetic modifications have been included in tissue
engineering to optimize the healing process (25). Modified cells are
transplanted into injured tissue to effect the repair with the introduced gene.
Bone and cartilage are the
tissues in which most tissue engineering techniques have been applied. Bone has
a high potential for repair. In large defects or when vascularization is
impaired, however, augmentation with scaffolds, genetically engineered cells,
and/or growth factors and cytokines can accelerate or enhance the healing (5).
Recently, autologous muscle tissue has been used as a delivery vehicle for
growth factor genes in treatment of bone defects (26), and laboratory-grown
skin has been recently approved for use in treatment of wounds (27,28).
In contrast, cartilage has
a poor intrinsic capacity for healing and therefore a limited ability to
regenerate (23). Intense investigations have focused on finding biomaterials
that would be capable of repairing cartilage defects, but no efficient
therapeutic approaches have yet been established (29); candidate materials
include fibrin, collagen, ceramics, alginate, polymers of lactic and glycolic
acid, hyaluronic acid, and synthetic materials.
Besides bone and cartilage
substitutes, biological scaffolds have been developed for other tissues of the
musculoskeletal system. The collagen meniscus implant is a biological scaffold
for meniscus regeneration made from bovine Achilles tendons. Short-term
clinical results were promising in 20 patients who had total meniscal loss
(21), but long-term data are not yet available. In addition, biomaterials for
ACL replacement have been used in trials but have never gained surgical
acceptance (30).
Another tissue engineering
approach is the use of autologous tissues or cells. Myoblasts become
post-mitotic by fusing with existing myofibers or fusing among themselves to
form postmitotic myotubes (31). Hence, muscle-derived cells that have been
transduced to express a therapeutic protein may differentiate within the host
tissues (muscle, ligament, bone, meniscus) and lead to a stable and persistent
expression of the desired protein at the injury site (32).
Skeletal muscle. Depending on the type of sport, the
prevalence of muscle injuries varies from 10% to 55% among all sustained
injuries (33). While minor muscle injuries such as strains can heal completely,
most severe muscle injuries often heal with dense scar tissue formation,
impairing muscle function and potentially leading to muscle contractures and
chronic pain. Other clinical problems include disparate limb length after
injury, disabling contractures from scar tissue, and post-compartment syndrome
muscle necrosis.
Growth factors may offer a
new avenue to treat muscle injuries. Recently, in vitro and in vivo experiments
with growth factors have shown improved healing, especially with basic
fibroblast growth factor (bFGF), nerve growth factor (NGF), and insulin-like
growth factor type 1 (IGF-1) (4,33,34). Promising gene therapy approaches have
already been used in treating inherited disorders such as Duchenne muscular
dystrophy (35). Gene therapy techniques are now being investigated to establish
an efficient treatment method for improved healing of sports-related muscle
injuries (4).
Cartilage. Damage to knee articular cartilage
is a common problem following sports injuries. It leads to premature arthritis,
causes a considerable decrease in quality of life, and has enormous long-term
healthcare costs (29). Regeneration of damaged articular cartilage is very
limited because in adults cartilage lacks a blood supply, lymphatic drainage,
and innervation. Furthermore, chondrocytes are sheltered from synovial fluid
nourishment and reparative recognition by their large extracellular matrix
(36).
The common operative
techniques for existing therapy of injured articular cartilage are subchondral
drilling or microfracture, transplantation of autologous or allogenic
chondrocytes, autogenous or allogenic osteochondral transplantation, and the
use of scaffolds (37). Despite some promising clinical results (38), new
strategies are required to obtain consistently good long-term results. Growth
factors, including BMP-2 (6), bFGF, transforming growth factor ß (TGF-beta),
epidermal growth factor (EGF), IGF-1 (39), and cartilage-derived morphogenic
proteins (CDMP) (39) have demonstrated both in vitro and in vivo positive
effects on chondrocyte growth and cartilage healing. Several gene therapy and
tissue engineering techniques are currently being investigated for treating
cartilage defects, but the most efficient methodology for solving this
sophisticated problem has not yet been established (39).
Anterior cruciate
ligament. The ACL
is the second most frequently injured knee ligament: More than 100,000 ruptures
are estimated to occur every year in the United States (40). Although tears of
the medial collateral ligament heal spontaneously in most cases, the ACL has a
low healing capacity (41). To restore the normal knee function after complete
ACL rupture, surgical reconstruction using autograft or allograft tendon is
required (41,42). For ACL replacement with autologous material, both the
bone-patella tendon-bone (BPTB) graft and hamstring tendon grafts represent the
standard choice. Although ACL replacement surgery has been significantly
improved in the last decade, there is still a remaining challenge to improve
and accelerate healing after ACL reconstruction.
Because ligaments can take
up to 3 years to reach full strength (43), the transplanted graft undergoes a
period of weakness, and rehabilitation after ACL reconstruction remains slow.
Even professional athletes are cautioned not to return to competitive sport
until at least 6 months after surgery (see "A Perioperative Rehabilitation
Program for Anterior Cruciate Ligament Surgery," January, page 31).
Recently, several studies
have shown a positive effect of growth factors (platelet-derived growth factor
AB [PDGF-AB], EGF, and bFGF) on ACL fibroblast metabolism (9). The data suggest
that these specific growth factors may improve healing of the ACL or
"ligamentization" of the ACL graft. Gene therapy holds promise for
delivering growth factors to the ligaments (44). The first feasibility studies
have demonstrated that ligaments can be transduced with viral vectors (45). The
next step is the transduction/transfection of the grafts with vectors
expressing appropriate growth factors and cytokines to improve the healing
process. Generally, the viral transduction could be performed directly at the
time of graft implantation or in vitro prior to the ACL reconstruction (graft
preconditioning).
Using the autologous
semitendinosus and/or gracilis tendon for ACL reconstruction, surgeons also
face disadvantages that stem from the tendon-to-bone healing in the femoral and
tibial tunnel, a result that seems inferior to the bone-to-bone fixation of the
BPTB graft. The bone morphogenetic protein type 2 (BMP-2) is one of the growth
factors that could solve this problem and improve the healing of the tendon in
the bone tunnel (46).
Meniscus. Meniscal tears due to twisting or
compression forces are common sports injuries. Several repair techniques,
including sutures, arrows, and staples, have been developed to preserve the
menisci, but only tears in the vascularized peripheral third of the meniscus
can heal (47). Meniscal lesions in the avascular central part do not heal and
present a critical clinical problem because even partial resection of the
meniscus leads over time to cartilage damage and osteoarthritis (48).
Experimental studies have shown that healing in the central meniscus might be
promoted by some chemotactic or mitogenic stimuli delivered by fibrin clot,
synovial tissue, or growth factors (transforming growth factor [TGF] -alpha,
bFGF, EGF, and PDGF-AB) (10,49,50). The goals of gene therapy for meniscal
healing are to transduce central meniscal tears with vectors directly or
transduce indirectly with autologous cells expressing growth factors and
cytokines that stimulate cell proliferation and matrix synthesis in meniscal
fibroblasts and promote efficient healing.
After a complete loss of
the meniscus from an extended injury or repeated resections, rapid impairment
of knee function occurs in most of the patients. Without therapy, osteoarthritis
develops in most patients in 5 to 10 years, faster than would occur as a
consequence of aging (51). The treatment after meniscal depletion is limited.
To find a therapeutic solution, meniscal transplantation (allograft) has been
performed (17). Clinical studies on this issue are rare and report failures of
up to 60% after less than 2 years (52,53). Experimental studies show a slow
immune rejection within the transplanted meniscal allografts (54). Pretreatment
of meniscus allografts with viral vectors expressing growth factors could lead
to acceleration of the graft healing and restructuring, and to suppression of
the immunogenicity.
Bone. Bone has sufficient potential to
heal, and mechanical fixation is an adequate method for healing most fractures;
however, in more than 10% of fractures, delayed unions or non-unions have been
described after trauma (55). The non-union presents a severe problem that has
to be addressed with sophisticated surgical practices (external fixation and/or
bone transplantation, microsurgical tissue or bone transfer, or bone grafting).
Another problem manifested
in sports medicine is stress fracture of the lower extremity, which constitutes
up to 15% of all injuries in runners (56). The treatment is prolonged and the
recovery time often demands 4 to 12 months or more (57).
For treatment of these bone
disorders, biological interventions by application of specific growth factors
to stimulate bone production have shown promising results (58). Recent in vivo
experiments have demonstrated the ability of BMPs, especially BMP-2, IGFs and
TGF-beta, to promote bone healing (table 2) (11). Gene therapy may be able to
deliver the specific gene to the site of repair followed by continuous
production of the desired growth factor (59,60).
|
TABLE
2. Effect of Growth Factors in Musculoskeletal Tissues |
|||||
|
Growth Factor |
Skeletal Muscle |
Hyaline Cartilage |
Meniscus |
Ligament |
Bone |
|
|
|||||
|
IGF-1 |
+ |
+ |
+ |
+ |
+ |
|
|
|||||
|
bFGF |
+ |
+ |
+ |
+ |
+ |
|
|
|||||
|
NGF |
+ |
|
- |
- |
|
|
|
|||||
|
PDGF |
- |
|
- |
+ |
|
|
|
|||||
|
EGF |
- |
+ |
+ |
+ |
|
|
|
|||||
|
TGF-alpha |
- |
|
+ |
- |
|
|
|
|||||
|
TGF-beta |
+ |
+ |
+ |
+ |
+ |
|
|
|||||
|
BMP-2 |
|
+ |
+ |
|
+ |
|
|
|||||
|
+ = positive
effect; - = no or negative effect; blank = not tested |
|||||
|
|
|||||
Muscle-derived gene therapy
could open another important avenue to treat bone defects (26), because muscle tissue
has demonstrated osteogenetic competence in response to osteoinductive stimuli,
and muscle-derived cells have been used to deliver therapeutic proteins in
nonorthopedic diseases (61).
Although gene therapy is
not yet established as an approved therapeutic technique, a great potential
exists for the treatment of musculoskeletal injuries in the future (figure 3).
Currently, only a few effective therapeutic gene therapy techniques have been
tested in human joints (13). At the experimental level, many studies have been
performed successfully to prove the feasibility of gene delivery into different
tissues of the musculoskeletal system. Beyond this stage, initial experimental
studies demonstrated positive effects of transduced genes (especially BMP-2,
IGF-1, TGF-beta in vitro and in vivo. The main obstacle today seems to be the
availability of vectors carrying effective genes, and some concern with the
safety of viral vectors after the death of a patient in a recent gene therapy
trial (62). However, great progress has been noticed in many laboratories
working on the engineering of these vectors.
In general, we believe that
the combination of gene therapy and tissue engineering will help us develop
effective therapies for tissues that has a low healing capacity (ie, cartilage,
meniscus, and ligament) and for other disorders such as osseous nonunion and
arthritis. With these new technologies, however, a large number of basic
science and preclinical studies still need to be performed before the
efficiency necessary for orthopedic applications and guaranteed safety are
reached (63,64).
*A complete reference
list will be available at www.physsportsmed.com beginning in March.
Dr Martinek is a sports medicine fellow in the
department of orthopaedic surgery, and Dr Fu is professor and chair in the
department of orthopaedic surgery at the University of Pittsburgh. Dr Huard is
an assistant professor in the department of orthopeadic surgery and molecular
genetics and biochemistry at the Children's Hospital of Pittsburgh and
University of Pittsburgh. Address correspondence to Johnny Huard, PhD, Dept of
Orthopaedic Surgery and Molecular Genetics and Biochemistry, Children's
Hospital of Pittsburgh and University of Pittsburgh, 3705 Fifth Ave,
Pittsburgh, PA 15213-2583; e-mail to jhuard+@pitt.edu.
___________________________________________________________________________________________________________________________________________
GENE
THERAPY OR DOPING OF THE FUTURE
STURBOIS
Xavier, MAIER Eddie, SCHAMASCH Patrick, CUMMISKEY Joseph
and Prince Alexandre de MERODE
Summary
In
theory everything seems to be in place regarding scientific research for a
probable confrontation in the sporting world between cell therapy and gene
therapy to enhance the performance of athletes. What are the stakes and what
are the means at the disposal of scientists to manipulate the genome? A review
of the situation is necessary.
Key words
Doping,
genome, DNA, gene manipulations, gene therapy, adenovirus, retrovirus and
liposomes.
Introduction
Today,
sport is very important in society with, as a corollary: many different
controversies. Athletes are easily suspected of doping and we must admit that
the virtues of competition are not always respected.
Since
the beginning of time, many doubtful practices have existed and the public
authorities as well as the International Olympic Committee and sports governing
bodies have condemned doping since the 60s: « Use of substances or methods
that are likely to enhance performance and that can be dangerous for health ».
Until today, the concerned substances were essentially clinical products from
the pharmaceutical industry. Nevertheless, the situation may evolve and concern
many biological laboratories that work on cell and gene therapy.
The human genome
The
human genome represents the whole gene message that gives to each individual
his/her own characteristics, thanks to a combination of communicable traits of
the cell and of its descendants. These properties lie in the DNA chromosome or Desoxyribonucleic
Acid and form a double helix of base composed nucleotides
(A=adenine, G=Guanine, T=Thymine and C=Cystosine) linked with hydrogen bonds
(A-G and T-C). The genetic information code is determined by the sequence of
the bases in the chains of nucleotides. This code owes its precision and
complexity to more than three billion base pairs. The human chromosomes (23
pairs) contain the genetic message in the cell nucleus, as each chromosome owns
a fragment of the DNA double helix. The message imposes the assembly order of
the amino acids to create a determined protein such as the enzymes, which are
necessary for the metabolism. The quantity of necessary information to obtain a
specific protein is called “gene”. The chromosomes assemble thousands of genes
that are now almost completely decoded (2002). The genetic information is not
always completely expressed from the origin of the cell line (primary or
differentiated cell) but progressively to acquire differentiation and maturity
(differentiated cell).
The manipulations of the human genome
Many
pathologies are caused by errors in the genetic code. They concern a single or
many genes at the same time. The idea of a possible correction of the
pathological gene comes from the observation of the development of certain
tumours by viral infection. It consists of the injection of viral DNA in
somatic cells. In the same era (1960), we understood the interest of the
viruses as the vectors of a new genetic material. Therefore, the problem was to
find how to allow viruses or other carriers to enter efficiently the desired
somatic cells. Friedmann’s work, who uses the in-vitro manipulations of
reimplanted cells in the organism, marked the beginning of tangible progress
towards an in-vivo approach, where the DNA could be directly introduced in the cells
of the organism. These modifications of the somatic cells do not lead to any
genetic transmission. This would not be the case for germ cells.
If
a gene is absent from the dysfunctional, it will lead to the appearance of
“genetic” diseases or cancers. Some functional disturbances will also appear if
there is the slightest transcription error of the gene.
The
principle of the gene transfer is based on the injection in the cell, of a
“therapeutic” gene into the place of the absent or abnormal gene. The vectors
of the therapeutic gene can be the adenovirus, the retrovirus and the liposome.
The adenovirus (cold) is made innocuous by the withdrawal of its own genetic
material and becomes vector by the injection of the therapeutic gene in its
nucleus. The pathogenicity of the retrovirus, which is smaller than the
previous one, must be depressed before introducing the therapeutic gene in its
DNA. The liposome is a lipid microcapsule in which the repairing gene is
positioned.
The
in vivo introduction of the gene material in the human pathological cells is
effectuated with aerosols or injection. It is also possible to collect somatic
cells and to reimplant them after introducing in vitro the adequate genetic
material. These methods over all concern the viruses and retroviruses. The
liposomes and the outer membrane are of the same nature. They fix themselves on
it and thanks to it; they will leave in the cell the genetic material of which
they are the vectors. Because of their cutaneous application, the liposomes are
part of the cosmetic substances.
In
the cell, the adenovirus acts no matter what biocellular phase. The new genetic
material stands apart from the deficient gene and replaces it as long as the
cell lives. The retrovirus integrates the genetic material of that (genome) of
the deficient cell, mainly during the cell division. This is a real genetic
graft. The action of the liposomes is little known and the issue of the
artificial chromosomes is still in its infancy.
In
theory, we can have the greatest hopes for cancer treatment, for instance, and
of certain pathologies of the osteoarticular system by a cell therapy. However,
we must note that current practice does not enable the successful clinical
exploitation of the efforts of research. Nevertheless, it turns out that the
sport community will probably be very soon confronted with this problem in the
years to come.
Applications of the gene therapy
The
inventory of research was effectuated in September 2001. It currently includes
311 studies against cancer, 52 against genetic diseases, 41 as part of the
cardiac diseases, 36 against AIDS and 3 respectively in the autoimmune, osseous
and neurological matters. Some people think that within 5 years, the sport
community could be concerned with gene therapies, which would aim at increasing
the auto production of EPO, of the growth factors, the muscles (angiogenic
factors of the cardiac and skeletal muscle), the vascular development
(endothelia-vascular growth factor), the analgesic peptides (endorphins,
enkephalins, etc.) and a great number of hormones (hypophysis), etc. Therefore,
the genetically modified man is looming on the horizon of the third millennium.
This phantasmagorical image becomes true when you learn that genetically
modified baboons endogenously produce fifteen times more EPO than the normal.
Screening methods
This
last observation raises the question of the screening methods that could
counter this kind of doping. Of course, some abnormally high concentrations of
hormones that are likely to enhance performance could be an important
incriminating piece of evidence. Moreover, certain vector viruses could leave,
in blood, the trace of an antigen and antibody reaction towards their specific
proteins. The use of chips that are able to analyse quantitatively a lot of
genes will enable to underscore the under or over expression of specific
proteins and to establish, therefore, a suspicion of doping. What emerges is
that in this field also, the part seems to be difficult but not insoluble
thanks to the evolution of certain complementary techniques such as, for
instance, the composed oligonucleotide probes.
Ethical considerations
Once
more, the fight against doping, in view of the current inadequacy of the
screening techniques, will mainly rest on the education in this field and on
the moral promotion of an ethical attitude. Indeed, we must recognise that the
transfer of genetic material, even if the objective is justified, can lead to
important risks for health. Indeed, those risks are caused by possible
abnormalities of the transferred genetic material that can lead to abnormal
gene expressions, which cause diseases. In the medical practice, there is no
question of harming his/her patient, especially when it is not a matter of
treating a sick person. Until now, sport has not been considered as a disease
and the physician has to treat the cause before the effects. Moreover, the
human being must be considered as a combination of coordinated systems and the
deterioration of a single piece of the jigsaw can ruin the harmony of the
combination. Similarly, human testing will have to be rigorously controlled by
the monitoring organisations, in view of the risks that patients are confronted
with, even if they volunteer. Nevertheless, the current testing only aims at
studying a therapy adapted to a pathological situation and strictly
distinguishes itself from any attempt to create an advantage for a non-medical
purpose. Competition must keep on providing a winner on the field and not by
gene manipulation in a laboratory, the dangers of which are evident and not yet
evaluated at their full magnitude. Nevertheless, we will have to accept that an
athlete, who decides to have gene therapy to treat a pathology he would suffer
from and thanks to which he would be cured, participates in the competition.
Conclusion
The
International Olympic Committee, after taking the opinion of six international
independent experts, wants here and now and in the current situation to
announce its recommendations about gene therapy:
- Gene
therapy is admittedly very promising for everyone, and even athletes
who participate in the Olympic Games ;
- The
IOC recognises the validity of the development and application of
gene therapy to prevent and treat the diseases ;
- The
IOC firmly warns against the potential abuse of gene therapy and will establish
as soon as possible any procedure and analytical method necessary to identify
the athletes who would be suitable for the appropriate use of these therapies;
- The
IOC is confident of acquiring the capacity to master the abuses and to set the
ethically acceptable use procedures;
- The
IOC makes a solemn call to each medical and scientific sports governing bodies
to support its stance about gene therapy in sports.
Xavier
STURBOIS is a Professor in the Medical Faculty of the Catholic University in
Louvain and a member of the Medical Commission of the International Olympic
Committee.
Eddie
MAIER, Research DG, European Commission.
Patrick
SCHAMASCH, medical Director of the International Olympic Committee.
Joseph
CUMMISKEY, MD coordinator of the CAFDIS project.
Prince
Alexandre de MERODE, President of the Medical Commission of the International
Olympic Committee.