Post on 17-Apr-2020
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Hormona del Crecimiento y
Dopaje Genético
Jordi SeguraLaboratorio Acreditado AMA, IMIM-Hospital del Mar,
Parque de Investigación Biomédica PRBB, Barcelona;
IOC Medical Commission, Games Group, Lausanne, IOC
• La llegada de Técnicas recombinantes ha revolucionado el abanico de sustancias dopantes
• Ha permitido el uso de proteínas y hormonas para curar enfermedades pero también su abuso en el deporte: EPO, hGH, LH, etc
• Ha propiciado el desarrollo de la Terapia Genética, pero al mismo tiempo la amenaza de su uso fraudulento en el ámbito deportivo
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Detección de substancias prohibidas:
1. Substancias exógenas: - Relativamente fácil, ya que se detectan compuestos
que el propio cuerpo no produce:
2. Substancias de estructura igual a las endógenas:- Mayor complicación por la necesidad de distinguir
el origen endógeno del exógeno
Control antidopaje – Dificultades
Detección de substancias de estructura igual a las endógenas:
Marcadores directos:
- Diferencias químicas sutiles entre la droga administrada y la hormona natural producida por el organismo
- Difícil obtener claros marcadores directos
Marcadores indirectos:
- Efectos fisiológicos
- Estudios poblacionales: probabilidad
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The World Anti-Doping CodeThe 2008 Prohibited List
PROHIBITEDSUBSTANCES• S1. Anabolic Agents • S2. Hormones and Related
Substances• S3. Beta-2 Agonists • S4. Hormones
anatagonists and modulators
• S5. Diuretics and other masking agents
• S6. Stimulants• S7. Narcotics• S8. Cannabinoids• S9. Glucocorticosteroids
PROHIBITED METHODS• M1. Enhancement of
oxygen transfer• M2. Chemical and physical
manipulation• M3. Gene Doping
SUBSTANCESPROHIBITED INPARTICULAR SPORTS• P1. Alcohol• P2. Beta blockers
S2. HORMONES AND RELATED SUBSTANCES (I)
The following substances, including other substances with a similar chemical structure or similar biological effects, and their releasing factors, are prohibited:
1. Erytropoietin (EPO)2. Growth hormone (hGH), Insulin-like Growth Factors
(e.g. IGF-1), Mecano Growth Factors (MGFs)3. Gonadotrophins (e.g. LH, hCG), prohibited in males
only4. Insulin5. Corticotrophins
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Detección indirecta de la GH
Proyecto GH 2000/4
Metabolismo hepático:• IGF-I• IGFBP-2• IGFBP-3• ALS
Metabolismo óseo:• Osteocalcina• P-III-P• PICP• ICTP
De entre ellos se buscaron marcadores sensibles al tratamiento con rhGH, pero relativamente inalterables con el tiempo o en respuesta al ejercicio.
Detección indirecta de la GH
-3 -2 -1 0 1 2 3 4 5 6 7
EM1_scal
Fig 5. Dotplots of scaled derived marker EM1, by day
d a y
0
21
28
30
33
42
84
Modelos obtenidos para la detección del abuso de rhGH
EM1=-2.269+0.7270·ln[P-III-P]+0.521·ln[IGF-I]
5
0
100
200
300
400
500
10 20 30 40
Age (years)
[IG
F-I]
(ng/
ml)
N=177
0
5
10
15
20
10 20 30 40
Age (years)[P
-III
-P] (
ng/m
l)
N=173
Effect of Age on GH-Responsive Markers
edad737.1011EM031.4560.2b1EM −⋅+=
Deportistas del CAR S.Cugat
• Implementación de la detección indirecta de hGH pendiente
• Diferentes métodos ofrecen diferentes resultados, aunque existen coeficientes de comparación
• Se busca reactivos de aplicación universal• Dependencia de la edad• Serán útiles para desarrollar un control
“inteligente”
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Detección de substáncias de estructura igual a las endógenas:
Marcadores directos:
- Diferencias químicas sutiles entre la droga administrada y la hormona natural producida por el organismo
- Difícil obtener claros marcadores directos
Marcadores indirectos:
- Efectos fisiológicos
- Estudios poblacionales: probabilidad
Gene Expression of GH Isoforms
transcription46
hGH mRNAimmature
-26-24 -23 31 32 71 72 126 127 191
splicing
22K-GH mRNAmature
20K-GH mRNAmature
1 2 3 4 5
1 2 3 4 5alternative splicing
hGH-N gene5‘ 3‘
Exon I II III IV V
B C DIntron
AB’
- 50Kb
5‘hGH-N hPL-1 hPL-4 hGH-V hPL-3 enhancerhGH gene cluster
Chromosome 17 (17q22-24)
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22K-GH vs 20K-GH
GH
HRP
anti-20K-GH
anti-GH
GH
HRP
anti-22K-GH
anti-GH
- Secreción normal de hGH:
-Nivel de fondo bajovariable- Picos intermitentesde gran magnitud de ambas isoformas
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22kDA hGH and 20kDa hGH following injection of0.2 IU/kg rhGH
Leung et al, Am. J. Physiol Endo Metabs, 283:836-843
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Human growth hormoneHuman growth hormone
191 aa – 22 kDa ~ 90 %176 aa – 20 kDa ~ 10 %
43 aa – 5 kDa (sequence 1-43)1
148 aa – 17 kDa (sequence 44-191)2
191 aa ? – 24 kDa (glycosylated)3
191 aa ? – 34 kDa (glycosylated)4
? – 12 kDa (glycosylated)4
Nα-acetylated formDeamidated form: Asp137/Glu152
Monomers, Dimers, Oligomers, Complexes
hGHhGH--N gene N gene –– pituitary glandpituitary gland
Immune cells Immune cells
Pituitary gland Pituitary gland
hGHhGH--V geneV gene
SyncytiotrophoblastSyncytiotrophoblastPlacenta Placenta
191 aa 22 kDa1 J.Immunol.Meth. 1998, 215, 1792 Trends Endocrinol.Metab. 1992, 3, 1173 Biochem.Biophys.Res.Commun. 1996, 228, 5494 Horm.Res. 2000, 53, 40
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HUMAN GROWTH HORMONE –Wu, Bidlingmaier & Strasburger, Munich University Hospital
rhGH InjectionOne isoform only:22kDA
Natural hGH SecretionMultiple isoforms:22kDa, 20kDa, 17kDa and others
Basis of a rhGH Doping Test1. Natural hGH is a mixture of types (isoforms), named for their relative sizes2. rhGH has only one GH isoform (22kDa)3. After injection of rhGH, the 22kDa isoformdominates in blood, and the other forms are missing. 4. Develop two GH tests:
Test 1: Pituitary GH Test (PitGH)Measures all the forms of GH made by the pituitary gland. Detects monomeric 22kDa but weakly
Test 2: Recombinant GH Test (RecGH)Measures only 22kDa
Doping or Not?Calculate the ratio GH test 2 (RecGH)
GH test 1 (PitGH)An increased ratio indicates dopingwith recombinant human hGH
Método directo para hGH• Método empleado en los Juegos Olímpicos de
Beijing• Empieza a ser utilizado de manera general• Limitaciones:
– Necesario recoger sangre– Corta ventana temporal de detección– Importante controles por sorpresa fuera de
competición• No se han detectado resultados positivos hasta
ahora
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Pero estas sustancias pueden también llegar a ser producidas “in vivo”
mediante la inserción de genes apropiados
Alerta sobre Dopaje Genético
• 2001: Gene Therapy Working Group, convened by the IOC-MC meeting:”Genetherapy and its future impact on sports”:
• Inclusion List, January 2003
WADA position
“the non-therapeutic use of cells, genes, genetic elements, or the modulation of gene expresion, having the capacity to enhance athletic performance, is prohibited in sport”
Probably not yet an actual problem. Gene therapy still mainly in clinical trials, but…
…what to do towards London Olympics?
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Degrees of potential geneticinterevention
• Genes and sports– Roger Bannister, first men to run one mile under 4 min.:
“Athletes are not born equal”• West African: short distance• East African: marathon• Caucasians; swimming
• Genetic screening: childs evolve top athletes• Genetic manipulation: gain genetic predisposition
• Marion Jones, Tim Montgomeny: child, genetic advantage?• Steffi Graf, Andre Agassi: child, genetic advantage?
• Gene doping: Gain advantatge through gene transfer
The World Anti-Doping CodeThe 2008 Prohibited List
PROHIBITEDSUBSTANCES• S1. Anabolic Agents • S2. Hormones and Related
Substances• S3. Beta-2 Agonists • S4. Hormones
anatagonists and modulators
• S5. Diuretics and other masking agents
• S6. Stimulants• S7. Narcotics• S8. Cannabinoids• S9. Glucocorticosteroids
PROHIBITED METHODS• M1. Enhancement of
oxygen transfer• M2. Chemical and physical
manipulation• M3. Gene Doping
SUBSTANCESPROHIBITED INPARTICULAR SPORTS• P1. Alcohol• P2. Beta blockers
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• Gene Therapy– 3000 patients, some (few) side effects. Trials
on going.• severe combined immunodeficiency disease• adenosine deaminase deficiency • haemophilia B• …..
– Few registered products so far: Vitraene(antisense technology); tumor supress gene p53 (reduce tumor growth; China); …
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Sport authorities Concerns
• 2001: Gene Therapy Working Group, convened by the IOC-MC meeting:”Genetherapy and its future impact on sports”: Inclusion List, January 2003
• 2004 / 2005 WADA Conferences: • 1st Conference at Banbury• 2nd Conference in Stockholm
Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
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Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
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Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
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Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
Stockholm declaration• Gene therapy now represents a proven, although very immature and still
experimental field of human medicine.
• Clinical research in human gene therapy is filled with many recognized andunrecognized pitfalls and dangers.
• The participation of physicians in gene transfer procedures that are not fullycompliant with standards of human clinical research should be consideredmedical malpractice.
• Greater interactions should be encouraged to stimulate awareness of thepotential illicit use of gene transfer techniques .
• New detection methods are likely to emerge and will help to prevent tainting ofsport by gene doping. Research programs should be supported.
• The use of genetic information to select for or discriminate against athletesshould be strongly discouraged.
• Sports organizations should promote knowledge about the potential dangersassociated with the misuse of genetic manipulations.
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Potential Sport Targets
• GH / IGF-1 Promote muscle mass• Myostatin (negative regulator of muscle
formation). Myostatin blockers increase skeletal muscle.
• EPO Gene therapy with EPO increases haematocrit in animals more than 80%
• VEGF (Vascular endolthelial growth factor): Increase blood supply
• Endorphins (for pain)
Laboratory mice transferred with IGF-1 gene at the University of Pennsylvania seem to holdthe “beneficial” effects of IGF-1 throughouttheir lives.
Growth Hormone / IGF-1
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A german boy had mutation with lack of myostatin production
(Schuelke M, N Eng J Med 2004, 350: 2682-2688)
Transgenic rats
In 1997, scientistsMcPherron and Lee revealed to thepublic the ‘secret’of an anomaly thatlivestock breedershave capitalizedsince the late 1800’s: the gene responsible for bigbeefy cows .
Myostatin inhibition
CMV mEpo SV40 Neo
Scheme of the mEpo vectorused in our animal gene transfer studies
Genes expressing rEPO
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Awaiting for gene transfer Anaesthesia Tibialis muscle exposed
Muscle width measurementPlasmid injection Applying a conductive gel
Setting electrical parameters Electroporation Work done!
Hematocrit
50
55
60
65
70
75
80
85
90
2 8 14
Time (days)
HC
T%
Control Transfected
0
2000
4000
6000
8000
10000
12000
14000
0 2 4 6 8 10 12 14 16
Time (days)
[EP
O] m
U/m
l
Serum EPO
Effects after gene transfer to mice muscle
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Monkeys injected with a virus carrying the gene for EPO
Gene therapy with EPO
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Possible strategies for detectiongene doping
Screening• Detect plasmid vector sequences and immune response to viral
vectors• Differentiate transgenic DNA from genomic DNA• Microarrays to detect changes in gene expression• Proteomics to detect changes in gene expression• Indirect physiological models• Different isoforms (promising for EPO)• Others
Possible strategies for detectiongene doping
Screening• Detect plasmid vector sequences and Iimune response to viral
vectors• Differentiate transgenic DNA from genomic DNA• Microarrays to detect changes in gene expression• Proteomics to detect changes in gene expression• DNA bar codes (difficult, it depends on multiple parties)• Indirect physiological models• Different isoforms (promising for EPO)• Others
Confirmation• Confirmation by non invasive imaging detection of
unexpected expression in an ectopic tissue
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IMAGENENon invasive molecular imaging of gene expression useful for doping control: Pilot study in animals after erythropoietin gene transfer
You may say I’m a dreamer, but I’m not the only oneImagine, John Lennon, 1974RESEARCH HYPOTHESIS AND OBJECTIVES
- The gene transfer processes may produce the expression of mRNA for the target hormone-protein in unusual cells or tissues.
Cap
AAA AAA
Cap
- mRNA molecules will hybridize with suitable antisense modified oligonucleotides available to the tissue expressing the ectopic hormone-protein.
- If a label of appropriate energy is associated to the modified oligonucleotides, detection of the unusual hybridization may be carried out non-invasively in real-time by suitable imaging technologies.
Labeled TAT-PNA preparation Quality control
Anaesthesia Preparing administration I Preparing administration II
Administration Image adquisition I Image adquisition II
Chromatograms
Imaging
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Final Aim : Sensitivity and Resolution of PositronEmission Tomography (PET) or Single Photon EmissionComputerized Tomography (SPECT)
PETfacility
Micro PET facility
CONCLUSIONES
- La detección óptima de la administración de hormona de crecimiento debería integrar marcadores directos e indirectos
- El Dopaje Genético en el deporte está prohibido por razones éticas, tanto médicas como deportivas, y de prevención de riesgos.
- Debe incrementasre el apoyo a la investigación para el desarrollo de métodos de detección del dopaje genético en el deporte, a ser posible de aplicación relativamente simple.
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