Transcript DIMENSIONS

Sandro Rusconi
1972-75
1975-79
1979-82
1982-84
1984-86
1987-94
1994-today
1996-02
Fribourg 06.11.2003
SSMI
Colloque Médecins-chefs
Primary school teacher (Locarno, Switzerland)
Graduation in Biology UNI Zuerich, Switzerland
PhD curriculum UNI Zuerich, molecular biology
Research assistant UNI Zuerich
Postdoc UCSF, K Yamamoto, (San Francisco)
Group leader, UNI Zuerich (mol. bio., PD)
Professor Biochemistry UNI Fribourg
Director Swiss National Research Program 37
'Somatic Gene Therapy'
2002-today Swiss Natl. Res. Program 50
'Endocrine disruptors'
2002-03
Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department
2002-05
President Union of Swiss Societies for
Experimental Biology (USGEB)
UNIFR
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Doping sportif
avec transfert de
gènes:
mythe ou réalité?
Schedule
Basic understanding of 'genes':
what is a gene, how many genes, molecular biology dogma
genetic diseases, environmental factors, ageing
Essential concepts on 'molecular medicine' & molecular doping:
applications and problems,
Techniques of gene transfer (Gene Therapy)
problems and solutions, vectors, clinical achievements
Gene-based doping
applications, comparison with other doping, detection
Conclusions
plausibility table
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1 gène-> 1 ou plusieures fonctions
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DNA
RNA(s)
Protein(s)
Transcription / translation
Gene expression
GENE
2-5 FUNCTIONS
100 ’000 genes
(50 ’000 genes?)
>300 ’000 functions
(>150 ’000 functions)
Un gène c'est quoi? une nanomachine pour la production de UNIFR
Rusconi
RNA gravée sur un segment de DNA
2003
DNA
GENE
RNA
Protein
Transcription / translation
FUNCTION
Ergo
 pour un transfert génique efficace
il faut transférér aussi les
RNA
séquences de régulation apprpriées
DNA
spacer
regulatory
coding
spacer
UNIFR
1 Organisme -> 1013 cellules,
spécialisées et organisées en tissus et organes
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2003
2 mm
2m
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
1 Cm3 de tissu
 1'000'000'000 cellules !
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Le paradigme réductionniste du biologiste moléculaire
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DNA
GENE
GENE OK
GENE KO
GENE transfer
Protein
FUNCTION(s)
le transfer génique impliquera:
 transfert
d'une fonction
FUNCTION
OK nouvelle , ou
 transfert d'une fonction compensatrice , ou
 transfert d'une fonction qui interfère
FUNCTION KO
FUNCTION transfer
L'état de santé/maladie ou des différences individuelles
ne dépend pas seulement du génome
genetics
Muscle distrophy
Familial Breast Cancer
Sporadic Breast Cancer
Lung Cancer
Obesity
Artherosclerosis
also acquired conditions
may have a genetic component
that modulates their healing/improvement
Alzheimer
 trauma
Parkinson ’s
 fractures
 burns
Drug Use/Abuse
 infections
Sexual orientation
behaviour
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environment
Les manipulations de base pour obtenir de DNA
récombinant sont relativement simples
segments of genomic DNA can be specifically cut and isolated
isolated segment can be recombined with a plasmid vector
Science-grade material
can be essentially prepared in your cellar
plasmid vector is transferred into bacteria where it can multiply
...not so clinical-grade material!
isolated recombinant DNA can be further recombined to obtain
the final desired molecule
Final molecule is transferred into cells or organisms
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Les quatres ères de la médecine moléculaire
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Eighties
Genes as probes
Nineties
Genes as factories
Y2K
Genes as drugs
1 2 3 4 5
ok ** ok ** **
50
10
3000
80 85 90 95 99
1000technologies
Y2K+n Post-genomic improvements
of former
80 85 90 95 00
voilà que l'on est prêts pour parler de
thérapie génique somatique
Definition of GT:
'Use genes as drugs':
Correcting disorders by
somatic gene transfer
NFP37 somatic gene therapy
www.unifr.ch/nfp37
Chronic treatment
Acute treatment
Preventive treatment
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
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Pouquoi 'somatique'?
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
Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise
to a descendant organism
Ergo:
le transfert génique somatique courant
cherche à éviter les cellules germinales
il ne porte pas à une trasmission héréditaire de
la modification

Somatic Cells: all the other cells of the body
Les quatre questions fondamentales
dans le transfert génique somatique
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
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Remember!
les questions pharmacologiques:
différent niveau de réversibilité
Classical Drugs







Mw 50- 500 Daltons
Synthetically prepared
Rapid diffusion/action
Oral delivery possible
Cellular delivery:
- act at cell surface
- permeate cell membrane
- imported through channels
Can be delivered as
soluble molecules
Ångstrom/nm size
rapidly reversible treatment
Protein Drugs







Mw 20 ’000- 100 ’000 Da
Biologically prepared
Slower diffusion/action
Oral delivery not possible
Cellular delivery:
- act extracellularly
Can be delivered as
soluble molecules
nm size
rapidly reversible treatment
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Nucleic Acids







Mw N x 1’000’000 Da
Biologically prepared
Slow diffusion
Oral delivery inconceivable
Cellular delivery:
- no membrane translocation
- no nuclear translocation
- no biological import
Must be delivered as
complex carrier particles
50-200 nm size
slowly or not reversible
Thérapie avec DNA:
 requère une formulation en particules
 est plus complexe que l'administration d'autres médicaments
 a un niveau inférieur de réversibilité
Trois classes de transfert anatomique
Ex-vivo
In-vivo
topical delivery
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In-vivo
systemic delivery
V
Examples:
- bone marrow
- liver cells
- skin cells
Examples:
- brain
- muscle
- eye
- joints
- tumors
Examples:
- intravenous
- intra-arterial
- intra-peritoneal
Deux classes de 'vecteurs': non-virales et virales
Non-viral transfer
(transfection)
Viral gene transfer
(Infection)
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a
b
Nuclear envelope barrier!
see, Nature Biotech
December 2001
Transfection (transfert non-viral)
comparée à infection (transfert viral)
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Transfection
exposed to
106 particles/cell
12 hours
Infection
exposed to
3 particle/cell
30 min
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Popular vectors for gene transfer:
r-Adenovirus (30 kb)
Naked r-plasmid DNA
r-Adeno-associated V. ( 4 kb)
Liposomes & Coformulated r-plasmid DNA
r- Retrovirus (incl. HIV)(10 Kb)
Oligonucleotides
Recap: current limitations of
popular gene transfer vectors
Adenovirus
- no persistence
- limited packaging
- toxicity
- immunogenicity
Retrovirus (incl. HIV)
- limited package
- random insertion
- unstable genome
General
- antibody response
- limited packaging
- gene silencing
Solutions:
- synthetic viruses
(“Virosomes”)
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Biolistic bombardment
or local direct injection
- limited area
Electroporation
- limited organ access
Liposomes, gene correction & Co.
- very inefficient transfer
General
- low transfer efficiency
1/10’000 of viruses’ in vivo
Solutions:
- improved liposomes
with viral properties (“Virosomes”)
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Thérapie génique dans la clinique
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trials
patients
As of Sept. 2003:
100
80
610 registered protocols
1500
~4000 treated patients
cancer
60
hered.
40
86% phase I
13% phase II
1 % phase III
500
vasc.
21% overall still pending
Infect.
or not yet Initiated !
20
www.wiley.com
1990 1992
1000
1994
1996
1998
2000
Faits marquants: anecdotiques mais importants
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Anderson, 1990
1990, 1993, 2000 // ADA deficiency
Isner, 1998
Dzau,
1999
F Anderson, M Blaese // C Bordignon
Kmiec, 1999
Fischer, 2000
1997, 2000, Critical limb ischemia
Dickson, 2000
J Isner († 4.11.2001), I Baumgartner, Circulation 1998
Aebischer, 2000
Kirn, 2001
1998, Restenosis
V Dzau, HGT 1998
1999, Crigler Njiar (animal)
C Steer, PNAS 1999
Clinical trials with ONYX-015,
2000, Hemophilia
what we learned?
M Kay, K High
2000, SCID
(Review)
A Fischer, Science April 2000
Bordignon, 2000 (ESGT, Stockholm)
2000, correction Apo E4 (animal model) proves efficacy of the same protocol
G. Dickson, ESGT congress, 7.10.2000 Stockholm
2000, correction Parkinson (animal model)
P Aebischer, Science, Nov 2000
2001, ONYX oncolytic Viruses
D Kirn (Gene Ther 8, p 89-98)
Les effets secondaires plus redoutés
dans le transfert génique somatique

Immune response to vector

immune response to new or foreign gene product

General toxicity of viral vectors

Adventitious contaminants in recombinant viruses

Random integration in genome
-> insertional mutagenesis (-> cancer risk)

side effects of newly acquired gene product

Contamination of germ line cells
Ergo
 beaucoup de ces effets sont liés au niveau prototypique
de la tecnologie de transfert génique
 couramment les risques associés limitent l'indication de
la TGS pour de maladies graves
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Quatre leçons amàres mais un seul cas de décès causé
directement par la Thérapie génique
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NY May 5, 1995, R. Crystal:
in a trial with adenovirus mediated gene transfer to treat cystic fibrosis
(lung) one patient developed a mild pneumonia-like condition and
recovered in two weeks. The trial interrupted and many others on hold.
UPenn, Sept. 19, 1999, J. Wilson:
in a trial with adenovirus mediated gene transfer to treat OTC deficiency
(liver) one patient (Jesse Gelsinger) died of a severe septic shock. Many
trials were put on hold for several months (years).
Paris, Oct 2, 2002, A Fischer:
in a trial with retrovirus mediated gene transfer to treat SCID (bone
marrow) one patient developed a leukemia-like condition. The trial has
been suspended to clarify the issue of insertional mutagenesis, and some
trials in US and Germany have been put on hold.
!! Most Recent
Paris, Jan 14, 2003, A Fischer:
Paris' Trial News
www.unifr.ch/nfp37/adverse.html
a second patient of the cohort of 9 comes up with a similar disease than
the one reported in october 2002. 30 trials in USA are temporarily
suspended
les hauts et les bas dans la thérapie génique:
comme les montagnes russes
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A. Fischer
M. Kay
high
lentivectors
in clinics?
R. Crystal
V.Dzau
Adeno I
C Bordignon
J. Isner
ADA
mood
NIH
Motulski
report
Ergo

Low
90
chaque fois qu'on était
convaincu avoir atteint une
'vitesse de croisière', un
évènement négatif a baissé les
enthousiasmes
91
92
93
94
95
AAV
germline
in mice?
Adeno III
Lentivectors
in pre-clinic
J. Wilson
J. Gelsinger
96
97
98
99
00
01
Adverse
events in
Paris
02 03
Finalement, doping 'génique':... possible?
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vu les difficultés de la thérapie génique on pourrait imaginer
qu'il est bien trop tôt pour se précoccuper des applications
possibles au doping sportif, cependant...
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Les trois niveaux de doping
+
Before the
competition
(anabolic enhancers)
'Molecular treatments
Application of the
know-how in
molecular genetics
to doping
+
During the competition
(performance enhancers)
+
After the
competition
(repair enhancers)
Quels transfert géniques seraient
envisageables pour des stratégies de dopage

ex vivo, hematopoietic tissue:
pro hematopoietic (Epo receptor, oxygen transport...)

in vivo local (example muscle):
metabolic enhancers, growth factors,
muscular fiber changers, cardio-modulators
(glucose/oxygen, MGF, IGF, anti-myostatin,...)

in vivo local (example joints):
pain reducers, inflammation inhibitors, recovery and
repair factors (anti-TNF, BMPs, ...)

in vivo systemic:
anabolic enhancers, endocrine factors, pain killers,
vascular controllers, (hormone metabolising
enzymes, proenkephalins, ...)
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Quels effets secondaires pourrait-on craindre lors de
dopage avec transfert génique?
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Short -mid term



Autoimmunity
Hyperimmunity
Toxic shock
Long term
dangers d'une attitude 'casse-cou'
 Fibrosis
 malpractice (unsuitable
vector/administration route)
 Cancer
 non-clinical grade material
 conventional side- effects of
administered factors
(pathogens or allergens)
 Inaccessibility to future gene
 lack of follow-up
therapy interventions (immunity)
Quelles seraient les limitations objectives dans des
stratégies de dopage avec transfert génique
Viral gene transfer
 immune problems
 limited readministration possibilities
 general toxicity, genotoxicity
Nonviral gene transfer
 generally inefficient
 lack of persistence, requires readministration
Strategy-independent problems
 laborious, not readily available
 long term gene expression difficult to control
 irreversible effects or permanent tagging
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Méthodes de détection plausibles
pour le dopage avec transfert génique
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
Antibody detection (viral antigens)

r-nucleic acids detection (PCR)

recombinant protein / post-translational
modification detection (MALDI-TOF )

Anatomically difficult to detect
Ergo
(if locally administered)
TG pas toujours analysable dans
-> but leaves permanent genetic marking
le sang ou l'urine, mais identifiable

indéniablement dans de biopsies
Detection of nucleic acids cannot be performed
in body fluids
même longtemps
après le traitement
(except in early phase after systemic administration)
-> might require specific tissue biopsy
Comparaison directe des 'avantages' / 'désavantages':
dopage 'génique' versus dopage conventionnel
Category
Ergo:
Drug/protein
Gene-based
Les chances parlent objectivement plutôt en défaveur
Rapidity
of effectsavec rapid
du dopage
transfert génique slow
mais:
Reversibility
rapid
slow
ces considérations se basent sur le bon sens, qui
semble être très straightforward
carent dans le secteur
du dopage
Dosage
difficult
et aussi:
simple
complex
...il y a plusieurs disciplines sportives (ou niveaux de
sport) dans lesquelles le dopage n'est pas vérifié
Associated risks
depends
high
le liaisons dangereuses:
... professionnelspossible
du dopage qui sedifficult
mettent
en
Concealability
/impossible
contact avec des chercheurs sans scrupules
Complexity of treatm.
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Le transfert génique dans le dopage:
conclusions





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somatic gene transfer has been originally developed for
the treatment of diseases (genetical orergo:
acquired)
le dopage
ne serait
has the potential to be applied for pre- duringandgénétique
postni plus dangereux ni plus moralement
performance enhancement
condannable du dopage conventionnel
currently dedicated to theapy, still experimental and not
mature for applications in non-serious conditions
 le danger majeur n'est pas
major risk in doping linked with improperleapplication
dopage génique en soi
(GCP non-compliant or non-clinical-grade
materials)
mais
l'ambition humaine incontrôlée
qui porte à répéter les même erreurs !
Has already raised the interest in the doping field

In spite of premature status is likely to be attempted
in the next years (perhaps already?)

the CIO and the WADA have already officially
included gene-based doping in the list of banned
practices (ww.wada.org) starting 1.1.2003
QuickTime™ et un décompresseur
None sont requis pour visualiser
cette image.
... MERCI et espérons que le sport puisse
rester un spectacle d'émotions et de fairplay
SSMI, Prof C. Regamey
My collaborators at UNIFR
Swiss National Research Foundation
NFP37
if you are too shy to ask
send an e-mail to:
[email protected]
or visit:
www.unifr.ch/nfp37
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2003
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2002