Transcript DIMENSIONS

Sandro Rusconi (9.3.52)
Lausanne 21.09.2005
AISTS 'genes and sport'
1972-75
School teacher (Locarno, Switzerland)
1975-79
Graduation in Biology UNI Zuerich, Switzerland
1979-82
PhD curriculum UNI Zuerich, molecular biology
1982-84
Research assistant UNI Zuerich
1984-86
Postdoc UCSF, K Yamamoto, (San Francisco)
1987-93
Principal Investigator, UNI Zuerich, PD
1994-today
Professor Biochemistry UNI Fribourg
1996-2002
Director Swiss National Research Program 37
'Somatic Gene Therapy'
2002-03
Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department
2002-05
President Union of Swiss Societies for
Experimental Biology (USGEB)
2002-06
Euregenethy Network (EU-harmonsiation of
biosafety and ethical aspects in gene therapy)
2005-xx
Director of Governmental Division for Culture
and University Affairs of Canton Ticino
UNIFR
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2005
'Gene doping':
Is it coming?
Is it there?
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Issues that will be addressed
2005
What is a 'gene'?
1 gene -> several fiunctions, genes language, gene expression,
gene manipulation
What is therapeutic gene transfer (gene therapy)?
functions transfer, objectiives, somatic
How far has gene therapy progressed ?
principles, goals, obstacles, clinical achievements
Which possibilities exist gor Gene-based Doping ?
doping in general, gene doping perspectives, obstacles,
detectability, side effects, risk-benefits
Conclusions
technically too early but recklessness and money
make it moving anyway
Quintessence
read it in this
orange box
Doping in top sports has some deadly aspects...
Name
Sport
Age
Date
Cause
Denis Zanette (Ita)
Fabrice Salanson (Fra)
Vivien Foe (Cam)
Jose Maria Jimenez(Spa)
Miklos Feher (Hun)
Raymond Junikis
Johan Sermon
Marco Pantani
???
cyclist
cyclist
football
ciclist
football
basket
cyclist
cyclist
...
33
23
28
32
24
23
21
28
...
10.01.2003
03.06.2003
26.06.2003
8.12.2003
25.01.2004
10.02.2004
13.02.1004
14,02.2004
...
cardiac arrest dentist room
cardiac arrest hotel room
cardiac arrest on the field
cardiac arrest hospital
cardiac arrest on the field
cardiac arrest at the match
cardiac arrest at home
cardiac arrest hotel room
...
Questions
 what is the perception of risk/benefit in sports?
 Can all this worsen when gene transfer enters the scene?
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2005
Mythos 'Gene': in the good and in the bad ...
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Medicine
Neue Medikamente, neue Heilungschancen,
neue Diagostik, ...
Myths 'Gene therapy':
- against hereditaryMedicine
diseases
Bio-Waffen, Monster-Generation,
- transmissible modification
Designer-babies, ...
Agriculture
Neue Eigenschaften von Nutzpflanzen/
Myths 'Gene doping':
Nutztieren ...
- better than conventional
- hereditary geneticAgriculture
modification
Oekologische Katastrophen,
- pre-natal designerGesundheitspropleme,
athletes
«GMO=Giftig»
Ergo:
we must first eliminate these myths
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1013
1 Organism ->
Cells,
distributed in specialised organs and tissues
2 mm
2m
2005
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
Ergo


at each cell division the genetic
marterial is copied
in 1 cm3 -> 1'000'000'000 cells
The old motto: 'one gene one function' is obsolete...
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2005
DNA
RNA(s)
Protein(s)
Transcription / translation
Gene expression
GENE
100 ’000 genes
(50 ’000 genes?)
Ergo:
2-5 FUNCTIONS
side effects in gene transfer might be
caused also by supplementary and
yet undiscovered functions of a given gene
>300 ’000 functions
(>150 ’000 functions)
But ...what is actually 'a gene'?:
...a regulated nano-machine for the production of RNA
DNA
GENE
RNA
Protein
Therefore,
to fullfil its FUNCTION
role,
Transcription
/ translation




a transferred gene segment must include:
regulatory sequences for Transcription
proper signals for RNA Maturation/transport
proper signals for mRNA Translation
proper signals for mRNA Degradation
RNA
DNA
spacer
regulatory
coding
spacer
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2005
The reductionist Paradigm of molecular biologists
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2005
DNA
GENE
GENE OK
Protein
Gene transfer can imply:
FUNCTION(s)
 Transfer of a new Function, or
 Transfer of a compensatory F., or
 Transfer
einer interfering
FUNCTION
OK Function
GENE KO
FUNCTION KO
GENE transfer
FUNCTION transfer
Gene transfer as logical consequence: the third era of
molecular biology
Eighties
Genes as probes
Nineties
Genes as factories
Y2K
Genes as drugs
1 2 3 4 5
ok ** ok ** **
50
3000
10
80 85 90 95 99

1000
80 85 90 95 00
Gene transfer (Gene therapy):
logical consequence of development in molecular biology
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Somatic Gene therapy (SGT)
Definition and applications
2005
Chronic treatment
Definition of SGT:
'Use genes as drugs':
Correcting disorders by
somatic gene transfer
NFP37 somatic gene therapy
www.unifr.ch/nfp37
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Acute treatment
Preventive treatment
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
Pharmacological considerations,
differences with conventional medication therapy
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


2005
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
Can be delivered as
 Must be delivered as
soluble molecules
complex carrier particles
nm size
50-200 nm size
rapidly reversible treatment slowly or not reversible
Theray with nucleic acids (DNA)

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Need special formulation (vectors)
more complex
less reversible

Why 'somatic'?
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2003

Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise
to a descendant organism

Ergo:
somatic gene transfer is
NOT aiming at GERM LINE cells
Somatic Cells: all the The
other cells
of the body is therefore
modification
NOT HERITABLE
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The four technical basic questions in SGT
2005
2003
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
The variables
 which disease?
 which gene?
 which vector?
 which target organ?
 which type of delivery?
Remember!
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Three anatomical delivery methods in SGT:
2005
Ex-vivo
In-vivo
topical delivery
Ergo


In-vivo
systemic delivery
ex vivo or local delivery are
currently preferred over
systemic delivery
V
Examples:
- bone marrow
- liver cells
- skin cells
Examples:
- brain
- muscle
- eye
- joints
- tumors
Examples:
- intravenous
- intra-arterial
- intra-peritoneal
Two classes of vectors:
viral / non viral
Transfert non viral
(transfection)
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A
Why are viruses 'better'?
viral transfer
(Infection)
direct nuclear shuttling!

viral transfer is much more efficient

nonviral transfer must solve a
B
number
of hurdles
- serum protection/stability
- target docking
Nuclear envelope barrier!
- endosomal escape
- nuclear trafficking
- genomic integration
- anti apoptotic functions
- immunological camouflage
- ...
Example: transfection (non viral) versus Infection (viral)
transfer of a reporter gene
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Transfection
cells exposed to
1'000'000 particles/cell
12 hours
Infection
cells exposed to
3 particle/cell
30 min
Ergo
 virally mediated gene transfer is millions of times more efficent than nonviral
transfer (when calculated in terms of transfer/particle)
Mini- List von current gene transfer methods
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r-Adenovirus
Naked DNA
r- Adeno-associated V.
Liposomes & Co.
r- Retrovirus
Oligonucleotides
r- Lentivirus
Recap: Limitations of current vectors
r-Adenovirus
- no persistence
- limited packaging
- toxicity, immunogenicity
r-AAV
- no integration in host g.
- very limited packaging
- autoimmunity?
r-Retrovirus (incl. HIV)
- limited packaging
- random insertion
- unstable genome
General
- antibody response
- limited packaging
- gene silencing
- Manufacturing limitations
Solutions:
- synthetic viruses
(“Virosomes”)
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2005
Biolistic bombardment
or local direct injection
- limited area
Electroporation
- limited organ access
Liposomes, gene correction & Co.
- rather inefficient transfer
General
- low transfer efficiency
- no or little genomic integration
Ergo
Solutions: see an increasing
 the future will probably
- improved liposomes
interest in viral-like,
but
artificial
particles
with viral
properties
(“Virosomes”)
The traditional clinical path:
lots of time/money
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2005
year
event
costs U$D
0
Idea
0
2
Cell culture assays
0.5 Mio
5
Pre-clinical tests
animal models
2 Mio
7
10
15
16>>
Clinical phase I
5-20 patients
verify side effects
6 Mio
Clinical phase II
30-100 patients
dosis escalation
12 Mio
Clinical Phase III
>300- 1000 patients
multicentric
double blind
80 Mio
Registration / Availability
Fazit:
on average only 1out of 5 drugs
makes it to approval
->
500 Mio U$D investment
per successful drug !!!
Gene therapy in the clinics:
Trials Worldwide (cumulative)
trials
80
60
40
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2005
patients
Ergo

100
UNIFR

in spite of 13 year- research only
less than 2% of the trials has
reached phase III
not necessarily due to the «novel»
'fail early, fail fast'cancer
paradigm
As of January 2005:
938 cumulative protocols
(90-2005)
1500
4700 treated /enrolled patients
! As of Jan 1, 2004:
1 approved product in China
(Gendicine, by Sibiono Inc. 2004)
hered.
2600 Patients treated in 2004
66% phase I
19% phase I-II
13% phase II
0.8% phase II-III
1.7% phase III
II
1000
I-II
I
500
vasc.
20% overall still pending
Infect.
or not yet Initiated !
20
www.wiley.com/genetherapy
1990 1992
1994
1996
1998
2000
clinical milestones in gene therapy
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2005
1990, 1993, 2000, // ADA deficiency
F Anderson, M Blaese 90/93/ C Bordignon 2000/2004
Anderson, 1990
Isner, 1998
Fischer,
Kirn, 2000
1997, 2000, Critical limb ischemia
2000, 2002
J Isner († 4.11.2001),
I
Baumgartner,
1998
25 lives
2001
Manuel Grez
were so far documentedly saved by GT in
2002
Sibiono
Hans Peter
Hossle
2000, Hemophilia
european
trials
(x-SCID,
ADA,
CGD)
2003
Shenzen
Reinhard
Seger
M Kay, K High
(France, UK, Italy) (all in phase I)
Intravascular adenoviral
2004/2005agents
2000, 2002, X-SCID
in cancer patients:
~200
lives
quality-improved
A Fischer, 2000/2002, Thrasher 2003
Lessons
from clinical
very
encouraging
data trials
from
in several other phase I and II trials
just initiated(review)
clinical trial,
dropped
inpatients
2004?
2001, 2003 ONYX oncolytic Viruses
prospected
>10
~nnn lives saved or quality-improved ?
licensed China 2005?
D Kirn (Cancer Gene Ther 9, p 979-86)
commercialisation of
by Gendicine (50'000 patients prospected
Bordignon, 2000Approved
(ESGT, Stockholm)
Gendicine
(Jan 2004) for cancer
for 2006)
2002,
science
296,
2410
ff)
2004, Chronic Granulomatous Disease
M Grez Frankfurt; R Seger Zürich
2004/2005 Gendicine (adeno-p53 vector)
L Peng, Sibiono Inc, Shenzen, China
treatment in China.
-> ! Hum Gene Ther 16, 1016 ff.
Die most feared side effects of Gene Therapy
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2005

Immune response to vector

immune response or long term side effects from
new or foreign gene product (-> autoimmunity)

General toxicity of viral vectors

Adventitious contaminants in recombinant viruses

Random integration in genome
-> insertional mutagenesis (-> cancer risk)

Contamination of germ line cells
Ergo
«The more effective is a drug, the more side effects
it will generate».
 Side-effect-free illusion in the 90ties is over
 Primitive state of the vectorology/delivery
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SAEs1: from Pennsylvania to Paris
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2005
NY May 5, 1995, R. Crystal:
adenovirus, cystic fibrosis (lung)
one patient mild pneumonia-like condition
Trial interrupted and many others on hold.
Most Recent Paris' Trial News
discussed under:
www.unifr.ch/nfp37/adverse03.html
UPenn, Sept. 19, 1999, J. Wilson:
adenovirus , 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:
retrovirus , x-SCID (bone marrow)
one patient developed a leukemia-like condition.
Trial suspended and some trials in US and Germany on hold until 2003.
Paris, Jan 14, 2003, A Fischer:
retrovirus X-SCID (bone marrow) same cohort
a second patient developed a similar leukemia
30 trials in USA were temporarily suspended
Ergo
gene therapy can produce both shortterm and long-term severe side effects
through acute immunogenicity or
insertional mutagenesis (cancer risk)
SAEs2: Recent Autoimmunity Reports in gene transfer...
should open the eyes of potential dopers
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2005
Blood (2004), Vol. 103, No. 9, comment: pp. 3248-3249
Autoimmunity in EPO gene transfer (macaques)
Els Verhoeyen and François-Loïc Cosset
Papers:
- Chenuaud and colleagues (page 3303)
- Gao and colleagues (page 3300)
inadvertent autoimmune response in nonhuman primates resulting from
transfer of a gene encoding a self-antigen.
- homologous EPO cDNA via AAV vectors
- muscle or lung,
- supra-physiologic serum levels of EPO
Ergo
K High, ASGT June meeting 2004
somatic gene transfer and ectopic
transgene expression
is detectable and
 can generate mid-term auto- immunity
 sounds quite risky for Epo doping

[Abstract1002] Immune Responses to AAV and to
Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed

Gene Transfer for Hemophilia B
Now, lets dedicate completely to gene doping:
.. is ist thinkable?
Gene therapy (features summary)
- treatment not hereditary
- principle works
- not yet clinically established
- high risk with todays vectorology
- applicable to virtually all disease status
- pioneer status
Ergo:
Realistically speaking, efforts in SGT
should be currently restricted to severe
diseases, and we should close the talk
here,
however...
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2005
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The three levels of Doping...
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2005
+
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)
Which gene-transfer-Strategies
could be concretely conceivable for doping?
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2005

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-1, anti-myostatin, Epo)

in vivo local (example joints):
Ergo:
pain reducers, inflammation inhibitors, recovery
Doping and
with gene transfer is
repair factors (anti-TNF, BMPs, ...)
conceivable at many
different levels

in vivo systemic:
anabolic enhancers, endocrine factors, pain killers,
vascular controllers, (hormone metabolising
enzymes, proenkephalins, ...)
The experiments of Lee Sweeney (2004)
have raised further smoke...
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2005
Gene transfer of IGF-1 (J. App Physiol 96, 1097 ff (2004))
The features


IGF-1 -> growth factor for muscles
AAV Vector, intra muscular
Rat model , + or - training
Results
muscle force and muscle mass increased
beyond levels obtained in training
Ergo
ok, Dr. Sweeney, transfer of IGF-1 in
rats significantly increases muscle
performance,, but...
muscle force

questions
- can it be extrapolated to humans?
- kg muscle to tranduce ?
- how to manufacture sufficient vector?
- symmetry of effects?
training:
+
- adverse side+effects?
IGF-1:
- -
-
+
+
Which side effects should we expect if gene transfer
would be currently applied in doping
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2005
Short -mid term



Autoimmunity
Hyperimmunity
Toxic shock
Long term
Specially dangerous: Recklessness...
 Fibrosis
 Improper technology
 Cancer

(unsuitable vector, low competence of doctors)
 conventional side- effects of
 Improper Material
administered factors
(contaminated with pathogens or pyrogens)
 Inaccessibility to future gene
 Insufficient follow-up
therapy interventions (immunity
to vectors)
Which would be the objective limitations
of gene doping?
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2005
Viral gene transfer
Ergo:
 immune problems
risks seem today
currently
 limited readministration possibilities
higher than benefits
 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
 asymmetry of effects
N
R
Detection possibilities
of gene doping
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2005

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)
 foreign genes detectable only short-> but leaves permanent genetic marking
term in blood or body fluids, but
- foreign genes detectable long term
Detection of nucleic acids cannot be performed
body fluids
in tissueinbiopsies,
and
(except in early phase after systemic administration)
- abnormal gene products detectable
-> might require specific tissue biopsy (example GT erythropoietin in
monkeys)

Comparison of advantages/disadvantages:
with respect to conventional Doping
Category
Ergo:
Drug/protein
Gene-based
the odds would speak actually against Gene doping
Rapidity of effects
rapid
slow
but:
Reversibility
rapid
slow
awareness of the above needs common sense,
a property that is rare in the doping field
Dosage
straightforward difficult
and: of treatm. simple
Complexity
complex
...there are several borderline sports lacking doping control
Associated risks
Question:
depends
high
...isnt it the high publicity over gene doping just a sort of
psychologically intimidating
some nations
(this has
Concealability
possiblestrategy by difficult
/impossible
happened before in doping)
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2005
Doping with Gene transfer:
Proust's questionnaire
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2005
Is someone in the field currently 'thinking GT' for doping ?
yes
Which sports will produce the first case ?
horse racing?
Which country may produce the first human cases?
...China?
Will gene doping be more effective than conventional?
QuickTime™ et un décompresseur
None sont requis pour visualiser
cette image.
No
Shall it be effective at all ?
probably little
Will some athlete suffer or even die from GT attempts?
yes
Which gene will be first transferred for doping purposes ?
Epo
Shall gene doping remain difficult to detect ?
No
Why is then gene doping still so attractive ?
M
Proust 1871-1922
ignorance
... Thank you, and let's hope that sports can
continue producing genuine emotions and fairplay
AISTS,
Prof. Bengt Kayser
my collaborators at UNIFR
for correspondence:
[email protected]
for info :
www.unifr.ch/nfp37
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2005
«That's all folks»
...looking forward to your questions
www.unifr.ch/nfp37
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2005
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2002