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UNIFR
Technical Remarks
Rusconi
2002
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S. Rusconi
Sandro Rusconi
UNIFR
Rusconi
2002
1972-75
1975-79
1979-82
1982-84
1984-86
1987-91
1994-today
1996-today
2001
2002
2002
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)
Principal Investigator, UNI Zuerich (mol. bio.)
Professor Biochemistry UNI Fribourg
Director Swiss National Research Program 37
'Somatic Gene Therapy'
Participant Swiss Natl. Res Program 50
'Endocrine disruptors'
Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department
President Union of Swiss Societies for
Experimental Biology (USGEB)
Bern, Nov 22, 2002
Swiss Olympic
gene doping workshop
Sports doping:
Is there a realistic
application for
gene transfer?
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
UNIFR
Rusconi
2002
Genetics has been used since millennia,
Molecular Biology, only since 30 years
100’000 b.C.
Empirical genetics
10’000 b.C.
Biotechnology
2000 a.d.
Molecular biology
2001 a.d, Genomics
UNIFR
Rusconi
2001
UNIFR
1 Gene -> 1 or more functions
Rusconi
2001
DNA
RNA
Protein
Transcription / translation
Gene expression
GENE
2-5 FUNCTIONS
100 ’000 genes
(50 ’000 genes?)
>300 ’000 functions
(>150 ’000 functions)
What is in fact a gene?: a segment of DNA acting as a
regulated machine for RNA production
DNA
GENE
RNA
Protein
Transcription / translation
FUNCTION
RNA
DNA
spacer
regulatory
coding
spacer
UNIFR
Rusconi
2002
UNIFR
1 Organism -> more than 105
genetically-controlled Functions
Rusconi
2002
2 mm
2m
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
Reductionistic molecular biology paradigm
(gene defects and gene transfer)
DNA
Protein
GENE
FUNCTION(s)
GENE OK
FUNCTION OK
GENE KO
FUNCTION KO
GENE transfer
FUNCTION transfer
UNIFR
Rusconi
2002
Gene amplification / manipulation techniques
(genetic engineering, recombinant DNA)
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
UNIFR
Rusconi
2002
UNIFR
The FOUR eras of molecular medicine
Rusconi
2002
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
UNIFR
Rusconi
2002
100%
10
1
E2/E
many treatments that slow down ageing
4
or age-related degenerative diseases
are also potential doping
treatments
20 40 60 80
80
70
60
1900
2000
20 40 60
50
1900
100
Alzheimer’s free %
Life expectancy (CH)
cancer incidence
The major disease of the 21st century: Ageing
1920
1940
1960
1980
199
1900
M
E3/E4
E4/E4
80
2000
Now, let's talk about Somatic Gene Therapy
(somatic gene transfer)
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
UNIFR
Rusconi
2002
Somatic gene therapy’s (gene transfer)
four fundamental questions
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
UNIFR
Rusconi
2002
Remember!
Why 'somatic'?
UNIFR
Rusconi
2001

Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise
to a descendant organism
i.e. somatic gene transfer
is a treatment aiming at
somatic cells and consequently does not lead to
a hereditary transmission
of the genetic alteration

Somatic Cells: all the other cells of the body
UNIFR
Rusconi
2001
Pharmacological considerations
Classical Drugs
Protein Drugs
Nucleic Acids
Mw 50- 500 Daltons
 Mw 20 ’000- 100 ’000
 Mw
DaN x 1’000’000 Da
 Synthetically prepared
 Biologically prepared
 Biologically prepared
 Rapid diffusion/action
 Slower diffusion/action
 Slow diffusion
 Oral delivery possible
 Oral delivery not possible
 Oral delivery inconceivable
 Cellular delivery:  Cellular delivery:  Cellular delivery:
- act at cell surface- act extracellularly - no membrane translocation
- permeate cell membrane
- no nuclear translocation
- imported through channels
- no biological import


Can be delivered as
Can be delivered as
 Must be delivered as
soluble molecules soluble molecules complex carrier particles
Ångstrom/nm size nm size
50-200 nm size
THREE classes of physiological gene delivery
Ex-vivo
In-vivo
topical delivery
UNIFR
Rusconi
2001
In-vivo
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 gene transfer vehicles: non-viral & viral
Non-viral transfer
(transfection)
Viral gene transfer
(Infection)
UNIFR
Rusconi
2001
a
b
Nuclear envelope barrier!
see, Nature Biotech
December 2001
Transfection with recombinant DNA
Vs Infection with recombinant viruses
UNIFR
Rusconi
2001
Transfection
exposed to
106 particles/cell
12 hours
Infection
exposed to
3 particle/cell
30 min
Quick parade of popular vectors/methods
Adenovirus
Naked DNA
Adeno-associated V.
Liposomes & Co.
Retrovirus (incl. HIV)
Oligonucleotides
UNIFR
Rusconi
2002
UNIFR
Rusconi
2002
Recombinant Adenoviruses
Approaches
Advantages / Limitations
Generation I
8 Kb capacity Generation I
>30 Kb capacity Generation III
Adeno can be grown at very high titers,
However
 Do not integrate
Generation III
Hybrid adenos:



Adeno-RV
Adeno-AAV
Adeno-Transposase

Can contain RCAs

Are toxic /immunogenic
Examples
 OTC deficiency (clin, ---)
 Cystic Fibrosis (clin, --- )
 Oncolytic viruses (clin, +++)
Recombinant AAV (adeno-associated-virus)
UNIFR
Rusconi
2002
Approaches
Advantages / Limitations
Helper-dependent production
Persistence in the genome permits longterm expression, high titers are easily
obtained, immunogenicity is very low,
However the major problem is:
Helper independent production

Cis-complementing vectors
Co-infection
Small capacity (<4.5 kb) which does
not allow to accommodate large genes
or gene clusters.
Examples
 Hemophilia A (clin, animal, +++)
 Gaucher (clin, animal, +++)
 Brain Ischemia (animal, +++)
 Cystic fibrosis (animal, +/-)
Recombinant Retroviruses (includes HIV-based)
Approaches
Advantages / Limitations
Murine Retroviruses
9 Kb capacity + integration through
transposition also in quiescent cells
(HIV), permit in principle long-term
treatments, however disturbed by:
 Insertional mutagenesis
VSV-pseudotyped RV
Lentiviruses !

Gene silencing

High mutation rate

Low titer of production
UUNIFR
Rusconi
2002
Self-inactivating RV
Combination viruses
Examples
 SCID (IL2R defect, Paris) (clin, +++)
 Adenosine Deaminase deficiency (clin, +++!!!)
 Parkinson (preclin, +++)
 Anti cancer (clin +/-)
UNIFR
Rusconi
2002
Naked / complexed DNA
Approaches
Advantages / Limitations
Naked DNA injection /biolistic
Unlimited size capacity + lower
immunogenicity and lower bio-risk
of non viral formulations is
disturbed by
Naked DNA + pressure
Naked DNA + electroporation
Liposomal formulations
Combinations

Low efficiency of gene transfer

Even lower stable integration
Examples
 Critical limb Ischemia (clin, +++)
 Cardiac Ischemia (clin, +/-)
 Vaccination (clin, +/-)
 Anti restenosis (preclin. +/-)
UNIFR
Rusconi
2002
Oligo-nucleotides
Approaches
Advantages / Limitations
Antisense
these procedures may be suitable for :
Ribozymes/DNAzymes

handling dominant defects

transient treatments (gene modulation)

permanent treatments (gene correction)
Triple helix
Decoy / competitors
Gene-correcting oligos
Examples
 Anti cancer (clin,preclin., +/-)
 Restenosis (clin, +++)
 Muscular Distrophy (animal, +++)
√!
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”)
UNIFR
Rusconi
2002
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”)
The most feared potential side-effects of gene transfer
UNIFR
Rusconi
2002

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)

Contamination of germ line cells
UNIFR
Gene Therapy in the clinic: Trials Wordldwide
Rusconi
2002
trials
patients
As of Sept. 2002:
100
80
599 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
Gene Therapy Milestones
UNIFR
Rusconi
2002
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)
Gene Therapy Adverse events:
NY 1995 // UPenn 1999 // Paris 2002
UNIFR
Rusconi
2002
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 was interrupted and many others were put 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.
Ups and Downs of Gene Therapy:
a true roller coaster ride!
UNIFR
Rusconi
2002
high
mood
A. Fischer lentivectors
in clinics?
M.
Kay
R. Crystal
Adeno I
V.Dzau
C Bordignon
J. Isner
ADA
AAV
NIH
Adeno III
germline
Motulski
in mice?
report Lentivectors
Paris
Ergo:
in pre-clinic
in spite of its respectable age,
J. Wilson
Low
gene transfer is still in its infancy
J. Gelsinger
and still produces more controversies
NFP37
than clinical results
90
91
92
93
94
95
96
97
98
99
00
01
02
UNIFR
Rusconi
2002
The THREE levels of doping
+
Before the
competition
(anabolic enhancers)
'Molecular treatments
Application of the
know-how in
molecular genetics
to doping
During the competition
(perfomance enhancers)
+
+
After the
competition
(repair enhancers)
Which gene transfer approaches would be
compatible with doping strategies

ex vivo, hematopoietic tissue:
erythropoietin?

in vivo local (example muscle):
metabolic enhancers, growth factors,
muscular fiber changers

in vivo local (example joints):
pain reducers, inflammation inhibitors, recovery and
repair factors

in vivo systemic:
anabolic factors, endocrine factors, pain killers
UNIFR
Rusconi
2002
Which are the objective current limitations in
gene-based doping strategies
Viral gene transfer
 immune problems
 limited readministration
 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
UNIFR
Rusconi
2002
Which side effects could be feared in
gene-based doping strategies
UNIFR
Rusconi
2002
Short -mid term



Autoimmunity
Hyperimmunity
Toxic shock
Long term
Intrinsic to reckless application
 Fibrosis
(probably the biggest danger)
 Cancer
 malpractice (unsuitable
 Conventional effects of
vector/administration route)
administered factors
 non-clinical grade material
 Inaccessibility to future gene
(adventitious pathogens
therapy interventions (immunity)
or allergens)
 lack of follow-up
Which detection methods would be (or not) evisageable
for gene-based doping strategies

Antibody detection (viral antigens or other epitopes)

recombinant-nucleic acids detection (PCR)

recombinant protein detection
(MALDI-TOF / proteomics)

Gene transfer may be anatomically difficult to
detect (if locally administered) but leaves
permanent genetic marking

the detection of nucleic acids cannot be
performed in body fluids (except for systemically
administered treatments) and might require
specific tissue biopsy
UNIFR
Rusconi
2002
Final side-by-side comparison:
gene-based doping versus drug- or protein-based doping
Category
Drug/protein
Gene-based
Rapidity of effects
rapid
slow
Reversibility
rapid
slow/none
UNIFR
Rusconi
2002
Ergo: straightforward difficult
The odds speak currently rather against the adoption of
Complexity of treatm.
simple
complex
gene-based
doping,
but this applies to common-sense clinical practice,
Associated risks
high
and thisdepends
aspect is not guaranteed
in the doping field
Dosage
Detectability
arduous
'straightforward'
...Thanks !
UNIFR
Rusconi
2002
Swissolympics
Our own project/goal may indeed
appear very small and harmless...
My collaborators at UNIFR
This does not necessarily apply
to its consequences...
Swiss National Research Foundation
Thank you all for the attention,
and... if you are too shy to ask
send an e-mail to:
[email protected]
or visit:
www.unifr.ch/nfp37
discussion slides
UNIFR
Rusconi
2002
UNIFR
Examples of inheritable gene defects
Rusconi
2002
Polygenic defects
(‘ frequent ’)
Type
estimated
min - max
genetics
Diabetes
poly
1
- 4%
Hyperurikemia
Multi defects
2
- 15 %
Monogenic
estimated
Glaucoma
poly
1
- 2%
(‘ rare ’)
min - max
Displasia
Multi
1
- 3%
Cystic fibrosis, muscular dystrophy
Hypercolesterolemia
Multi
1
- 5%
immodeficiencies, metabolic diseases, all together
Syn-& Polydactyly
poly
0.1
- 1%
Hemophilia...
0.4
- 0.7%
Congenital cardiac defects
Multi
0.5
- 0.8 %
Manic-depressive psychosis
Multi
0.4
- 3%
Predispositions
Type
Miopy
poly
3
- 4%
Polycystic kidney
poly
0.1
- 1%
Multi
Psoriasis
Multi (*)
2 Alzheimer
- 3%
Multi
Schizofrenia
Multi (*)
0.5Parkinson
- 1%
Multi
Scoliosis
Multi (*)
3 Breast
- cancer
5%
(*) Colon Carcinoma
Multi
(*) Obesity
Multi
(*) Alcolholism/ drug addiction Multi
Sum of incidences
(all defects)
behaviour
environment
estimated
min - max
7
1
4
0.1
0.5
0.5
- 27 %
- 3%
- 8%
- 1%
- 2%
3%
min - max
32
- 83%
The long way to drug/procedure registration is the
principal cause of financial burden, but we cannot avoid it
year
event
costs U$D
0
Idea
0
2
Cell culture assays
0.5 Mio
5
Pre-clinical tests
animal models
2 Mio
Clinical phase I
5-20 patients
verify side effects
6 Mio
Clinical phase II
30-100 patients
dosis escalation
12 Mio
Clinical Phase III
>300- 1000 patients
multicentric
double blind
80 Mio
7
10
15
16>>
Registration / Availability
UNIFR
Rusconi
2002
This means:
assuming 20% of new developments
makes it to final registration,
the average investment is
300-500 Mio U$D
for each approved drug/procedure
UNIFR
Not only the genome determines the health status...
Rusconi
2002
genetics
Muscle distrophy
Familial Breast Cancer
Sporadic Breast Cancer
Lung Cancer
Obesity
Artherosclerosis
Alzheimer
Parkinson ’s
Drug Abuse
Homosexuality
behaviour
environment
Recap: what is a virus ? ->
A superbly efficient replicating machine
UUNIFR
Rusconi
2002
100 nm
docking
entry
disassembly
genome replication
early genes exp
capsid
replication
E L1 L2
E L1 L2
assembly
Spread
standard viral genome
Etc...
late genes
exp
Engineering of replication-defective, recombinant viruses
(Principle)
rp
E
L1 L2
UNIFR
Rusconi
2002
rp
Wild type genome
X
Normal target cells
E
E
E
E
E
Recombinant genome
Virions
E
E
Packaging cells
Normal target cells
R-Virions
UNIFR
'Classical' GT models and strategies
Rusconi
2002

Disease

transferred function

Clinical Results

ADA deficiency
(Immunodeficiency)

ADA normal gene
(enzyme)

1990 F. Anderson,
2002 C. Bordignon

Cystic Fibrosis
(Lung, Pancreas)

CFTR gene
(chlorine transporter)

no significant results
in spite of several trials

Haemophilia B
(Blood)

Factor IX gene
(blood clotting factor

1999-2000 M. Kay and K.
Horwitz

SCID
(Immunodeficiency)

IL2R gene
(gamma-C receptor)

2000 A. Fischer

Limb ischaemia
(Hands, Feet)

VEGF gene
(vascular promoter)

1998 J. Isner

Cardiac ischaemia
(Heart)

VEGF gene
(vascular promoter)

2000 J. Isner