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UNIFR
Technical Remarks Rusconi
2002
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S. Rusconi
UNIFR
Sandro Rusconi Rusconi
2002
1972-75 Primary school teacher (Locarno, Switzerland) Bern, Nov 22, 2002
1975-79 Graduation in Biology UNI Zuerich, Switzerland Swiss Olympic
1979-82 PhD curriculum UNI Zuerich, molecular biology gene doping workshop
1982-84 Research assistant UNI Zuerich
1984-86 Postdoc UCSF, K Yamamoto, (San Francisco) Sports doping:
1987-91 Principal Investigator, UNI Zuerich (mol. bio.) Is there a realistic
1994-today Professor Biochemistry UNI Fribourg application for
1996-today Director Swiss National Research Program 37
gene transfer?
'Somatic Gene Therapy'
2001 Participant Swiss Natl. Res Program 50
'Endocrine disruptors'
2002 Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department
2002 President Union of Swiss Societies for
Experimental Biology (USGEB)
UNIFR
Schedule Rusconi
2002
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
Genetics has been used since millennia, Rusconi
Molecular Biology, only since 30 years 2001
100’000 b.C.
Empirical genetics
10’000 b.C.
Biotechnology
2000 a.d.
Molecular biology
2001 a.d, Genomics
UNIFR
1 Gene -> 1 or more functions Rusconi
2001
DNA RNA Protein
Transcription / translation
Gene expression
GENE 2-5 FUNCTIONS
100 ’000 genes >300 ’000 functions
(50 ’000 genes?) (>150 ’000 functions)
UNIFR
What is in fact a gene?: a segment of DNA acting as a Rusconi
regulated machine for RNA production 2002
DNA RNA Protein
GENE Transcription / translation FUNCTION
RNA
DNA
spacer regulatory coding spacer
UNIFR
1 Organism -> more than 105 Rusconi
genetically-controlled Functions 2002
2m 2 mm 0.2mm
0.02mm
0.001mm
DNA RNA Protein
UNIFR
Reductionistic molecular biology paradigm Rusconi
(gene defects and gene transfer) 2002
DNA Protein
GENE FUNCTION(s)
GENE OK FUNCTION OK
GENE KO FUNCTION KO
GENE transfer FUNCTION transfer
UNIFR
Gene amplification / manipulation techniques Rusconi
(genetic engineering, recombinant DNA) 2002
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
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
3000
10
80 85 90 95
Y2K+n Post-genomic improvements 99 former technologies
of 1000
80 85 90 95 00
UNIFR
The major disease of the 21st century: Ageing Rusconi
2002
100
cancer incidence
1 0 0%
Alzheimer’s free %
10
1 E2 /E
many treatments that slow down ageing
Life expectancy (CH)
80
M
70 or age-related degenerative diseases
4
E3 /E4
treatments
are also potential doping 4 0 6 0 8 0
20 E4 /E4
60
50 1900 2000
20 40 60 80
1900 1920 1940 1960 1980 199 1900 2000
Now, let's talk about Somatic Gene Therapy UNIFR
Rusconi
(somatic gene transfer) 2002
Definition of GT: Chronic treatment
'Use genes as drugs': Acute treatment
Correcting disorders by
somatic gene transfer Preventive treatment
NFP37 somatic gene therapy
www.unifr.ch/nfp37
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
Somatic gene therapy’s (gene transfer) UNIFR
Rusconi
four fundamental questions 2002
Remember!
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
UNIFR
Why 'somatic'? 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 conse-
quently does not lead to
a hereditary transmission
of the genetic alteration
Somatic Cells: all the other cells of the body
UNIFR
Pharmacological considerations Rusconi
2001
Classical Drugs Protein Drugs Nucleic Acids
Mw N
Mw 50- 500 Daltons 20 ’000- 100 ’000 Da x 1’000’000 Da
Mw
Biologically prepared
Synthetically prepared Biologically prepared
Slower diffusion/action
Rapid diffusion/action Slow diffusion
Oral delivery not possible
Oral delivery 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
as
Can be delivered Can be delivered asMust be delivered as
soluble molecules soluble molecules complex carrier particles
Ångstrom/nm size nm size 50-200 nm size
UNIFR
THREE classes of physiological gene delivery Rusconi
2001
Ex-vivo In-vivo In-vivo
topical delivery systemic delivery
V
Examples: Examples: Examples:
- bone marrow - brain - intravenous
- liver cells - muscle - intra-arterial
- skin cells - eye - intra-peritoneal
- joints
- tumors
UNIFR
TWO classes of gene transfer vehicles: non-viral & viral Rusconi
2001
Non-viral transfer a
(transfection)
Viral gene transfer
(Infection) b
Nuclear envelope barrier!
see, Nature Biotech
December 2001
Transfection with recombinant DNA UNIFR
Rusconi
Vs Infection with recombinant viruses 2001
Transfection
exposed to
106 particles/cell
12 hours
Infection
exposed to
3 particle/cell
30 min
UNIFR
Quick parade of popular vectors/methods Rusconi
2002
Adenovirus Naked DNA
Adeno-associated V. Liposomes & Co.
Retrovirus (incl. HIV)
Oligonucleotides
UNIFR
Recombinant Adenoviruses Rusconi
2002
Approaches Advantages / Limitations
Generation I 8 Kb capacity Generation I
>30 Kb capacity Generation III
Adeno can be grown at very high titers,
Generation III However
Do not integrate
Can contain RCAs
Hybrid adenos: Are toxic /immunogenic
Adeno-RV
Adeno-AAV Examples
Adeno-Transposase OTC deficiency (clin, ---)
Cystic Fibrosis (clin, --- )
Oncolytic viruses (clin, +++)
UNIFR
Recombinant AAV (adeno-associated-virus) Rusconi
2002
Approaches Advantages / Limitations
Helper-dependent production Persistence in the genome permits long-
term expression, high titers are easily
obtained, immunogenicity is very low,
Helper independent production However the major problem is:
Small capacity (<4.5 kb) which does
not allow to accommodate large genes
Cis-complementing vectors
or gene clusters.
Co-infection Examples
Hemophilia A (clin, animal, +++)
Gaucher (clin, animal, +++)
Brain Ischemia (animal, +++)
Cystic fibrosis (animal, +/-)
UUNIFR
Recombinant Retroviruses (includes HIV-based) Rusconi
2002
Approaches Advantages / Limitations
Murine Retroviruses 9 Kb capacity + integration through
transposition also in quiescent cells
(HIV), permit in principle long-term
VSV-pseudotyped RV treatments, however disturbed by:
Insertional mutagenesis
Gene silencing
Lentiviruses ! High mutation rate
Low titer of production
Self-inactivating RV
Examples
SCID (IL2R defect, Paris) (clin, +++)
Combination viruses Adenosine Deaminase deficiency (clin, +++!!!)
Parkinson (preclin, +++)
Anti cancer (clin +/-)
UNIFR
Naked / complexed DNA Rusconi
2002
Approaches Advantages / Limitations
Naked DNA injection /biolistic Unlimited size capacity + lower
immunogenicity and lower bio-risk
of non viral formulations is
Naked DNA + pressure disturbed by
Low efficiency of gene transfer
Naked DNA + electroporation Even lower stable integration
Examples
Liposomal formulations Critical limb Ischemia (clin, +++)
Cardiac Ischemia (clin, +/-)
Vaccination (clin, +/-)
Combinations Anti restenosis (preclin. +/-)
UNIFR
Oligo-nucleotides Rusconi
2002
Approaches Advantages / Limitations
Antisense these procedures may be suitable for :
handling dominant defects
Ribozymes/DNAzymes transient treatments (gene modulation)
permanent treatments (gene correction)
Triple helix
Examples
Decoy / competitors Anti cancer (clin,preclin., +/-)
Restenosis (clin, +++)
Muscular Distrophy (animal, +++)
Gene-correcting oligos √!
Recap: current limitations of popular UNIFR
Rusconi
gene transfer vectors 2002
Adenovirus
Biolistic bombardment
- no persistence
or local direct injection
- limited packaging
- limited area
- toxicity
- immunogenicity
Electroporation
Retrovirus (incl. HIV) - limited organ access
- limited package
- random insertion Liposomes, gene correction & Co.
- unstable genome - very inefficient transfer
General
- antibody response General
- limited packaging - low transfer efficiency
- gene silencing 1/10’000 of viruses’ in vivo
Solutions: Solutions:
- synthetic viruses - improved liposomes
(“Virosomes”) with viral properties (“Virosomes”)
UNIFR
The most feared potential side-effects of gene transfer 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 1500
599 registered protocols
4000 treated patients
cancer
80
60 86% phase I 1000
13% phase II
1 % phase III
40 hered.
500
20 vasc.overall still pending
21%
Infect.
or not yet Initiated !
www.wiley.com
1990 1992 1994 1996 1998 2000
UNIFR
Gene Therapy Milestones Rusconi
2002
1990, 1993, 2000 // ADA deficiency Anderson, 1990
Isner, 1998
F Anderson, M Blaese // C Bordignon Dzau, 1999
Kmiec, 1999
1997, 2000, Critical limb ischemia Fischer, 2000
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: UNIFR
Rusconi
NY 1995 // UPenn 1999 // Paris 2002 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: UNIFR
Rusconi
a true roller coaster ride! 2002
high A. Fischer lentivectors
M. Kay in clinics?
R. Crystal
Adeno I V.Dzau C Bordignon
J. Isner
ADA AAV
NIH Adeno III
germline
mood
Motulski
report Lentivectors in mice?
Ergo: in pre-clinic Paris
in spite of its respectable age, J. Wilson
Low gene transfer is still in its infancy J. Gelsinger
and still produces more controversies
than clinical results NFP37
90 91 92 93 94 95 96 97 98 99 00 01 02
UNIFR
The THREE levels of doping Rusconi
2002
Before the +
competition
(anabolic enhancers) 'Molecular treatments
Application of the
know-how in
molecular genetics
During the competition
+
(perfomance enhancers)
to doping
+
After the
competition
(repair enhancers)
Which gene transfer approaches would be UNIFR
Rusconi
compatible with doping strategies 2002
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
Which are the objective current limitations in UNIFR
Rusconi
gene-based doping strategies 2002
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
Which side effects could be feared in UNIFR
Rusconi
gene-based doping strategies 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 UNIFR
Rusconi
for gene-based doping strategies 2002
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
Final side-by-side comparison: UNIFR
Rusconi
gene-based doping versus drug- or protein-based doping 2002
Category Drug/protein Gene-based
Rapidity of effects rapid slow
Reversibility rapid slow/none
Dosage Ergo: straightforward difficult
The odds speak currently rather against the adoption of
Complexity of treatm. simple
gene-based doping, complex
but this applies to common-sense clinical practice,
high
Associated risks thisdepends not guaranteed in the doping field
and aspect is
Detectability arduous 'straightforward'
UNIFR
...Thanks ! 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:
sandro.rusconi@unifr.ch
or visit:
www.unifr.ch/nfp37
UNIFR
discussion slides Rusconi
2002
UNIFR
Examples of inheritable gene defects Rusconi
2002
Polygenic defects Type estimated genetics behaviour environment
(‘ frequent ’) min - max
Diabetes poly 1 - 4%
Hyperurikemia Multi 2
Monogenic defects- 15 % 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%
Miopy poly Predispositions
3 - 4% Type estimated
Polycystic kidney poly 0.1 - 1% min - max
Psoriasis Multi (*) Alzheimer%
2 - 3 Multi 7 - 27 %
Schizofrenia Multi (*) Parkinson%
0.5 - 1 Multi 1 - 3%
Scoliosis Multi (*) Breast cancer
3 - 5% Multi 4 - 8%
(*) Colon Carcinoma Multi 0.1 - 1%
(*) Obesity Multi 0.5 - 2%
(*) Alcolholism/ drug addiction Multi 0.5 - 3%
Sum of incidences min - max
(all defects) 32 - 83%
The long way to drug/procedure registration is the UNIFR
Rusconi
principal cause of financial burden, but we cannot avoid it 2002
year event costs U$D
0 Idea 0
2 Cell culture assays 0.5 Mio This means:
assuming 20% of new developments
5 Pre-clinical tests makes it to final registration,
animal models 2 Mio
the average investment is
7 Clinical phase I 300-500 Mio U$D
5-20 patients for each approved drug/procedure
verify side effects 6 Mio
10 Clinical phase II
30-100 patients
dosis escalation 12 Mio
15 Clinical Phase III
>300- 1000 patients
multicentric
double blind 80 Mio
16>> Registration / Availability
UNIFR
Not only the genome determines the health status... Rusconi
2002
genetics behaviour environment
Muscle distrophy
Familial Breast Cancer
Sporadic Breast Cancer
Lung Cancer
Obesity
Artherosclerosis
Alzheimer
Parkinson ’s
Drug Abuse
Homosexuality
Recap: what is a virus ? -> UUNIFR
Rusconi
A superbly efficient replicating machine 2002
100 nm
docking entry disassembly genome replication
early genes exp
capsid
replication
E L1 L2 assembly late genes
E L1 L2 exp
standard viral genome Spread
Etc...
Engineering of replication-defective, recombinant viruses UNIFR
Rusconi
(Principle) 2002
rp E L1 L2 rp
Wild type genome Normal target cells Virions
X E E E
E E
E
E
Recombinant genome Packaging cells R-Virions
Normal target cells
UNIFR
'Classical' GT models and strategies Rusconi
2002
Disease transferred function Clinical Results
ADA deficiency ADA normal gene 1990 F. Anderson,
(Immunodeficiency) (enzyme) 2002 C. Bordignon
Cystic Fibrosis CFTR gene no significant results
(Lung, Pancreas) (chlorine transporter) in spite of several trials
Haemophilia B Factor IX gene 1999-2000 M. Kay and K.
(Blood) (blood clotting factor Horwitz
SCID IL2R gene 2000 A. Fischer
(Immunodeficiency) (gamma-C receptor)
Limb ischaemia VEGF gene 1998 J. Isner
(Hands, Feet) (vascular promoter)
Cardiac ischaemia VEGF gene 2000 J. Isner
(Heart) (vascular promoter)
