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UNIFR
Sandro Rusconi
Rusconi
2003
1972-75
1975-79
1979-82
1982-84
1984-86
1987-91
1994-today
1995-today
2002-03
2002-05
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
Professor Biochemistry UNI Fribourg
Director Swiss National Research Program 37
'Somatic Gene Therapy'
Sabbatical, Tufts Med. School Boston and
Univ. Milano, Pharmacology Department
President Union of Swiss Societies for
Experimental Biology (USGEB)
Feb 19, 2003
ECPM Basel
2003: Gene
therapy turning
teenage, what
have we learned?
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
2003
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1 Gene -> 1 or more functions
Rusconi
2003
DNA
RNA
Protein
Transcription / translation
Gene expression
GENE
2-5 FUNCTIONS
100 ’000 genes
(50 ’000 genes?)
>300 ’000 functions
(>150 ’000 functions)
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Recap: what is a gene?:
a regulated machine for RNA production
DNA
GENE
RNA
Rusconi
2003
Protein
To fulfil its role, a transferred gene must include:
Transcription / translation
FUNCTION



regulatory sequences for Tx initiation
proper signals for RNA maturation/transport
proper signals for mRNA translation
RNA
DNA
spacer
regulatory
coding
spacer
UNIFR
1 Organism -> more than 105
genetically-controlled Functions
Rusconi
2003
2 mm
2m
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
1 Cm3 of tissue
 1'000'000'000 cells!
Reductionistic molecular biology paradigm
(gene defects and gene transfer)
DNA
UNIFR
Rusconi
2003
Protein
Gene transfer implies either:
 transfer of new function, or
 transfer of restoring function, or
 transfer of interfering function
GENE
FUNCTION(s)
GENE OK
FUNCTION OK
GENE KO
FUNCTION KO
GENE transfer
FUNCTION transfer
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Examples of inheritable gene defects
Rusconi
2003
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%
Ergo:
Multi
Schizofrenia
Multi (*)
0.5Parkinson
- 1%
Breast
Multi
Scoliosis
Multi or(*)
3more
- cancer
5%
 every person bears one
(*) Colon Carcinoma
Multi
latent genetic defects
(*) Obesity
Multi
(*) Alcolholism/ drug addiction Multi
 many defects are not manifest

but lead to predispositions
Sum of incidences
there are also protective predispositions
(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%
UNIFR
Not only the genome determines the health status...
Rusconi
2003
genetics
Muscle distrophy
Familial Breast Cancer
Sporadic Breast Cancer
Lung Cancer
Obesity
also acquired conditions
may have a genetic component
that modulates their healing
 trauma
 fractures
 burns
 infections
Artherosclerosis
Alzheimer
Parkinson ’s
Drug Abuse
Homosexuality
behaviour
environment
UNIFR
Rusconi
2003
The major disease of the 21st century: Ageing
80
70
100%
10
1
20
60
E2/E
4
40
60
80
1900
2000
20 40 60
50
1900
100
Alzheimer’s free %
Life expectancy (CH)
cancer incidence
This major challenge means:
 higher investments
 more financial returns
 long term treatment
 customised treatment
 social security dilemma
1920
1940
1960
1980
199
1900
M
E3/E4
E4/E4
80
2000
UNIFR
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2003
The THREE missions of medicine
Prevention
+
'Molecular Medicine'
Diagnosis
Application of the
know-how in
molecular genetics
to medicine
+
+
Therapy
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The FOUR eras of molecular medicine
Rusconi
2003
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
Now, let's talk about Somatic Gene Therapy (SGT)
UNIFR
Rusconi
2003
Definition of SGT:
'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
The
SGTtherapy
principle
is teenage
simple Yes,...
Gene
turns
in 2003, but:
buthas
theitdevil
often in
the details
reallyisgrown
up?
UNIFR
Rusconi
2003
There are many things that First
are clinical
simpletrial
in of
principle,
like...
a monogenic
disease
1990
F. Anderson & Co: ADA deficiency
getting a train ticket...
...does
work
! try thisnot
5 min
before departure
and with a group of Chinese tourists in front
parking
2002 your car...
!Same
try this
at noon,
given day
protocol
asany
Anderson's
for ADA
in
Zuerich
or Geneva
...
gene
therapy
(C. Bordignon)
counting votes...
...it
works!
! ask
Florida's officials ...
gene therapy...
look at progress in 13 years...
Why 'somatic'?
UNIFR
Rusconi
2003

Germ Line Cells: the cells (spermatocytes and oocytes and their precursors) that
upon fertilisation can give rise to a descendant organism
Ergo
 transformation of
germ line cells is
avoided, to exclude
risk of erratic
mutations due to
insertional
mutagenesis

Somatic Cells: all the other cells of the body
i.e. somatic gene therapy
is a treatment aiming at
somatic cells and consequently does not lead to
a hereditary transmission
of the genetic alteration
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When/where/ may be SGT indicated?
Rusconi
2003
No existing cure or treatment

most monogenic diseases
Side effects and limitations of protein injection



interleukin 12 (cancer)
-> toxic effects and rapid degradation
VEGF (ischemias)
-> angiomas
Factor VIII or IV (hemophilia)
-> insufficient basal level
Ergo:
 there are many indications
for SGT as stand-alone or
as complementary therapy
Complement to conventional


increase specificity of conventional therapy (cancer)
increase efficacly of conventional therapy (hemophilia)
Life quality burden of patient


costs of enzyme therapy (ex. ADA)
burden of daily injections (ex. Insulin)
SGT's four fundamental questions & players
UNIFR
Rusconi
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!
The SGT acrobatics:
matching vectors / delivery system / disease
Chronic Conditions






Slow onset of expression acceptable
Initiation of the treatment
weeks/months/years before
'point of no return' (ex. cystic fibrosis)
persisting expression of the transgene or
re-administration required (example
hemophilia)
Usually based on compensation of
'genetic loss-of-function' (permanent regain of function; ex. ADA)
Regulation of gene expression often
necessary (because of persistence)
For some diseases even a small % of
tissue transformation is already
therapeutic
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2003
Acute Conditions






Rapid onset of expression necessary
Initiation of the treatment
minutes/hours/days before
'point of no return' (ex. brain ischemia)
persisting expression of the transgene not
required, occasional re-administration
(example
Usually based on augmentation of resident
function (transient gain of function; ex.
VEGF)
Regulation of gene expression not
necessary (because of transiency)
For most diseases even a small % of
transformation is already therapeutic
Ergo
 many divergent variables must be matched for each case
 an advantage for one purpose becomes a disadvantage for another (viceversa)
Pharmacological considerations for DNA transfer
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
UNIFR
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2003
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

Therapy with nucleic acids
 requires particulated formulation
 is much more complex than previous drug deliveries
 has a different degree of reversibility (dosage problem)
THREE classes of anatomical gene delivery
Ex-vivo
In-vivo
topical delivery
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2003
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 vectors:
non-viral & viral delivery
Non-viral transfer
(transfection of plasmids)
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2003
a
Viral gene transfer
(Infection by r-vectors)
b
Nuclear envelope barrier!
see, Nature Biotech
December 2001
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2003
Transfection versus Infection
Transfection
exposed to
106 particles/cell
12 hours
Infection
exposed to
1 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)
Most relevant issues in the
two main 'vectorology' sectors (viral versus nonviral)
Viral vectors







Packaging capacity from 4 to 30 kb problem for some
large genes (ex. dystrophin gene or CFTR gene)
important toxic load: ratio infectious/non-infectious
particles from 1/10 to 1/100
strong immunogenicity: capsid and envelope
proteins, residual viral genes
contaminants: replication-competent viruses (ex. wild
type revertant viruses)
Viral amount (titre) obtainable with recombinants (ex.
10exp5 = poor, 10exp10=excellent)
Complexity of production (existence or not of
packaging cell systems)
Emotional problems linked to pathogenicity of donor
vectors (ex. lentiviruses)
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2003
Nonviral vectors







Packaging capacity not an issue, even very large
constructs can be used (example entire loci up to 150
kb)
minor toxic load: small percentage of non relevant
adventitious materials
moderate immunogenicity: methylation status of DNA
(example CpG motifs)
contaminants: adventitious pathogens from poor DNA
purification (ex endotoxins)
Amount of DNA molecules is usually not a problem, the
other components depends on chemical synthesis
No particular complexity, except for specially formulated
liposomes
no particular emotional problems linked to the nature of
the reagents
Ergo
 problems that must be solved to be suitable for clinical treatment and for industrial
production are different between viral and non-viral vectors
 when ignoring thir low efficiency, nonviral vectors appears largely superior
Ideal properties of a systemically delivered
non-viral formulation
Stability


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2003
Ergo
several independent
problems must be solved for
a nonviral formulation to be
Addressability
suitable for clinical treatment
 particle should possess a vascular addressing signature
and for industrial production
 particle should bear a tissue-docking specificity
 most viral vectors include
 DNA construct should include tissue-specific regulatory elements many, if not all those
properties
particle should resist serum inactivation
particle should be inert to immune inactivation

Efficiency




cargo should be protected from cytoplasmic inactivation (ex. lysosomes)
cargo should contain nuclear-translocating signals
DNA cargo should include genome-integration functions
DNA element must be guaranteed to function after genomic integration
(no silencing)
Other properties



Particle should not include immunogenic/toxic surfaces
cargo should not encode immunogenic/toxic products
Cargo should include anti-apoptotic functions
Small parade of popular vectors/methods
Adenovirus
Naked DNA
Adeno-associated V.
Liposomes & Co.
Retrovirus (incl. HIV)
Oligonucleotides
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2003
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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 adeno-associated-virus (AAV)
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2003
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)
UUNIFR
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2003
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
Self-inactivating RV
Combination viruses
Examples
 SCID (IL2R defect, Paris) (clin, +++)
 Adenosine Deaminase deficiency (clin, +++!!!)
 Parkinson (preclin, +++)
 Anti cancer (clin +/-)
UNIFR
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2003
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. +/-)
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2003
Oligonucleotides
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 vectors
Adenovirus
- no persistence
- limited packaging
- toxicity, immunogenicity
Retrovirus (incl. HIV)
- limited packaging
- random insertion
- unstable genome
General
- antibody response
- limited packaging
- gene silencing
Solutions:
- synthetic viruses
(“Virosomes”)
UNIFR
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2003
Biolistic bombardment
or local direct injection
- limited area
Electroporation
- limited organ access
Liposomes, gene correction & Co.
- very inefficient transfer
General
- low transfer efficiency
- no or little genomic integration
Solutions:
- improved liposomes
with viral properties (“Virosomes”)
Ergo
 the future will see increasing interest in viral-like, but artificial particles
Not all gene therapy approaches are 'random shooting'
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2003
Ergo
 genotoxic
 non-genotoxic
Random integrating vectors





r-lentiviruses
r-retroviruses
r-AAV
plasmids (low frequency)
plasmids + transposase (eg 'sleeping beauty')
Specifically integrating vectors
Transient, non integrating vectors





adenovirus
plasmid
RNA virus based
oligonucleotides (SiRNA, antisense, ribozymes)
artificial chromosomes



hybrid vectors (HSV-AAV)
Phage 31 integrase-based
designer integrase
Gene correction vectors


chimeroplasts (RNA-DNA chimeric oligos)
single stranded DNA (homologous recom)
Which vector for which disease category
UNIFR
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2003
Disease Type
Most suitable vector
Justifications /Issues
Chronic Metabolic
AAV, Lenti, Adeno III, rretroviruses, repair oligo
persistence of expression of
the transferred gene,
minimize readministration
AAV, nonviral, Lenti
No rapid expression
necessary, persistence
required, low toxicity
Adeno II, Plasmid, oncolytic
recombinant viruses
rapid & transient expression
of cytotoxic or
immunomodulators
Adeno II, Plasmid,
modulatory oligonucleotides
Rapid and transient action
required
(ex. OTC, Gaucher,
Haemophilia, hematopoietic)
Local chronic or progressive
(ex. CNS, joints, eyes)
Solid tumors +/- metastat.
(cervical, breast, brain, skin)
Trauma or infection
(Ischemia, fracture, burn, wound,
acute infection, anaphyllaxis)
Technologies related to-, but not genuinely
definable as 'gene therapy'
Bioactive oligonucleotides





antisense
decoy dsDNA
decoy RNA
ribozymes DNAzymes
Si RNA
Oncolytic viruses



ONYX-15, ONYX-638 (r-adeno)
r-HSV
r-FSV
Implants of encapsulated cells


neurotrophic factor producer cell implants
hormone-producing cells
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2003
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'Classical' SGT models and strategies
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2003
Disease
transferred function
Clinical Results
ADA deficiency
ADA normal gene (enzyme)
(Immunodeficiency)
retrovirus, ex-vivo BM
1990 F. Anderson,
2002 C. Bordignon
Cystic Fibrosis
CFTR gene (chlorine transpor-
(Lung, Pancreas)
ter), retrov., aav, adenoII, local
Haemophilia B
Factor IX gene (clotting factor),
(Blood)
aav, adenoIII, intramuscular
1999-2000 M. Kay,
K. High
SCID
IL2R gene (gamma-C receptor)
2000 A. Fischer
(Immunodeficiency)
retrov., ex vivo BM
Limb ischaemia
VEGF gene (vascular growth
(Hands, Feet)
factor), plasmid, intramuscular
Cardiac ischaemia
VEGF gene (vascular growth
(Heart)
factor), plasmid, intracardiac
no significant results
in spite of several trials
1998 J. Isner
2000 J. Isner
additional 'popular' and emerging examples:
Morbus Gaucher, Morbus Parkinson, Crigler Njiar, OTC deficiency, Duchenne's MD, Restenosis control
UNIFR
Gene Therapy in the clinic: Trials Wordldwide
Rusconi
2003
trials
100
Ergo
 in spite of 13 yearresearch only less than
1% of the trials has
reached phase III
80
patients
As of December 2002:
632 registered protocols
1500
3472 treated patients
cancer
60
hered.
40
66% phase I
21% phase I-II
11% phase II
0.8% phase II-III
0.7% phase III
II
1000
I-II
I
500
vasc.
21% overall still pending
Infect.
or not yet Initiated !
20
www.wiley.com/genetherapy
1990 1992
1994
1996
1998
2000
Gene therapy in Switzerland: the 30 projects
financed by the NFP37 programme (1996-2001)
NFP37
Submissions
Granted
Total requested
Granted
phase A
(96-99)
30
19
32 Mio
7.6 Mio
phase B
(99-01)
26
18
9 Mio
6 Mio
DISEASE ORIENTATION
Cancer
Acquired disorders
Vector development
Hereditary disorders
Infectious diseases
8
2
5
2
1
10
7
3
4
2
RESEARCH LEVEL
Fundamental
Preclinical (animal models)
Clinical phase I
Clinical Phase II
Clinical Phase III
Ethical/social aspects
10
5
2
0
0
1
7
9
3
1
0
1
UNIFR
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2003
Nationales
Forschungsprogramm 37
NFP37
« somatic gene therapy »
www.unifr.ch/nfp37
Please Note
 the NFP37 represented at
most 30% of the Swissbased experimentation in
SGT during 1996-2001
Gene Therapy Clinical and Preclinical Milestones
UNIFR
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2003
Anderson, 1990
1990, 1993, 2000 // ADA deficiency
Isner, 1998
Dzau,
1999
F Anderson, M Blaese // C Bordignon
Kmiec,
1999
Fischer,
1997, 2000, Critical limb ischemia
Dickson, 2000
2000
J Isner († 4.11.2001), I Baumgartner, Circulation 1998
Aebischer, 2000
2002
Kirn,
1998, Restenosis
2000,
V Dzau, HGT 1998
2001
1999, Crigler Njiar (animal)
2002
C Steer, PNAS 1999
2000, Hemophilia
Intravascular adenoviral agents
M Kay, K High
in cancer patients:
2000, SCID
Lessons from clinical trials
A Fischer, Science April 2000
(review)
Bordignon, 2000 (ESGT, Stockholm)
2000, correction Apo E4 (animal model) 2002, science 296, 2410 ff)
G. Dickson, 2000 esgt, 2002 BBA
2000, correction Parkinson (animal model)
P Aebischer, Science, Nov 2000
2001, ONYX oncolytic Viruses
D Kirn (Cancer Gene Ther 9, p 979-86)
Two major SGT frustration cases
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2003
Muscular dystrophy
(incidence 1: 3000 newborn males)




requires persistence of expression
extremely large gene (14 kb transcript, 2 megaBP gene
unclear whether regulation necessary
unclear at which point disease is irreversible
Cystic fibrosis
(incidence 1: 2500 newborns)




luminal attempts failed because of anatomical /
biochemical barrier: no receptors, mucus layer
large gene that requires probably regulation
requires long term regulation
unclear at which point disease becomes irreversible
Although genes discovered in
the 90ties:
 no suitable vector
 no satisfactory delivery
method
The most feared potential side-effects of gene transfer
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2003

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
Ergo
 Most side effects are still related to the rather
primitive state of the vectorology/delivery
Three (four) bitter lessons, but only one
treatment-related death so far
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.
Paris, Jan 14, 2003, A Fischer:
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
UNIFR
Rusconi
2003
Public perception problems
UNIFR
Rusconi
2003
Negative perception of manipulative genetics


general aversion of genetic manipulation
fear of catastrophic scenarios
Confusion with other gene-based and
non-gene-based technologies



stem cell technology
human cloning procedures
genetically modified food
Deception after excessive promises


hopes reinforced by media spectacularisation and
over-simplification
deception after non-complied deadline
Other factors that have negatively influenced
the public perception and progress of gene therapy






UNIFR
Rusconi
2003
Naive statements by some good-willing scientists in the early 90ties
Not-so-naive statements by not-so-naive scientists in search of fame
Huge amount of money that flowed into the research and development
that attracted many incompetent researchers.
Concomitance with stock-market euphoria (little attention to realism)
Reckless statements or misreporting by greedy scientists or company managers
to increase the value of their stock options (memorandum by the ASGT on
conflict of interest 2000, www.asgt..org)
Tendency by the media to spectacularise good news and/or bad news
Ergo
 An explosive cocktail, just like for sports or arts,...
 the field tends to degenerate as soon as huge amounts of money are involved
and when the mass media become interested in it.
Ups and Downs of Gene Therapy:
a true roller coaster ride!
UNIFR
Rusconi
2003
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
whenever a reasonable cruise
speed was achieved, a major
adverse event has brought us
back square one
AAV
germline
in mice?
Adeno III
Lentivectors
in pre-clinic
NFP37
J. Wilson
J. Gelsinger
90
91
92
93
94
95
96
97
98
99
00
01
Adverse
events in
Paris
02 03
Genes, cells, tissue transplants...
some people fear possible negative developments
UNIFR
Rusconi
2003
Amelioration instead of
therapy?
Too High-tech
too expensive
Bioweapons?
Somatic Gene Therapy is facing fierce competition
UNIFR
Rusconi
2003
1. Cell Therapy (Stem cells (SC))




identified in many tissues
cell transfer could be combined with gene transfer
there would be no anatomical barriers for gene transfer
Selection /amplification of desired transformants
Current limitations of SC


Lack of control on differentiation and trans-determination
Difficulties in complex organ-reconstruction
2. Breakthroughs from the
small/medium molecules
 STI571 (Glivec)
 anti HER2 (Herceptin)
 Si RNA?
 ...
Future of SC:



Increasing number of SC types will be characterised
culturing conditions will be perfectioned
May replace in vivo gene transfer for treatment of chronic
conditions?
V
3. Challengers from
the biomechanics world
 bone reconstruction
 intelligent protheses (stents)
 micropumps
 artificial organs
UNIFR
Conclusions
Rusconi
2003
Fundamentally



a gene encodes usually more than one function
The therapeutic gene transfer in somatic cells must cope
with: efficiency, specificity, persistence and toxicity
many genes with potential therapeutic value have been
identified, and essentially all types of diseases can be
treated by gene transfer
Vectors and models



There is the choice of a certain number of viral and non
viral vectors, none of them being generally applicable
viral vectors have the advantage of efficiency and nonviral
vector the advantage of lower toxicity/danger.
viral vectors have the disadvantage of limited packaging
and some toxicity, while nonviral vector have the major
disadvantage of low efficiency of transfer
Clinically



over 600 trials and 3500 patients in 12 years
only a handful of trials is now reaching phase III
Progress further slowed down by periodical pitfalls
UNIFR
Perspectives
Rusconi
2003
Fundamental level & vectorology



the better understanding of gene interactions and
networking (functional genomics) could improve the
utilisation of gene-based or gene targeted strategies
novel paradigms can become available (Si RNA, PNA
triplex etc...)
specifically integrating gene constructs or artificial
chromosomes becoime more realistic
Preclinically


scaling up to larger animal models (dog and monkey)
permits better appreciation of dosage requirements
new transgenic models may give improved similarities to
human diseases
Clinically




Use of recombinant lentiviruses may be imminent
Increase of Phase III procedures over the next 5 years
First therapeutical applications may be registered within
3-5 years
challenge by other emerging therapies
...Thanks !
UNIFR
Rusconi
2003
ECPM
My collaborators at UNIFR
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: The Paris' trial
(see also www.unifr.ch/nfp37/adverse.html)
Disease
 deficiency of the receptor gamma(c)
 incapacity of maturing lymphocytes
 severe combined immunodeficiency
 lethal at 4 months if untreated
 survival 10 years under sterile conditions
Conventional treatments
 maintenance under sterile condition
 treatment with antibiotics
 transplant of matching bone marrow
Gene Therapeutical approach
 explant BM (3-6 month old)
 select CD34+
 transduce with retroviral vector encoding gamma(c)
 re-infusion, follow-up
UNIFR
Rusconi
2003
Discussion: The Paris' odyssey
(see also www.unifr.ch/nfp37/adverse.html)
UNIFR
Rusconi
2003
Chronology




1998 start treatment of patients
2000 publication results first 2 patients
2001/2002 publication further 8 patients
9 out of 10 responded well, back home, normal life
Adverse 1




summer 2002, high WBC in a 36 months patient
september 2002, hyper-proliferatory cells with insertion in proximity of LMO2 gene, notification authorities
October 2003, public disclosure, chemotherapy, good response, report at ESGT congress.
October 2003 3 US and 3 EU trials on hold
Adverse 2




december 2002, T cell hyper-proliferation in a second, 36 months patient
hyper-proliferatory cells also contain insertion of transgene close to LMO2 gene
January 2003, notification to authorities, public disclosure, treatment chemotherapy
January 2003, 27 US and 5 EU trials on hold
Discussion: Questions & hypotheses from the Paris' Trial
(see also www.unifr.ch/nfp37/adverse.html)
UNIFR
Rusconi
2003
Facts






in both patients insertion of the transgene in proximity of LMO2
this type of insertion not found in CD34+ cells in these patients
LMO2 expression is apparently increased in these patients
LMO2 gene already known as proto-oncogene involved in
some chromosomal-translocations found in some leukaemias
gamma(c) receptor can respond to IL-2, IL-5, IL-7, IL-9, IL-15,
Il-21 and ...
gamma(c) receptor is therefore itself a pro-proliferatory and
anti-apoptotic signaling molecule
Questions/hypotheses




is this adverse event specific for the disease status?
is the transgene contributing to the hyper-proliferatory potential?
is the gamma(c) synergising with LMO2?
Has there been such an adverse event in the over 20 retrovirally
transduced patients treated so far for other diseases?
Perspectives if the answers are
'YES'
'NO'
'UNK'
good
good
good
bad
bad
bad
bad
good
not good
not good
not good
not good
Discussion: 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
Discussion: 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