Transcript DIMENSIONS

UNIFR
Sandro Rusconi (09.03.52)
Rusconi
2005
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
Sept 08, 2005
GTRV Debio
What have we
learned from 15
years in gene
therapy?
Gene therapy: A 15-years hailstorm of
highly emotionalised good and bad news
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2005
BBC, NBC, CNN,...
New York Times
Washington Post
Times
No previous medical procedure
Le Monde
generated
that many
discussions
so long Allgemeine
before
Frankfurter
C Bordignon, Milano trial May 2002
being ever clinically applicable
...
How manyNature
of you have heard
mostlyScience
bad news... ?
mostlyNEJM
good news...?
Internet
...
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1 Gene -> 1 or more functions
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2005
DNA
RNA(s)
Protein(s)
Transcription / translation
Gene expression
Ergo
GENE
100 ’000 genes
(50 ’000 genes?)

to say
2-5 FUNCTIONS
'one gene -> one function'
is like pretending
'one disease -> one drug'
Multifunctional character of genes implies:
>300
’000
functions
 cross talk with
different
pathways
(>150 ’000
functions)
 unclarified hyerarchical
position
 unclarified side-effects potential
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Recap: what is a gene?:
a regulated nanodevice for RNA production
DNA
GENE
RNA(s)
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2005
Protein(s)
Therefore, to fullfil its role,
Transcription / translation
a transferredFUNCTION
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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1 Organism -> more than 105
developmentally and genetically-controlled functions
2 mm
2m
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2005
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
Remember
1 Cm3 of tissue
 1'000'000'000 cells!
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Reductionistic molecular biology paradigm
(gene defects and gene transfer)
DNA
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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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Gene therapy as logical consequence: 'the third era'
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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
Ergo

1000
85 90 95 00
gene transfer is a80
logical
development of molecular biology
Somatic Gene Therapy (SGT) definition
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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
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Why 'somatic'?
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
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
 germline changes are
avoided also because of
ethical problems
Requestioned?
 whenever genomic repair
systems will be perfectioned
the issue of germ line
will probably
be the other cells of the body
 therapy
Somatic
Cells: all
readdressed.

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
When/where/ may be SGT (currently) indicated?
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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


increases specificity of conventionalPerverse
therapy (cancer)
deviation dreams
increases efficacy of conventional therapy
(with (hemophilia)
current technologyI:
gene-based sports doping
Life quality burden of patient
 performance amelioration
 costs of enzyme therapy (ex. ADA)
 cosmetics


burden of daily injections (ex. Insulin)
Pharmacological considerations for DNA transfer
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Classical Drugs





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
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
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
Ergo: Therapy with nucleic acids
 requires particulated formulation
 is much more complex than previous drug deliveries
 has a different degree of reversibility (intrinsic dosage / titration problem)
SGT's FOUR fundamental questions & players
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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?
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THREE classes of anatomical gene delivery
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Ex-vivo
In-vivo
topical delivery
In-vivo
systemic delivery
Ergo

V
Examples:
- bone marrow
- liver cells
- skin cells
ex vivo or local delivery are
currently preferred over systemic
delivery
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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Ergo
a


viral transfer is much more efficient
nonviral transfer must solve a
number of hurdles
Viral gene transfer
(Infection by r-vectors)
b
Nuclear envelope barrier!
see, Nature Biotech
December 2001
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Transfection versus Infection
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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)
Comparing 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 manufacturing (existence or not of
packaging cell systems)
Emotional problems linked to pathogenicity of donor
vectors (ex. lentiviruses)
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Nonviral vectors

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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
manufacturing are different between viral and non-viral vectors
 when ignoring thir low efficiency, nonviral vectors appears largely superior
Short list of popular vectors/methods
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r-Adenovirus
Naked DNA
r-Adeno-associated V.
Liposomes & Co.
r-Retrovirus (incl. HIV)
Oligonucleotides
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Recombinant Adenoviruses
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Manufacturing
Advantages / Limitations
Generation I/ II
8 Kb capacity Generation I / II
>30 Kb capacity Generation III
Adeno can be grown at very high titers,
However
 Do not integrate in host genome
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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Manufacturing
Advantages / Limitations
Helper-dependent production
Persistence in the genome permits longterm expression, high titers are easily
obtained, immunogenicity is very low,
However the major problems are:
 insertional mutagenesis
 Promotes autoimmunity?
 Small capacity (<4.5 kb) which does
not allow to accommodate large genes
or gene clusters.
Helper independent production
Cis-complementing vectors
Co-infection
Examples
 Hemophilia A (clin, animal, +++(autoimm?)
 Gaucher (clin, animal, +++)
 Brain Ischemia (animal, +++)
 Cystic fibrosis (animal, +/-)
 retinopathy (animal (+/-)
Recombinant retroviruses (incl. HIV)
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Manufacturing
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 in manufacturing
Self-inactivating RV
Combination viruses
Examples
 SCID (IL2R defect, Paris) (clin, +++)
 Adenosine Deaminase deficiency (clin, +++!!!)
 Parkinson (preclin, +++)
 Anti cancer (clin +/-)
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Naked or complexed DNA
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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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Oligonucleotides
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Approaches
Antisense
Ribozymes
DNAzymes
Advantages / Limitations
reversible (except gene correcting oligos),
easy manufacturing, easy delivery
these procedures may be suitable for :

handling dominant defects

transient treatments (gene modulation)
Triple helix

permanent treatments (gene correction)
Aptamers
efficacy still questionable in most cases
Decoy / competitors
Examples
 Anti cancer (clin,preclin., +/-)
 Restenosis (clin, +++)
√!
 Muscular Distrophy (animal, +++)
SiRNA
Gene-correcting oligos
Recap: current limitations of popular 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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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”)
Technologies related to-,
but not all genuinely definable as 'gene therapy'
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Transiently bioactive oligonucleotides




antisense
decoy dsDNA, decoy RNA
ribozymes DNAzymes
Si RNA oligonucleotides
Genuine gene therapy oligos

chimeroplasts (*gene correction induction)
Ergo
Oncolytic
viruses
among
all the above,



SiRNA is among
from www.nature.com
theONYX-638
most promising
inhibitor factors,
ONYX-15,
(r-adeno)
r-HSV and can conceived as transienttly
acting oligo (improper gene therapy)
Implants of encapsulated cells
r-FSV or as permanently expressed from
 neurotrophic factor producer cell implants
DNA vectors

hormone-producing cells
Gene Therapy in the clinics: Trials Worldwide
(cumulative)
trials
80
60
40
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2005
patients
Ergo

100
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
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.
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
Gene Therapy Clinical and Preclinical Milestones
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1990, 1993, 2000, 2004 // ADA deficiency
F Anderson, M Blaese // C Bordignon
1997, 2000, Critical limb ischemia
J Isner († 4.11.2001), I Baumgartner, Circulation 1998
1998, Restenosis
V Dzau, HGT 1998
2000, Hemophilia
M Kay, K High
Anderson, 1990
Isner, 1998
Dzau, 1999
Fischer,
Kirn, 2000
2000, 2002
Manuel
Grez
2001 Sibiono
Hans
Hossle
2002Peter
Shenzen
Reinhard
Seger
2003
Intravascular 2004
adenoviral agents
in cancer patients:
21 lives
veryLessons
encouraging
data
from
from
clinical
trials
were
so
far
documentedly
saved
by
GT
in
2000, 2002, X-SCID
just initiated (review)
clinical trial,
european
A Fischer, Science April 2000, UK trials
2003 trials (x-SCID, ADA, CGD)
(France, UK, Italy)prospected
(all in phase>10
I) patients
2000Approved
(ESGT, Stockholm)
commercialisation
lives quality-improved
2001, 2003 ONYX oncolytic Viruses ~200 Bordignon,
2002,other
science
296,
2410
ff)
ofI Gendicine
(Jan 2004) for
in several
phase
and
II trial
D Kirn (Cancer Gene Ther 9, p 979-86)
~nnn lives saved or quality-improved
? in China
cancer treatment
2004, Chronic Granulomatous Disease
by Gendicine (still undocumented)
M Grez Frankfurt; R Seger Zürich
2004, Gendicine (adeno-p53 vector)
L Peng, Sibiono Inc, Shenzen, China
Two persisting major SGT frustration cases
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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)




most 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
In spite of genes discovered
in the 90ties:
 lacking suitable vector
 no satisfactory delivery
method
 no persistence
 treatment 'too late'
The most feared potential side-effects of gene transfer
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
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».
 SGT enjoyed a side-effect-free illusion during its
first 10-year of non-working early period
 Many side effects are still related to the rather
primitive state of the vectorology/delivery
SAEs1: established cases: acute and long term SAEs:
from Gelsingers' death to Paris' Leukaemias
NY May 5, 1995, R. Crystal:
adenovirus, cystic fibrosis (lung)
one patient mild pneumonia-like condition
Trial interrupted and many others on hold.
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:
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Most Recent Paris' Trial News
discussed at:
www.unifr.ch/nfp37/adverse03.html
it is now rather established (2004) that the
Paris' leukaemia events were caused by
treatment-specific circumstances (type of
transferred gene, dosing, type of vector,
predisposition)
The third SAE might delay the nextly
restart of patients recruitment
retrovirus , x-SCID (bone marrow)
planned
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)
Parenthesis: future solutions to insertional mutagenesis:
targeted gene transfer approaches




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2005
Ergo
 genotoxic
 non-genotoxic
Random integrating vectors

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r-retroviruses
r-lentiviruses
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
Ergochromosomes
vector systems that allow specific or at least
better location-controlled gene delivery are
experimentally well advanced (see
accompanying text)



hybrid vectors (HSV-AAV)
Phage 31 integrase-based
designer integrases (ZnFinger proteins)
Gene correction vectors


chimeroplasts (RNA-DNA chimeric oligos)
single stranded DNA (homologous recom)
SAEs2: emerging cases mid-term effects documented by
recent Autoimmunity Reports
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Blood, 1 May 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.
- delivered the homologous EPO cDNA driven by ubiquitous and/or regulatable
promoters via AAV vectors injected in muscle or aerosolized in lung, resulting in
supra-physiologic serum levels of EPO, from 10- to 100 000- fold over the baseline
K High, ASGT June meeting 2004
Ergo
somatic gene transfer can
generate mid-term auto- immunity
under certain circumstances
[Abstract1002] Immune Responses to AAV and to
Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed
Gene Transfer for Hemophilia B
SAEs3: Non-science factors that have disturbed progress
and image of gene therapy

'Naive' statements in the early 90ties

Excess of speculative financing in mid-late 90ties.

Concomitance with stock-market euphoria

Reckless statements/promises or misreporting in late 90ties

Tendency by the media to spectacularise good and/or bad news
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Ergo
 too much money, too much time pressure, too much media
exposure among the image killer factors.
 The fundamental error: we pretended making a business issue
out of a scientific issue
Ups and Downs of Gene Therapy:
a true roller-coaster ride!
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>90
high
Ergo
R. Crystal
V.Dzau
whenever
a reasonable cruise
Adeno I
speed was achieved, a major
adverse event has brought us
F Anderson
back «square one» or even
NIH
below
Motulski
mood

C Bordignon
Adeno III
AAV
germline
in mice?
Lentivectors
16
Low
4
companies
2005
J. Isner
report
25
A. Fischer
M. Kay
J. Gelsinger
Paris I and II
Leukaemias
lentivectors
hopes
gendi
?
cine
Auto?
immunity
?
5
Paris III
90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05
Conclusions: GT has proven several concepts, has several
tools, but is still in the pioneering phase
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Fundamentally



many new potentially therapeutic genes identified
All types of diseases can be virtually treated by gene
transfer
we start to manage efficiency, specificity, persistence and
toxicity
Vectors and models





Choice of among a number of viral and non viral vectors
Viral vectors have the advantage of efficiency
nonviral vector the advantage of lower toxicity/danger.
Viral vectors have the disadvantage of limited packaging
and some toxicity
nonviral vectors have the major disadvantage of low
efficiency of transfer
Clinically




over 600 trials and >4000 patients in 15 years
only a handful of trials is now reaching phase III
Progress further slowed down by periodical pitfalls
1 product/treatment approved in China 2004 (gendicine)
Ergo
 we are somewhat ahead but still
in the pioneering phase !

«failure of evidence» does not
mean «evidence of failure» !
Perspectives: somatic gene therapy will progress
in spite of all past, present and future incidents/accidents
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Fundamental level & vectorology





Better understanding of gene interactions and networking
Gene inhibition through Si RNA, Zn finger
specifically integrating gene constructs
artificial chromosomes become more realistic
novel, semi-artificial particles
Preclinically


scaling up to larger animal models (dog and monkey)
new transgenic models may give improved similarities to
human diseases
Ergo

Clinically




Use of recombinant lentiviruses
Increase of Phase III procedures over the next 5 years
therapeutical applications may be registered within 3-5
years
challenge by other emerging therapies



many adverse events were due rather
to human errors than to intrinsic
dangers
other undesired effects are due to
prototypic state of tools
hurdles can be overcome
the genuine potential of SGT is
intact
Proust's questionnaire to myself and to you,
concerning gene therapy
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2005
will GT ever make it into routine clinical practice ?
yes
The most worrying side-effect?
immunity
Is insertional mutagenesis an important hurdle?
No
Which will bloom: viral or non viral transfer?
combination thereof
Who will 'win' the race: gene transfer or cell therapy?
both or neither
Will GT be applicable also for non-severe conditions?
yes
Which will be the best inhibitor function: antisense,
intrabodies, aptamers, ribozymes, SiRNA, designer Zn
Fingers, triple helix, small drugs, ...whatever ?
...whatever
...Thanks, and let's remain optimistic
UNIFR
Rusconi
2005
GTRV Debio summer school
Sergio Capancioni, Christiane Damgé
The other organisers
Ergo
Thank you all for the patience and
attention,
[email protected]
or visit:
www.unifr.ch/nfp37/

let's look forward
to a safe landing
UNIFR
That's all, folks!
Rusconi
2005
www.unifr.ch/nfp37
UNIFR
Rusconi
2004