Transcript DIMENSIONS

Sandro Rusconi (09.03.52)
UNIFR
Rusconi
2004
1972-75
1975-79
1979-82
1982-84
1984-86
1987-93
1994-today
1995-today
2002-03
2002-05
2003-06
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, PD
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)
Euregenethy Network
Oct. 10, 2004
SUK education Program
'genetic regulation of
cardiovascular functions'
Tools for genetic
modification
Gene therapy: A 14-years hailstorm of
highly emotionalised good and bad news
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2004
BBC, NBC, CNN,...
New York Times
Washington Post
Times
No previous medical procedure
Le
Monde
generated so many discussions
Frankfurter
Allgemeine
Cever
Bordignon, Milano trial May 2002
so
long before being
... clinically applicable
Nature
Science
NEJM
Internet
...
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1 Gene -> 1 or more functions
Rusconi
2004
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 implies:
>300
’000pathways
functions
 cross talk with
different
(>150 ’000position
functions)
 unclarified hyerarchical
 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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2004
Protein(s)
Therefore, to fulfil its role,
Transcription / translation
a transferredFUNCTION
gene 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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2004
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
Remember
1 Cm3 of tissue
 1'000'000'000 cells!
Reductionistic molecular biology paradigm
(gene defects and gene transfer)
DNA
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2004
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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Somatic Gene Therapy (SGT)
Rusconi
2004
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 SGT principle is simple Yes,...
but the devil is often in the details
There are many things that are simple in principle, like...
getting a train ticket...
! try this 5 min before departure
and with a group of Chinese
tourists in front
parking your car...
! try this at noon, any
given day
in Zuerich or Geneva
...
counting votes...
! ask Florida's
officials ...
gene therapy...
look at progress
in 13 years...
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2004
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Why 'somatic'?
Rusconi
2004

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
When/where/ may be SGT (currently) indicated?
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2004
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 conventionalPerfid
therapy
(cancer) dreams
deviation
increases efficacy of conventional therapy
(with (hemophilia)
current technologyI:
gene-based doping
Life quality burden of patient
 performance amelioration
 costs of enzyme therapy (ex. ADA)
 cosmetics


burden of daily injections (ex. Insulin)
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?
Pharmacological considerations for DNA transfer
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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
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
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)
THREE classes of anatomical gene delivery
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Ex-vivo
In-vivo
topical delivery
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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a
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) ('MAD' !)
Emotional problems linked to pathogenicity of donor
vectors (ex. lentiviruses)
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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
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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2004
r-Adenovirus
Naked DNA
r-Adeno-associated V.
Liposomes & Co.
r-Retrovirus (incl. HIV)
Oligonucleotides
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2003
Recombinant Adenoviruses
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, +++)
Recombiant 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 of production
Self-inactivating RV
Combination viruses
Examples
 SCID (IL2R defect, Paris) (clin, +++)
 Adenosine Deaminase deficiency (clin, +++!!!)
 Parkinson (preclin, +++)
 Anti cancer (clin +/-)
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2003
Naked or 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
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2004
r-Adenovirus
- no persistence
- limited packaging
- toxicity, immunogenicity
r-AAV
- no integration in host g.
- very limited packaging
- promotes autoimmunity?
r-Retrovirus (incl. HIV)
- limited packaging
- random insertion
- unstable genome
General
- antibody response
- limited packaging
- gene silencing
- Manufacturing limitations
Solutions:
- synthetic viruses
(“Virosomes”)
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
Solutions:
- improved liposomes
with viral properties (“Virosomes”)
Ergo
 the future will see increasing interest in viral-like, but artificial particles
Technologies related to-,
but not all genuinely definable as 'gene therapy'
Transiently bioactive oligonucleotides




antisense
decoy dsDNA
decoy RNA
ribozymes DNAzymes Si RNA
oligonucleotides
Genuine gene therapy oligos

chimeroplasts (*gene correction induction)
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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Which vector for which disease category
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2004
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)
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'Classical' SGT models and strategies
Rusconi
2004
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
SCID
IL2R gene (gamma-C receptor)
(Immunodeficiency)
retrov., ex vivo BM
2000 A. Fischer
2002, UK trials
Limb ischaemia
VEGF gene (vascular growth
1998 J. Isner
(Hands, Feet)
factor), plasmid, intramuscular
Cardiac ischaemia
VEGF gee (vascular growth
(Heart)
factor), plasmid, intracardiac
no significant results
in spite of several trials
1999-2000 M. Kay,
K. High
2000 J. Isner
additional 'popular' and emerging examples:
Morbus Gaucher, Morbus Parkinson, Crigler Njiar, OTC deficiency, Duchenne's MD, Restenosis control
Gene Therapy in the clinics: Trials Worldwide
(cumulative)
trials
80
60
40
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2004
patients
Ergo

100
UNIFR

in spite of 13 year- research only
less than 2% of the trials has
reached phase III
not necessarily due to the «novel»
'fail early, fail fast'cancer
paradigm
As of April 2004:
918 cumulative protocols
(90-04)
1500
4500 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.
21% 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 saved
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
(ESGT, Stockholm)
Approved
commercialisation
life qualities2000
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)
cancer treatment in China
2004, Chronic Granulomatous Disease
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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2004
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
Although genes discovered in
the 90ties:
 lacking suitable vector
 no satisfactory delivery
method
The most feared potential side-effects of gene transfer
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2004

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
can produce.
 SGT enjoyed a side-effect-free illusion during its
10-year non-working early period
 Many side effects are still related to the rather
primitive state of the vectorology/delivery
SAEs1: earlier cases: acute and long term SAEs: from
Gelsinger to Paris Leukaemiaa
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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:
!! Most Recent Paris' Trial News
In the Discussion or at:
www.unifr.ch/nfp37/adverse.html
adenovirus , OTC deficiency (liver)
one patient (Jesse Gelsinger) died of a severe septic shock. it is now rather established (2004) that the
Many trials were put on hold for several months (years).
leukaemia events were caused by treatment
Paris, Oct 2, 2002, A Fischer:
specific circumstances (Type of transferred
gene, dosing, type of vector, predisposition)
retrovirus , x-SCID (bone marrow)
one patient developed a leukemia-like condition.
Ergo
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
gene therapy can produce short
term and long term effetcs
Not all gene therapy approaches are 'random shooting'
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2004
Ergo
 genotoxic
 non-genotoxic
Random integrating vectors





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 chromosomes



hybrid vectors (HSV-AAV)
Phage 31 integrase-based
designer integrase
Gene correction vectors


chimeroplasts (RNA-DNA chimeric oligos)
single stranded DNA (homologous recom)
SAEs 2:mid-term effects:
Recent Autoimmunity Reports
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2004
Blood, 1 May 2004, Vol. 103, No. 9, pp. 3248-3249
Autoimmunity in EPO gene transfer (macaques)
Els Verhoeyen and François-Loïc Cosset
- 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.
Ergo
K High, ASGT June meeting 2004
somatic gene transfer can
[Abstract1002] Immune Responses to AAV andgenerate
to Factor mid-term self immunity
IX in a Phase I Study of AAV-Mediated, Liver-Directed
under inappropriate
Gene Transfer for Hemophilia B
circumstances
Non-science factors that have negatively influenced
the public perception and progress of gene therapy

'Naive' statements in the early 90ties

Excess of speculative funds in mid-late 90ties.

Concomitance with stock-market euphoria (little attention to realism)

Reckless statements/promises or misreporting in late 90ties

Tendency by the media to spectacularise good and/or bad news
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2004
Ergo
 An explosive cocktail, just like for sports or arts,...
 the field tends to degenerate as soon as excessive amounts
of money are involved and when the mass media become
overly interested in it.
 The 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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high
R. Crystal
Adeno I
V.Dzau
mood
NIH
Motulski
report
Ergo

C Bordignon
J. Isner
F Anderson
Low
2004
A. Fischer
M. Kay
whenever a reasonable cruise
speed was achieved, a major
adverse event has brought us
back square one
Adeno III
AAV
germline
in mice?
lentivectors
in clinics?
promising
results
2003-2004
Lentivectors
J. Gelsinger
Paris
Leukaemias
Autoimmunity
?
Epo
Factor IX?
90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05
?
Conclusions 1: in spite of the many hurdles, GT has
already saved >20 otherwise condemned lives
and keeps producing positive signals
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X- SCID trials


France: 9/10 patients permanently cured of the lethal
disease X-SCID
UK: 8/8 patients cured of X-SCID lethal condition
ADA deficiency

C Bordignon trials 4/4 patients permanently corrected +
detoxified
Others




significant amelioration of CLI condition in Phase II trials
important therapeutic benefit with oncolytic viruses
promising amelioration in hemophilia patients
First gene medicine product registered in China by
Sibiono Inc. (see www.unifr.ch/sibiono.html
Ergo

gene therapy's principle works
Conclusions 2: GT has proven several concepts, has
several tools, but is still in the pioneering phase
UNIFR
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2004
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
Ergo

we are somewhat ahead but still in
the pioneering phase !

«failure of evidence» does not mean
«evidence of failure» !
Clinically



over 900 trials and >4000 patients in 14 years
only a handful of trials is now reaching phase III
Progress further slowed down by periodical pitfalls
Perspectives: somatic gene therapy will progress
in spite of all past, present and future adverse events
UNIFR
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2004
Fundamental level & vectorology




Better understanding of gene interactions and networking
Gene inhibition through Si RNA
specifically integrating gene constructs
artificial chromosomes become more realistic
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
First therapeutical applications may be registered within
3-5 years
challenge by other emerging therapies




many adverse events were due more
to human errors than to intrinsic
dangers
other undesired effects are due to
primitive state of tools
hurdles can be overcome
the genuine potential of SGT is intact
...thanks, and let's remain optimistic
UNIFR
Rusconi
2004
SUK education program
Zhihong Yang
Swiss National Research Foundation
Thank you all for the attention,
[email protected]
or visit:
www.unifr.ch/nfp37/
UNIFR
That's all, folks!
Rusconi
2004
www.unifr.ch/nfp37
UNIFR
Rusconi
2004
Discussion: Recap: what is a virus ? ->
A superbly efficient replicating nanomachine
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