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Sandro Rusconi (09.03.52)
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
1995-today
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
Societies for
*essentielle
wiederholung
in Swiss
Genetik*
Experimental
Biology (USGEB)
*grundkonzepte
der Gentherapie*
*Klinische experimentiering in SGT*
Courmayeur,
*Hohe und Tiefe in der SGT*
March 2003
*Schlussfolgerungen und Perspektiven*
20 Oktober 2003
Liestal Biovalley
UNIFR
Rusconi
2003
2003: wohin führt uns
Gentherapie?
UNIFR
Rusconi
2003
*essentielle Wiederholungen in Genetik*
UNIFR
1 Gen
-> 1 oder mehrere Funktionen
1 Genom -> über Nx100'000 Funktionen
DNA
RNA
Rusconi
2003
Protein
Transcription / translation
Gene expression
GENE
2-5 FUNCTIONS
100 ’000 genes
(50 ’000 genes?)
>300 ’000 functions
(>150 ’000 functions)
UNIFR
1 Organismus -> 1013 Zellen,
verteilt und spezialisiert in Organe und Gewebe
Rusconi
2003
2 mm
2m
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
1 Cm3 Gewebe
 1'000'000'000 Zellen
Aber was istr eigentlich 'ein Gen'?:
eine regulierbare Nano-machine zur Herstellung von RNA
DNA
GENE
RNA
Rusconi
2003
Protein
Um wirksam
zu sein sollte ein transferierter Gen beinhalten:
Transcription
/ translation
FUNCTION



Sequenzen fuer Genregulation
Signale fuer reifung/transport der RNA
Signale fuer Uebersetzung in proteinen
RNA
DNA
spacer
UNIFR
regulatory
coding
spacer
UNIFR
Das reduktionistische Paradigma des Molekularbiologes
Rusconi
2003
DNA
GENE
Protein
Gentransfer kann beinhalte:
 transfer einer neuen Funktion, oder
 transfer einer kompensierenden F., oder
 transfer einer interferierenden Funktion
FUNCTION(s)
GENE OK
FUNCTION OK
GENE KO
FUNCTION KO
GENE transfer
FUNCTION transfer
UNIFR
Beispiele von Vererbbare Defekte
Rusconi
2003
Polygenic defects
(‘ frequent ’)
Type
estimated
min - max
genetics
behaviour
environment
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
estimated
Miopy
poly
3
- 4%
min - max
Polycystic kidney
poly
0.1
- 1%
Multi
7
- 27 %
Psoriasis
Multi (*)
2 Alzheimer
- 3%
Ergo:
Multi
1
- 3%
Schizofrenia
Multi (*)
0.5Parkinson
- 1%
Multi
4
- 8%
Scoliosis
Multi
3 Breast
- cancer
5 % einen Defekt
 Jedermann ist Träger
von (*)
mindestens
(*) Colon Carcinoma
Multi
0.1
- 1%
 Viele Defekte manifestieren sich erst spät im Leben (Anfälligkeiten)
(*) Obesity
Multi
0.5
- 2%
Alcolholism/
drug addiction Infektionsresistenz
Multi
0.5
3% etc...)
 Einige Anfälligkeiten sind (*)
positiv
(langlebigkeit,
Sum of incidences
(all defects)
min - max
32
- 83%
UNIFR
Das genom ist nicht das einzige determinant
des gesund-krang gleichgewichtes
genetics
Muscle distrophy
Familial Breast Cancer
Sporadic Breast Cancer
Lung Cancer
Obesity
Sogar die heilung von 'erworbene' Artherosclerosis
krankheiten kann genetisch bedingt sein:
 Trauma, Wunde
Alzheimer
 Brüche
Parkinson ’s
 Verbrennungen, Infektionen
 Vergiftungen
Drug Abuse
Homosexuality
Rusconi
2003
behaviour
environment
UNIFR
Rusconi
2003
*grundkonzepte in der Somatischen Gentherapie (SGT)*
UNIFR
Die 4 Aeren der molekularen Medizin
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
Somatische Gentherapie (SGT)
Definierung und Anwendungsbereich
Rusconi
2003
Chronic treatment
Definition of SGT:
'Use genes as drugs':
Correcting disorders by
somatic gene transfer
NFP37 somatic gene therapy
www.unifr.ch/nfp37
UNIFR
Acute treatment
Preventive treatment
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
Das Prinzip darf einfach sein,
aber der Teufel liegt häufig in den Details...
UNIFR
Rusconi
2003
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 Paris ...
counting votes...
! ask Florida's officials ...
gene therapy...
look at progress in 13 years...
UNIFR
Rusconi
2003
Pharmakologische Betrachtungen
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
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

Thérapies avec acides nucléiques



nécessitent de formulation en micro-particules
bien plus complexes que la pharmacologie conventionnelle
différent niveau de reversibilité (problème de dosage et de maitrise des effets indésirables
Wieso 'somatisch'?
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
UNIFR
Die vier Grundfragen bei der SGT
Rusconi
2003
Efficiency of gene transfer
Specificity of gene transfer
Persistence of gene transfer
Toxicity of gene transfer
Le variables
 welche Krankheit?
 Welches Gen?
 Welches Vektor?
 Welches Organ / Gewebe?
 Welche Transfermethode?
Remember!
Die drei Transfer-wege bei der SGT:
Ex-vivo
In-vivo
topical delivery
UNIFR
Rusconi
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
Die zwei klassen von 'Vektoren':
virale / nicht-virale
Transfert non viral
(transfection)
UNIFR
Rusconi
2003
A
B
Nuclear envelope barrier!
viral transfer
(Infection)
direct nuclear shuttling!
Effizienz der Transfektion mit rekombinante DNA
im vergleich zur Infektion mit rekombinante Viren
UNIFR
Rusconi
2003
Transfection
cells exposed to
1'000'000 particles/cell
12 hours
Infection
cells exposed to
3 particle/cell
30 min
Ergo
 das gentransfer mittels rekombinante Viren ist ueber 1'000'000-fach effizienter
als jene nicht-viral transfer methode
kleine Parade von Genransfervektoren
Adenovirus
Naked DNA
Adeno-associated V.
Liposomes & Co.
Retrovirus (incl. HIV)
Oligonucleotides
UNIFR
Rusconi
2003
UNIFR
Rusconi
2003
rekombinante Adenoviren
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, +++)
r4ekombinante Adeno-associated-virus (AAV)
UNIFR
Rusconi
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 problems are:
 insertional mutagenesis
 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, +++)
 Gaucher (clin, animal, +++)
 Brain Ischemia (animal, +++)
 Cystic fibrosis (animal, +/-)
Rekombinante Retroviren (inkl. HIV)
UUNIFR
Rusconi
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
Rusconi
2003
Reine oder komplexierte DNS
Approaches
Advantages / Limitations
Naked DNA injection /biolistic
Unlimited size capacity + lower
immunogenicity and lower bio-risk
of non viral formulations is
disturbed by
Naked DNA + pressure
Naked DNA + electroporation
Liposomal formulations
Combinations

Low efficiency of gene transfer

Even lower stable integration
Examples
 Critical limb Ischemia (clin, +++)
 Cardiac Ischemia (clin, +/-)
 Vaccination (clin, +/-)
 Anti restenosis (preclin. +/-)
UNIFR
Rusconi
2003
Oligonuklotide
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: Limitierungen der heutigen Genvektoren
Adenovirus
- no persistence
- limited packaging
- toxicity, immunogenicity
Retrovirus (incl. HIV) & AAV
- limited packaging
- random insertion
- unstable genome
General
- antibody response
- limited packaging
- gene silencing
- random insertion
Solutions:
- synthetic viruses
(“Virosomes”)
UNIFR
Rusconi
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”)
UNIFR
Rusconi
2003
*klinische Versuche in der SGT*
Der klassische klinische Weg: viel Zeit und Geld
year
event
costs U$D
0
Idea
0
2
Cell culture assays
0.5 Mio
5
Pre-clinical tests
animal models
2 Mio
Clinical phase I
5-20 patients
verify side effects
6 Mio
Clinical phase II
30-100 patients
dosis escalation
12 Mio
Clinical Phase III
>300- 1000 patients
multicentric
double blind
80 Mio
7
10
15
16>>
Registration / Availability
UNIFR
Rusconi
2003
This means:
assuming 20% of new developments
makes it to final registration,
the average investment is
300-500 Mio U$D
for each approved drug/procedure
Vernichten wir mindestens vier Mythen bei der
Gentherapie
Classical Gene Therapy Image

Hereditary disease
UNIFR
Rusconi
2003
Reality

Many acquired diseases can be treated
(ex. infections, traumatic lesions, tumors,...)

culprit gene must be known

'Short circuit' or symptomatic treatments
(ex. neurodeg. conditions with trophic factors)

requires 100% efficiency of transfer/expr.
 Few % sufficient for many diseases
(ex. hemophilia, limb ischaemia ...)

gene transfer/expression must persist  No persistence required in many cases
(ex. vaccination, cytotoxic antitumoral factors,
restenosis prevention, acute rejection prevention..
UNIFR
Trends bei der klinischen SGT experimentierung
Rusconi
2003
trials
100
Ergo
 en dépit de son age la
TGS peut compter
couramment seulement
1% d'essais en phase III
80
patients
As of August 2003:
660 registered protocols
1500
3672 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
Einige Meilensteine
UNIFR
Rusconi
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)
UNIFR
Rusconi
2003
*Die hoch- und tief-punkte ... *
Zwei besonders frustrierende Faelle:
Muskelschwund und Mucoviszidose
UNIFR
Rusconi
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
Trotz Isolierung der entsprechenden Genen in 1984
 kein geeignetes Vektor
 keine geeignete
Lieferungsmethode
Die mesit befuerchtete Nebeneffekte der Gentherapie
UNIFR
Rusconi
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
 die Nebeneffekte waren nicht so scvheinbar wenn
SGT ineffizient war
 Heute muessen wir diese Nebeneffekte seriös
betrachten
4 bittere Feststellungen aber nur einen Patient bis jetzt
direkt an SGT gestorben
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
Der klinische Versuch in Paris X-SCID
(A. Fischer, Hôpital Necker)
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
Die Odyssee des klinischen Versuchs in Paris
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.
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
UNIFR
Die Fragen
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
UNIFR
Anhäufung von hoch- und tief-Punkte: ein Rollercoaster!
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
Weitere Faktoren die zum schlechten 'Image'
der Gentherapie beigetragen haben

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
 zuviel geld und spekulation: ein explosiver cocktail,
wie beim Sport oder Kunst...
UNIFR
Rusconi
2003
UNIFR
Rusconi
2003
*Conclusions & Perspectives*
Schlussfolgerungen
UNIFR
Rusconi
2003
Grundkonzepte


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
Vektoren und Modelle



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
Klinische Versuche



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
QuickTime™ et un décompresseur
Sorenson Video 3 sont requis pour visualiser
cette image.
Perspectiven: SGT wird weiter fortschreiten
trotz schwieruigkeiten und unvermeidbare Unfälle
UNIFR
Rusconi
2003
Grundlage Forschung und 'Vektorologie'



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 become more realistic
Praeklinische Forschung


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
Klinische Forschung




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
Ergo


der grösste teil der fehler waren
menschliche Fehler
Die Huerde koennen bewältigt
werden.
...Danke ... und bleiben wir optimistisch
UNIFR
Rusconi
2003
Biovalley Program
Gymnasium Liestal
My collaborators at UNIFR
Swiss National Research Foundation
Danke fuer die aufmerksamkeit
und fuer spezielle Fragen, bitte
schreiben sie an:
[email protected]
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END, let's open the Discussion
UNIFR
Rusconi
2003
***Diskussions-slides...
UNIFR
Rusconi
2002
text
 ttt
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
Rusconi
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
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
UNIFR
Rusconi
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 ischemia)
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)
UNIFR
Rusconi
2003
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
Die Hauptkrankheit des 21. Jahrhunderts: Veralterung
1920
1940
1960
1980
199
1900
M
E3/E4
E4/E4
80
2000
Pas toutes les stratégies de transfert se basent sur un
ancrage au hasard




Rusconi
2003
Ergo
 genotoxic
 non-genotoxic
Random integrating vectors

UNIFR
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)
Mais un virus c'est quoi?
Une machine auto-réplicative extrèmement efficace
UUNIFR
Rusconi
2003ß
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
UNIFR
Rusconi
2002
Comment peut-on construire des virus récombinants?
rp
E
L1 L2
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
D'autres tecnolgies émergentes entrent en compétition
brutale avec la thérapie génique
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
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?
Rusconi
2003
2. Challengers from the
small/medium molecules




STI571 (Glivec)
anti HER2 (Herceptin)
Si RNA?
...
3. Challengers from
the biomechanics world




V
UNIFR
bone reconstruction
intelligent protheses (stents)
micropumps
artificial organs
La génétique est practiquée depuis de millénaires, la
biologie moléculaire seulement depuis 30 ans
100’000 b.C.
Empirical genetics
10’000 b.C.
Biotechnology
2000 a.d.
Molecular biology
2001 a.d, Genomics
UNIFR
Rusconi
2003
UNIFR
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


increases specificity of conventional therapy (cancer)
increases efficacy of conventional therapy (hemophilia)
Life quality burden of patient


costs of enzyme therapy (ex. ADA)
burden of daily injections (ex. Insulin)
Which vector for which disease category
UNIFR
Rusconi
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)
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 production (existence or not of
packaging cell systems)
Emotional problems linked to pathogenicity of donor
vectors (ex. lentiviruses)
UNIFR
Rusconi
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
UNIFR
Rusconi
2003
The THREE missions of medicine
Prevention
+
'Molecular Medicine'
Diagnosis
Application of the
know-how in
molecular genetics
to medicine
+
+
Therapy
Ideal properties of a systemically delivered
non-viral formulation
Stability


UNIFR
Rusconi
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
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
*d Why so many cancer trials?
UNIFR
Rusconi
2002
Better benfit/risk balance and high emotional acceptance
(terminal patients, ethical committees)
Market potential higher than monogenic diseases
(most thereof being orphan diseases)
Many more diversified approaches envisageable than in
monogenic diseases
Much higher number of patients/center than in
monogenic diseases
*d Ethical dilemmas can sometimes slow down progress
and hamper objective appreciations
ADA gene therapy 1990-1999
Feeling:
It ’s time we face reality, my friends…
We ’re not exactly rocket scientists
vector
« it does not work »
ADA gene therapy 2000
« it always worked but we couldn ’t know »
The PEG-ADA ethical dilemma has prevented earlier results recognition
(Bordignon, ESGT meeting Stockholm, 9.10.2000, Science July 2002 )
UNIFR
Rusconi
2002
*d Cancer molecular treatment Example:
Oncolytic viruses on the example of ONYX-015
UNIFR
Rusconi
2002
A) Normal Adenovirus
 can propagate in virtually all cells
B) ONYX-015
 deleted E1B function
 can propagate efficiently only in
P53 -deficient cells (e.g. most cancer cells)

Clinical success Head & Neck Cancer

Awaiting for further successes
(currently in Phase II and III)

expected to be useful in combination with
conventional therapy




ADVANTAGE:
the 'drug' has its own dynamics
DISADVANTAGE:
danger of evolving viruses
unclear if it works in adeno-immune
patients
unclear if if works in immunocompromised patients (chemotherapy)
*d 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
*d Examples of gene transfer treatments against cancer
UNIFR
Rusconi
2002
Type of treatment





31% of protocols
(very strong potential, low
relapse chances)

5 % of protocols
(very limited application,
laborious, not widely explored)

26 % of protocols
(good effect but low bystander
and likely relapse)
Transfer chemoresistance genes in
immune cells
Tumor modulation

38% of protocols
(strong complementary effect,
possible relapse)
Transfer of Immuno-attracting functions
Instruction of immune cells
Immunoprotective


Prodrug activation
Tumor-specific cytotoxic expression
Immunostimulatory

percentage of trials
(therapeutic potential)
examples
Directly cytotoxic


Restore tumor-suppressor functions
down-regulate pro-oncogenic functions
*d Gene correction strategies
(independent of delivery vectors)
UUNIFR
Rusconi
2002
Approaches
Advantages / Limitations
Trans-splicing mRNA
All extremely promising approaches
which could permit the treatment of
dominant defects. They do not require
specificity of delivery, and are not
subject to gene silencing. Also they
would permit from the biosafety point of
view germ line correction, However the
major issue today is:
Ribozymic splicing of RNA
Chimeroplasts-induced repair

Controversial efficiency of repair
Triple-helix-guided repair
Homologous recombination
Examples
 Crigler Njiar (animal, +++ 30%)
 M. Distrophy (animal, +++ 20%)
 Correction Apo E 4 (animal, +++ 20%)
 Correction Albino (animal, +/- <1%)
*d Gene therapy ‘ FAM ’ (Frequently Asserted Myths…)
UNIFR
Rusconi
2002
Gene therapy ...
Isner, 1998
Kmiec, 2000
1999
Fischer,
...is withdrawing huge public funding
...will be invariably expensive
...is solely targeting hereditary diseases
Wrong: 1998: USA < 2% of NIH budget,
CH less than 1% of NF budget
Wrong: example DNA based
vaccination
Wrong: Cancer, Cardiovascular,
Restenosis, Ischemia, ...
...is based only on a ‘shotgun insertion'
Wrong: J. Isner/ I. Baumgartner
(ischemia), A. Fischer (SCID), C
Bordignon (ADA); M. Kay (Haemophilia,
Wrong:
Gene(cancer),
correction
F McCormick
…strategies,
...is a dangerous procedure
Wrong: 1 death out of >3000 patients
...will be imediately boosted by genomics
Wrong: it will take long time to benefit
from genomics knowledge
...has not yet proven its therapeutic value