Transcript DIMENSIONS

Sandro Rusconi (09.03.52)
UNIFR
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 15, 2005
Uniklinik Balgrist
'Therapeutische'
Gentherapie:
Stand 2005
Eigentlich gemeint war:
Therapeutischer
Gentransfer: Stand 2005
Gene therapy: A 15-years hailstorm of
highly emotionalised good and bad news
UNIFR
Rusconi
2005
BBC, NBC, CNN,...
New York Times
Washington Post
Times
Selten hat eine medizinische
LesoMonde
Technik
viel Rauch und so
wenig
Feuer produziert
Frankfurter
Allgemeine
C Bordignon, Milano trial May 2002
...
How many of you have heard
mostlyNature
bad news... ?
mostlyScience
good news...?
NEJM
Internet
...
UNIFR
1 Gen -> 1 oder mehrere Funktionen
Rusconi
2005
DNA
RNA(s)
Protein(s)
Transcription / translation
Gene expression
Ergo
GENE
100 ’000 genes
(50 ’000 genes?)

'ein Gen -> eine 2-5
Function'
FUNCTIONS
ist so falsch wie
'eine Krakheit -> ein Medikament'
Multifunctional character of genes implies:
>300
’000
functions
 cross talk with
different
pathways
(>150 ’000
functions)
 unclarified hyerarchical
position
 unclarified side-effects potential
UNIFR
Recap: was ist ein Gen?:
ein grosses Molekuel mit informativem Inhalt
DNA
GENE
RNA(s)
Rusconi
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
UNIFR
1 Organismus -> mehr als 105
entwicklungsgenetisch kontrollierte Funktionen
Rusconi
2005
2 mm
2m
0.2mm
0.02mm
0.001mm
DNA
RNA
Protein
Zur Erinnerung
1 Cm3 Gewebe
 1'000'000'000 Zellen!
UNIFR
Gentherapie als logische Folge: die dritte Aera
Rusconi
2003
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
Somatische Gentherapie (SGT): Definition
UNIFR
Rusconi
2005
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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Wieso 'somatisch'?
Rusconi
2005

Germ Line Cells: the cells (spermatocytes and oocytes and their precursors)
that upon fertilisation can give rise to a descendant organism
Ergo


tranformierung von
keimbahnzellen ist
zur Zeit
vermieden (technische und
ethische Probleme)
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
Wann gibt heute eine Indikation fuer SGT ?
UNIFR
Rusconi
2005
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:
 viele Indikationen
Complement to conventional


deviation dreams
increases specificity of conventionalPerverse
therapy (cancer)
(even(hemophilia)
with current
increases efficacy of conventional therapy
technologyI:
 gene-based sports doping
Life quality burden of patient
 costs of enzyme therapy 
(ex.
ADA)
performance
amelioration
 burden of daily injections(ex.
Insulin)
cosmetics
UNIFR
Pharmacologische Betrachtungen
Rusconi
2005
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
Ergo: Therapy with nucleic acids
 Spezielle Formulierung
 Viel komplexer als konventionelle Medikament-Therapie
 Geringere Reversibilitaet
VIER grundlegende Fragen der SGT
UNIFR
Rusconi
2005
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?
UNIFR
DREI Kategorien von anatomische Gen-Lieferung
Rusconi
2005
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
ZWEI Vektor-Typen:
non-viral & viral
Non-viral transfer
(transfection of plasmids)
Viral gene transfer
(Infection by r-vectors)
UNIFR
Rusconi
2005
a
Ergo


viral transfer is much more efficient
b
nonviral transfer
must solve a
number of hurdles
- serum protection/stability
Nuclear envelope barrier!
- target docking see, Nature Biotech
- endosomal escape
December 2001
- nuclear trafficking
- genomic integration
- anti apoptotic functions
- immunological camouflage
- ...
UNIFR
Transfection versus Infection
Rusconi
2005
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)
UNIFR
Kurze Liste von Vektoren
Rusconi
2005
r-Adenovirus
Naked DNA
r-Adeno-Associated V.
Liposomes & Co.
r-Retrovirus (incl. HIV)
Oligonucleotides
Recap: Limitierungen heutiger Vektoren
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”)
UNIFR
Rusconi
2004
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”)
UNIFR
Gentherapie in der Klinik: Trials Worldwide (cumulative)
Rusconi
2005
trials

100
80
60
40
patients
Ergo

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.
2600 Patients treated in 2004
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
Klinische Meilensteine der Gentherapie
UNIFR
Rusconi
2005
1990, 1993, 2000, // ADA deficiency
F Anderson, M Blaese 90/93/ C Bordignon 2000/2004
Anderson, 1990
Isner, 1998
Fischer,
Kirn, 2000
1997, 2000, Critical limb ischemia
2000, 2002
J Isner († 4.11.2001),
I
Baumgartner,
1998
25 lives
2001
Manuel Grez
were so far documentedly saved by GT in
2002
Sibiono
Hans Peter
Hossle
2000, Hemophilia
european
trials
(x-SCID,
ADA,
CGD)
2003
Shenzen
Reinhard
Seger
M Kay, K High
(France, UK, Italy) (all in phase I)
Intravascular adenoviral
2004/2005agents
2000, 2002, X-SCID
in cancer patients:
~200
lives
quality-improved
A Fischer, 2000/2002, Thrasher 2003
Lessons
from clinical
very
encouraging
data trials
from
in several other phase I and II trials
just initiated(review)
clinical trial,
dropped
inpatients
2004?
2001, 2003 ONYX oncolytic Viruses
prospected
>10
~nnn lives saved or quality-improved ?
licensed China 2005?
D Kirn (Cancer Gene Ther 9, p 979-86)
commercialisation of
by Gendicine (50'000 patients prospected
Bordignon, 2000Approved
(ESGT, Stockholm)
Gendicine
(Jan 2004) for cancer
for 2006)
2002,
science
296,
2410
ff)
2004, Chronic Granulomatous Disease
M Grez Frankfurt; R Seger Zürich
2004/2005 Gendicine (adeno-p53 vector)
L Peng, Sibiono Inc, Shenzen, China
treatment in China.
-> ! Hum Gene Ther 16, 1016 ff.
Zwei persistierende Frustrationsfälle
UNIFR
Rusconi
2005
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'
Die meist befürchtete Nebeneffekte der Gentherapie
UNIFR
Rusconi
2005

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».
 Side-effect-free illusion in the 90ties is over
 Primitive state of the vectorology/delivery
SAEs1: Vom Gelsingers' Tod bis zu den Paris'
Leukaemias
adenovirus, cystic fibrosis (lung)
one patient mild pneumonia-like condition
Trial interrupted and many others on hold.
adenovirus , OTC deficiency (liver)
one patient (Jesse Gelsinger) died of a severe septic shock.
Many trials were put on hold for several months (years).
Rusconi
2005
Most Recent Paris' Trial News
discussed under:
www.unifr.ch/nfp37/adverse03.html
NY May 5, 1995, R. Crystal:
UPenn, Sept. 19, 1999, J. Wilson:
UNIFR
Tomorrow (16.09.05)
A Fischer will talk at the Kontderspital
(Workshop organised by R Seger)
Paris, Oct 2, 2002, A Fischer:
retrovirus , x-SCID (bone marrow)
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)
SAEs2: Recent Autoimmunity Reports
UNIFR
Rusconi
2005
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.
- homologous EPO cDNA via AAV vectors
- muscle or lung,
- supra-physiologic serum levels of EPO
K High, ASGT June meeting 2004
Ergo
somatic gene transfer and ectopic
transgene expression can
generate mid-term auto- immunity
[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: Nicht-wissenschaftliche Faktoren die Fortschritt
und Image von GT negativ beeinflusst haben

'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
UNIFR
Rusconi
2005
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
Gentherapie Hoehe und Tiefe:
a true roller-coaster ride!
UNIFR
Rusconi
>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
Schlussfolgerungen: GT hat Konzepte bewiesen aber
bleibt immer noch im Pionierzustand
UNIFR
Rusconi
2005
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
NonViral vectors lower toxicity/danger
BUT -> inefficient
Viral vectors limited packaging and high toxicity
BUT -> efficient
Clinically




Over 1000 trials and >4000 patients in 15 years
Only a handful phase III
Periodical pitfalls
Gendicine approved in China (2004)
Ergo
 we are somewhat ahead but still
in the pioneering phase !
Aussichte: GT wird fortschreiten trotz den gängigen und
zhukuenftiken Zwischenfälle
UNIFR
Rusconi
2005
Fundamental level & vectorology





Better understanding of gene interactions and networking
Gene inhibition through Si RNA, designed 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
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
Ergo



Accidents typical of prototypic status
hurdles can be overcome
the genuine potential of SGT is
intact
Meinen 'Proust's questionnaire' bezüglich Gentherapie
UNIFR
Rusconi
2005
will GT ever make it into routine clinical practice ?
yes
when will GT widely established ?
not tomorrow
The most worrying adverse-effect?
immunity
Is insertional mutagenesis an important hurdle?
No
Which will bloom: viral or non viral transfer?
combination thereof
Who shall '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, DNAzymes, SiRNA,
designer Zn Fingers, triple helix, small drugs, ...whatever?
...whatever
M Proust 1871-1922
...Danke, und ... let's remain optimistic
UNIFR
Rusconi
2005
Orthopedics Update
Ch. Gerber, B. Fuchs
My UNIFR and TI collaborators
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