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Sandro Rusconi (09.03.52)

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Title: Sandro Rusconi (09.03.52)


1
Sandro Rusconi (09.03.52)
UNIFR Rusconi 2003
20 Oktober 2003 Liestal Biovalley
2003 wohin führt uns Gentherapie?
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 Swiss
Societies for Experimental Biology (USGEB)
essentielle wiederholung in Genetik grundkonzep
te der Gentherapie Klinische experimentiering
in SGT Hohe und Tiefe in der SGTSchlussfolger
ungen und Perspektiven
Courmayeur, March 2003
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essentielle Wiederholungen in Genetik
UNIFR Rusconi2003
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1 Gen -gt 1 oder mehrere Funktionen1 Genom -gt
über Nx100'000 Funktionen
UNIFR Rusconi 2003
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1 Organismus -gt 1013 Zellen, verteilt und
spezialisiert in Organe und Gewebe
UNIFR Rusconi 2003
  • 1 Cm3 Gewebe
  • 1'000'000'000 Zellen

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Aber was istr eigentlich 'ein Gen'?eine
regulierbare Nano-machine zur Herstellung von RNA
UNIFR Rusconi 2003
  • Um wirksam zu sein sollte ein transferierter Gen
    beinhalten
  • Sequenzen fuer Genregulation
  • Signale fuer reifung/transport der RNA
  • Signale fuer Uebersetzung in proteinen

coding
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spacer
regulatory
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Das reduktionistische Paradigma des
Molekularbiologes
UNIFR Rusconi 2003
  • Gentransfer kann beinhalte
  • transfer einer neuen Funktion, oder
  • transfer einer kompensierenden F., oder
  • transfer einer interferierenden Funktion

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Beispiele von Vererbbare Defekte
UNIFR Rusconi 2003
Polygenic defects Type estimated ( frequent )
min - max Diabetes poly 1 - 4
Hyperurikemia Multi 2 - 15 Glaucoma poly 1 - 2
Displasia Multi 1 - 3 Hypercolesterolemia Mul
ti 1 - 5 Syn- Polydactyly poly 0.1 - 1
Congenital cardiac defects Multi 0.5 - 0.8
Manic-depressive psychosis Multi 0.4 - 3
Miopy poly 3 - 4 Polycystic
kidney poly 0.1 - 1 Psoriasis Multi 2 - 3
Schizofrenia Multi 0.5 - 1 Scoliosis Multi 3
- 5
Monogenic defects estimated ( rare )
min - max Cystic fibrosis, muscular
dystrophy immodeficiencies, metabolic diseases,
all together Hemophilia... 0.4 - 0.7
Predispositions Type estimated min - max ()
Alzheimer Multi 7 - 27 () Parkinson Multi 1 -
3 () Breast cancer Multi 4 - 8 () Colon
Carcinoma Multi 0.1 - 1 ()
Obesity Multi 0.5 - 2 () Alcolholism/ drug
addiction Multi 0.5 - 3
  • Ergo
  • Jedermann ist Träger von mindestens einen Defekt
  • Viele Defekte manifestieren sich erst spät im
    Leben (Anfälligkeiten)
  • Einige Anfälligkeiten sind positiv
    (langlebigkeit, Infektionsresistenz etc...)

Sum of incidences min - max (all
defects) 32 - 83
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Das genom ist nicht das einzige determinant des
gesund-krang gleichgewichtes
UNIFR Rusconi 2003
  • Sogar die heilung von 'erworbene' krankheiten
    kann genetisch bedingt sein
  • Trauma, Wunde
  • Brüche
  • Verbrennungen, Infektionen
  • Vergiftungen

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grundkonzepte in der Somatischen Gentherapie
(SGT)
UNIFR Rusconi2003
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Die 4 Aeren der molekularen Medizin
UNIFR Rusconi 2003
genomeABC.mov
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Somatische Gentherapie (SGT) Definierung und
Anwendungsbereich
UNIFR Rusconi2003
Chronic treatment
Definition of SGT 'Use genes as
drugs' Correcting disorders by somatic gene
transfer
Acute treatment
Preventive treatment
NFP37 somatic gene therapy www.unifr.ch/nfp37
Hereditary disorders
Acquired disorders
Loss-of-function
Gain-of-function
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Das Prinzip darf einfach sein, aber der Teufel
liegt häufig in den Details...
UNIFR Rusconi2003
There are many things that are simple in
principle, like...
getting a train ticket...
! try this 5 min before departureand 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...
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Pharmakologische Betrachtungen
UNIFR Rusconi 2003
Classical Drugs
Protein Drugs
Nucleic Acids
  • Mw 20 000- 100 000 Da
  • Biologically prepared
  • Slower diffusion/action
  • Oral delivery not possible
  • Cellular delivery - act extracellularly
  • Can be delivered as soluble moleculesnm size
  • rapidly reversible treatment
  • 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 moleculesÅngstrom/nm
    size
  • rapidly reversible treatment
  • Mw N x 1000000 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
    particles50-200 nm size
  • slowly or not reversible

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  • 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

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Wieso 'somatisch'?
UNIFR Rusconi2003
  • 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

i.e. somatic gene therapy is a treatment aiming
at somatic cells and conse-quently does not lead
to a hereditary transmission of the genetic
alteration
  • Somatic Cells all the other cells of the body

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Die vier Grundfragen bei der SGT
UNIFR Rusconi2003
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?

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Die drei Transfer-wege bei der SGT
UNIFR Rusconi 2003
Ex-vivo
In-vivo topical delivery
In-vivo systemic delivery
Examples - bone marrow - liver cells - skin cells
Examples - brain - muscle - eye - joints - tumors
Examples - intravenous - intra-arterial -
intra-peritoneal
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Die zwei klassen von 'Vektoren'virale /
nicht-virale
UNIFR Rusconi 2003
Transfert non viral (transfection)
Nuclear envelope barrier!
viral transfer (Infection)
direct nuclear shuttling!
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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

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kleine Parade von Genransfervektoren
UNIFR Rusconi 2003
Naked DNA Liposomes Co. Oligonucleotides
Adenovirus Adeno-associated V. Retrovirus
(incl. HIV)
but remember... "Nobody's perfect "!
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rekombinante Adenoviren
UNIFR Rusconi 2003
  • Approaches
  • Generation I
  • Generation III
  • Hybrid adenos
  • Adeno-RV
  • Adeno-AAV
  • Adeno-Transposase
  • Advantages / Limitations
  • 8 Kb capacity Generation I gt30 Kb capacity
    Generation IIIAdeno can be grown at very high
    titers,However
  • Do not integrate
  • Can contain RCAs
  • Are toxic /immunogenic
  • Examples
  • OTC deficiency (clin, ---)
  • Cystic Fibrosis (clin, --- )
  • Oncolytic viruses (clin, )

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r4ekombinante Adeno-associated-virus (AAV)
UNIFR Rusconi 2003
  • Advantages / Limitations
  • Persistence in the genome permits long-
  • term expression, high titers are easily
  • obtained, immunogenicity is very low,
  • However the major problems are
  • insertional mutagenesis
  • Small capacity (lt4.5 kb) which does not allow to
    accommodate large genes or gene clusters.

Approaches Helper-dependent production Helper
independent production Cis-complementing
vectors Co-infection
  • Examples
  • Hemophilia A (clin, animal, )
  • Gaucher (clin, animal, )
  • Brain Ischemia (animal, )
  • Cystic fibrosis (animal, /-)

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Rekombinante Retroviren (inkl. HIV)
UUNIFR Rusconi 2003
Approaches Murine Retroviruses VSV-pseudotyped
RV Lentiviruses ! Self-inactivating
RV Combination viruses
  • Advantages / Limitations
  • 9 Kb capacity integration through
  • transposition also in quiescent cells
  • (HIV), permit in principle long-term
  • treatments, however disturbed by
  • Insertional mutagenesis
  • Gene silencing
  • High mutation rate
  • Low titer of production
  • Examples
  • SCID (IL2R defect, Paris) (clin, )
  • Adenosine Deaminase deficiency (clin, !!!)
  • Parkinson (preclin, )
  • Anti cancer (clin /-)

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Reine oder komplexierte DNS
UNIFR Rusconi 2003
Approaches Naked DNA injection /biolistic Naked
DNA pressure Naked DNA electroporation Liposom
al formulations Combinations
  • Advantages / Limitations
  • Unlimited size capacity lower
  • immunogenicity and lower bio-risk
  • of non viral formulations is
  • disturbed by
  • 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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Oligonuklotide
UNIFR Rusconi 2003
Approaches Antisense Ribozymes/DNAzymes Triple
helix Decoy / competitors Gene-correcting oligos
  • Advantages / Limitations
  • these procedures may be suitable for
  • handling dominant defects
  • transient treatments (gene modulation)
  • permanent treatments (gene correction)
  • Examples
  • Anti cancer (clin,preclin., /-)
  • Restenosis (clin, )
  • Muscular Distrophy (animal, )

v !
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Recap Limitierungen der heutigen Genvektoren
UNIFR Rusconi 2003
Adenovirus - no persistence - limited packaging -
toxicity, immunogenicity
Biolistic bombardment or local direct injection -
limited area
Electroporation - limited organ access
Retrovirus (incl. HIV) AAV - limited
packaging - random insertion - unstable genome
Liposomes, gene correction Co. - very
inefficient transfer
General - antibody response - limited packaging -
gene silencing- random insertion
General - low transfer efficiency - no or little
genomic integration
Solutions - improved liposomes with viral
properties (Virosomes)
Solutions - synthetic viruses (Virosomes)
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klinische Versuche in der SGT
UNIFR Rusconi2003
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Der klassische klinische Weg viel Zeit und Geld
UNIFR Rusconi 2003
year event costs UD
0 Idea 0 2 Cell culture assays 0.5
Mio 5 Pre-clinical tests animal models 2
Mio 7 Clinical phase I 5-20 patients verify
side effects 6 Mio 10 Clinical phase
II 30-100 patients dosis escalation 12
Mio 15 Clinical Phase III gt300- 1000
patients multicentric double blind 80
Mio 16gtgt Registration / Availability
This means assuming 20 of new
developments makes it to final registration, the
average investment is 300-500 Mio UD for each
approved drug/procedure
Molecular therapy is NO EXCEPTION !
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Vernichten wir mindestens vier Mythen bei der
Gentherapie
UNIFR Rusconi2003
  • Classical Gene Therapy Image
  • Hereditary disease
  • culprit gene must be known
  • requires 100 efficiency of transfer/expr.
  • gene transfer/expression must persist
  • Reality
  • Many acquired diseases can be treated(ex.
    infections, traumatic lesions, tumors,...)
  • 'Short circuit' or symptomatic treatments (ex.
    neurodeg. conditions with trophic factors)
  • Few sufficient for many diseases(ex.
    hemophilia, limb ischaemia ...)
  • No persistence required in many cases (ex.
    vaccination, cytotoxic antitumoral factors,
    restenosis prevention, acute rejection
    prevention...)

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Trends bei der klinischen SGT experimentierung
UNIFR Rusconi2003
  • Ergo
  • en dépit de son age la TGS peut compter
    couramment seulement 1 d'essais en phase III

As of August 2003660 registered protocols 3672
treated patients
66 phase I 21 phase I-II 11 phase II 0.8
phase II-III 0.7 phase III
21 overall still pending or not yet Initiated
! www.wiley.com/genetherapy
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Einige Meilensteine
UNIFR Rusconi2003
1990, 1993, 2000 // 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
1999, Crigler Njiar (animal) C Steer, PNAS 1999
2000, Hemophilia M Kay, K High
2000, SCID A Fischer, Science April 2000
2000, correction Apo E4 (animal model) 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)
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Die hoch- und tief-punkte ...
UNIFR Rusconi2003
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Zwei besonders frustrierende FaelleMuskelschwund
und Mucoviszidose
UNIFR Rusconi2003
  • 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 entspre-chenden Genen in
    1984
  • kein geeignetes Vektor
  • keine geeignete Lieferungsmethode

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Die mesit befuerchtete Nebeneffekte der
Gentherapie
UNIFR Rusconi2003
  • 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-gt insertional
    mutagenesis (-gt cancer risk)
  • Contamination of germ line cells
  • Random integration in genome-gt insertional
    mutagenesis (-gt cancer risk)
  • Ergo
  • die Nebeneffekte waren nicht so scvheinbar wenn
    SGT ineffizient war
  • Heute muessen wir diese Nebeneffekte seriös
    betrachten

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4 bittere Feststellungen aber nur einen Patient
bis jetzt direkt an SGT gestorben
UNIFR Rusconi2003
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
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Der klinische Versuch in Paris X-SCID (A.
Fischer, Hôpital Necker)
UNIFR Rusconi2003
  • 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

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Die Odyssee des klinischen Versuchs in Paris
UNIFR Rusconi2003
  • 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

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Die Fragen
UNIFR Rusconi2003
  • 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
  • Perspectives if the answers are
  • 'YES' 'NO' 'UNK'
  • good bad not good
  • good bad not good
  • good bad not good
  • bad good not good
  • 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?

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Anhäufung von hoch- und tief-Punkte ein
Rollercoaster!
UNIFR Rusconi2003
A. Fischer M. Kay
lentivectors in clinics?
R. Crystal
V.Dzau
Adeno I
C Bordignon
J. Isner
ADA
AAV germline in mice?
NIH Motulski report
Adeno III
  • Ergo
  • whenever a reasonable cruise speed was achieved,
    a major adverse event has brought us back square
    one

Lentivectors in pre-clinic
Adverse events in Paris
J. Wilson J. Gelsinger
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Weitere Faktoren die zum schlechten 'Image' der
Gentherapie beigetragen haben
UNIFR Rusconi2003
  • 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...

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Conclusions Perspectives
UNIFR Rusconi2003
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Schlussfolgerungen
UNIFR Rusconi2003
  • 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

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Perspectiven SGT wird weiter fortschreitentrotz
schwieruigkeiten und unvermeidbare Unfälle
UNIFR Rusconi2003
  • 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.

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...Danke ... und bleiben wir optimistisch
UNIFR Rusconi2003

Biovalley Program Gymnasium Liestal
My collaborators at UNIFR
Swiss National Research Foundation
Danke fuer die aufmerksamkeit und fuer spezielle
Fragen, bitte schreiben sie an sandro.rusconi_at_uni
fr.ch oder besuchen sie die WEB
seite www.unifr.ch/nfp37
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END, let's open the Discussion
UNIFR Rusconi2003
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Diskussions-slides...
UNIFR Rusconi2002
  • text
  • ttt

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Gene therapy in Switzerland the 30 projects
financed by the NFP37 programme (1996-2001)
UNIFR Rusconi2003
NFP37 phase A phase B (96-99) (99-01)
Submissions 30 26 Granted 19 18 Total requested
32 Mio 9 Mio Granted 7.6 Mio 6 Mio
DISEASE ORIENTATION Cancer 8 10 Acquired
disorders 2 7 Vector development 5 3
Hereditary disorders 2 4 Infectious diseases
1 2 RESEARCH LEVEL Fundamental 10 7
Preclinical (animal models) 5 9 Clinical phase
I 2 3 Clinical Phase II 0 1 Clinical Phase
III 0 0 Ethical/social aspects 1 1
Nationales Forschungsprogramm 37 NFP37  somatic
gene therapy www.unifr.ch/nfp37
  • Please Note
  • the NFP37 represented at most 30 of the
    Swiss-based experimentation in SGT during
    1996-2001

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The SGT acrobatics matching vectors / delivery
system / disease
UNIFR Rusconi2003
  • 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 re-gain 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
  • 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)

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Die Hauptkrankheit des 21. Jahrhunderts
Veralterung
UNIFR Rusconi 2003
aa getting oldcomp2.mov
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Pas toutes les stratégies de transfert se basent
sur un ancrage au hasard
UNIFR Rusconi2003
  • Ergo
  • genotoxic
  • non-genotoxic
  • Random integrating vectors
  • r-retroviruses
  • r-lentiviruses
  • r-AAV
  • plasmids (low frequency)
  • plasmids transposase (eg 'sleeping beauty')
  • Specifically integrating vectors
  • hybrid vectors (HSV-AAV)
  • Phage 31 integrase-based
  • designer integrase
  • Transient, non integrating vectors
  • adenovirus
  • plasmid
  • RNA virus based
  • oligonucleotides (SiRNA, antisense, ribozymes)
  • artificial chromosomes
  • Gene correction vectors
  • chimeroplasts (RNA-DNA chimeric oligos)
  • single stranded DNA (homologous recom)

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Mais un virus c'est quoi?Une machine
auto-réplicative extrèmement efficace
UUNIFR Rusconi 2003ß
entry
disassembly
docking
genome replication
early genes exp
replication
late genes exp
assembly
standard viral genome
Spread
Etc...
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Comment peut-on construire des virus récombinants?
UNIFR Rusconi 2002
E
Wild type genome
Virions
R-Virions
Packaging
Packaging
Packaging
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D'autres tecnolgies émergentes entrent en
compétition brutale avec la thérapie génique
UNIFR Rusconi2003
  • 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?
  • 2. Challengers from the small/medium molecules
  • STI571 (Glivec)
  • anti HER2 (Herceptin)
  • Si RNA?
  • ...
  • 3. Challengers from the biomechanics world
  • bone reconstruction
  • intelligent protheses (stents)
  • micropumps
  • artificial organs

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La génétique est practiquée depuis de
millénaires, la biologie moléculaire seulement
depuis 30 ans
UNIFR Rusconi 2003
100000 b.C. Empirical genetics
10000 b.C. Biotechnology
2000 a.d. Molecular biology
2001 a.d, Genomics
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When/where/ may be SGT indicated?
UNIFR Rusconi2003
  • No existing cure or treatment
  • most monogenic diseases
  • Side effects and limitations of protein injection
  • interleukin 12 (cancer)-gt toxic effects and
    rapid degradation
  • VEGF (ischemias)-gt angiomas
  • Factor VIII or IV (hemophilia)-gt 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)

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Which vector for which disease category
UNIFR Rusconi2003
Justifications /Issues
Most suitable vector
Disease Type
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Comparing relevant issues in the two main
'vectorology' sectors (viral versus nonviral)
UNIFR Rusconi2003
  • 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-infecti
    ous 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, 10exp10excellent)
  • Complexity of production (existence or not of
    packaging cell systems)
  • Emotional problems linked to pathogenicity of
    donor vectors (ex. lentiviruses)
  • 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

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The THREE missions of medicine
UNIFR Rusconi 2003
Prevention

'Molecular Medicine' Application of the
know-how in molecular genetics to medicine
Diagnosis


Therapy
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Ideal properties of a systemically delivered
non-viral formulation
UNIFR Rusconi2003
  • Stability
  • particle should resist serum inactivation
  • particle should be inert to immune inactivation
  • Ergo
  • several independent problems must be solved for a
    nonviral formulation to be suitable for clinical
    treatment and for industrial production
  • most viral vectors include many, if not all those
    properties
  • Addressability
  • particle should possess a vascular addressing
    signature
  • particle should bear a tissue-docking specificity
  • DNA construct should include tissue-specific
    regulatory elements
  • 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

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Public perception problems
UNIFR Rusconi2003
  • 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

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d Why so many cancer trials?
UNIFR Rusconi2002
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
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d Ethical dilemmas can sometimes slow down
progress and hamper objective appreciations
UNIFR Rusconi2002
ADA gene therapy 1990-1999  it does not work
Feeling It s time we face reality, my
friends We re not exactly rocket scientists
vector
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 )
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d Cancer molecular treatment ExampleOncolytic
viruses on the example of ONYX-015
UNIFR Rusconi2002
  • 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 dynamicsDISADVANTAGE
  • danger of evolving viruses
  • unclear if it works in adeno-immune patients
  • unclear if if works in immuno-compromised
    patients (chemotherapy)

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d Recap what is a virus ? -gt A superbly
efficient replicating machine
UUNIFR Rusconi 2002
entry
disassembly
docking
genome replication
early genes exp
replication
late genes exp
assembly
standard viral genome
Spread
Etc...
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d Examples of gene transfer treatments against
cancer
UNIFR Rusconi2002
  • percentage of trials(therapeutic potential)
  • Type of treatment
  • examples

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d Gene correction strategies (independent of
delivery vectors)
UUNIFR Rusconi 2002
Approaches Trans-splicing mRNA Ribozymic splicing
of RNA Chimeroplasts-induced repair Triple-helix-g
uided repair Homologous recombination
  • Advantages / Limitations
  • 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
  • Controversial efficiency of repair

Specificity
  • Examples
  • Crigler Njiar (animal, 30)
  • M. Distrophy (animal, 20)
  • Correction Apo E 4 (animal, 20)
  • Correction Albino (animal, /- lt1)

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d Gene therapy  FAM  (Frequently Asserted
Myths)
UNIFR Rusconi2002
Gene therapy ...
Wrong 1998 USA lt 2 of NIH budget, CH less
than 1 of NF budget
...is withdrawing huge public funding
Wrong example DNA based vaccination
...will be invariably expensive
Wrong Cancer, Cardiovascular, Restenosis,
Ischemia, ...
...is solely targeting hereditary diseases
Wrong J. Isner/ I. Baumgartner (ischemia), A.
Fischer (SCID), C Bordignon (ADA) M. Kay
(Haemophilia, F McCormick (cancer),
...has not yet proven its therapeutic value
...is based only on a shotgun insertion'
Wrong Gene correction strategies,
Wrong 1 death out of gt3000 patients
...is a dangerous procedure
Wrong it will take long time to benefit from
genomics knowledge
...will be imediately boosted by genomics
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