Sandro Rusconi (09.03.52)Sandro Rusconi (09.03.52)UNIFRRusconi

2003

UNIFRRusconi

2003

2003: wohin führt uns Gentherapie?

20 Oktober 2003Liestal Biovalley

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 andUniv. Milano, Pharmacology Department

2002-05 President Union of Swiss Societies for

Experimental Biology (USGEB)

Courmayeur,March 2003

*essentielle wiederholung in Genetik**grundkonzepte der Gentherapie**Klinische experimentiering in SGT**Hohe und Tiefe in der SGT**Schlussfolgerungen und Perspektiven*

*essentielle Wiederholungen in Genetik**essentielle Wiederholungen in Genetik*

UNIFRRusconi2003

UNIFRRusconi2003

1 Gen -> 1 oder mehrere Funktionen1 Genom -> über Nx100'000 Funktionen

1 Gen -> 1 oder mehrere Funktionen1 Genom -> über Nx100'000 Funktionen

UNIFRRusconi

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UNIFRRusconi

2003

RNADNA

GENE

Protein

2-5 FUNCTIONS

Gene expression

Transcription / translation

>300 ’000 functions(>150 ’000 functions)

100 ’000 genes(50 ’000 genes?)

1 Organismus -> 1013 Zellen, verteilt und spezialisiert in Organe und Gewebe

1 Organismus -> 1013 Zellen, verteilt und spezialisiert in Organe und Gewebe

UNIFRRusconi

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UNIFRRusconi

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2m 2 mm 0.2mm

0.02mm

DNA RNA Protein

0.001mm 1 Cm3 Gewebe 1'000'000'000 Zellen

Aber was istr eigentlich 'ein Gen'?:eine regulierbare Nano-machine zur Herstellung von RNA

Aber was istr eigentlich 'ein Gen'?:eine regulierbare Nano-machine zur Herstellung von RNA

UNIFRRusconi

2003

UNIFRRusconi

2003

RNADNA Protein

GENE FUNCTIONTranscription / translation

codingspacer spacerregulatoryDNA

RNA

Um wirksam zu sein sollte ein transferierter Gen beinhalten: Sequenzen fuer Genregulation Signale fuer reifung/transport der RNA Signale fuer Uebersetzung in proteinen

Das reduktionistische Paradigma des MolekularbiologesDas reduktionistische Paradigma des MolekularbiologesUNIFRRusconi

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UNIFRRusconi

2003

GENE transfer FUNCTION transfer

GENE KO FUNCTION KO

GENE OK FUNCTION OK

DNA

GENE

Protein

FUNCTION(s)

Gentransfer kann beinhalte: transfer einer neuen Funktion, oder transfer einer kompensierenden F., oder transfer einer interferierenden Funktion

Beispiele von Vererbbare DefekteBeispiele von Vererbbare DefekteUNIFRRusconi

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UNIFRRusconi

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 Multi 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 - maxCystic fibrosis, muscular dystrophyimmodeficiencies, metabolic diseases, all togetherHemophilia... 0.4 - 0.7%

Predispositions Type estimatedmin - 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%

Sum of incidences min -max (all defects) 32 - 83%

genetics behaviour environment

Ergo: Jedermann ist Träger von mindestens einen Defekt Viele Defekte manifestieren sich erst spät im Leben (Anfälligkeiten) Einige Anfälligkeiten sind positiv (langlebigkeit, Infektionsresistenz etc...)

Das genom ist nicht das einzige determinant des gesund-krang gleichgewichtes

Das genom ist nicht das einzige determinant des gesund-krang gleichgewichtes

UNIFRRusconi

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UNIFRRusconi

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genetics behaviour environment

Muscle distrophy

Obesity

Artherosclerosis

Alzheimer

Parkinson ’s

Drug AbuseHomosexuality

Familial Breast Cancer

Lung Cancer

Sporadic Breast Cancer

Sogar die heilung von 'erworbene' krankheiten kann genetisch bedingt sein:

Trauma, Wunde Brüche Verbrennungen, Infektionen Vergiftungen

*grundkonzepte in der Somatischen Gentherapie (SGT)**grundkonzepte in der Somatischen Gentherapie (SGT)*

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Die 4 Aeren der molekularen MedizinDie 4 Aeren der molekularen MedizinUNIFRRusconi

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UNIFRRusconi

2003EightiesGenes as probes

ok ** ** **ok1 2 4 53

NinetiesGenes as factories

80 85 90 95 99

10

50

Y2KGenes as drugs

80 85 90 95 00

1000

3000

Y2K+n Post-genomic improvements of former technologies

Somatische Gentherapie (SGT) Definierung und Anwendungsbereich

Somatische Gentherapie (SGT) Definierung und Anwendungsbereich

UNIFRRusconi2003

UNIFRRusconi2003

Definition of SGT:'Use genes as drugs':Correcting disorders by somatic gene transfer

Chronic treatment

Acute treatment

Preventive treatment

Hereditary disorders

Acquired disorders

Loss-of-function

Gain-of-function

NFP37 somatic gene therapywww.unifr.ch/nfp37

Das Prinzip darf einfach sein, aber der Teufel liegt häufig in den Details...

Das Prinzip darf einfach sein, aber der Teufel liegt häufig in den Details...

UNIFRRusconi2003

UNIFRRusconi2003

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...

Pharmakologische BetrachtungenPharmakologische BetrachtungenUNIFR

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OHOH

O

OHOH

O

O

OHOH

O

O

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

Classical 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 moleculesnm size

rapidly reversible treatment

Protein Drugs

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 particles50-200 nm size

slowly or not reversible

Nucleic Acids

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

Germ Line Cells: the cells (spermatocytes and oocytes and their precursors) that upon fertilisation can give rise to a descendant organism

Somatic Cells: all the other cells of the body

i.e. somatic gene therapyis a treatment aiming atsomatic cells and conse-quently does not lead to a hereditary transmission of the genetic alteration

Ergo transformation of

germ line cells is avoided, to exclude risk of erratic mutations due to insertional mutagenesis

Wieso 'somatisch'?Wieso 'somatisch'?UNIFRRusconi2003

UNIFRRusconi2003

Die vier Grundfragen bei der SGTDie vier Grundfragen bei der SGTUNIFRRusconi2003

UNIFRRusconi2003

Efficiency of gene transfer

Specificity of gene transfer

Persistence of gene transfer

Toxicity of gene transfer

Remember!

Le variables welche Krankheit? Welches Gen? Welches Vektor? Welches Organ / Gewebe? Welche Transfermethode?

Die drei Transfer-wege bei der SGT:Die drei Transfer-wege bei der SGT:UNIFR

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Ex-vivo In-vivotopical delivery

In-vivosystemic 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

Die zwei klassen von 'Vektoren':virale / nicht-virale

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A

B

Transfert non viral(transfection)

viral transfer(Infection)

Nuclear envelope barrier!

direct nuclear shuttling!

Effizienz der Transfektion mit rekombinante DNA im vergleich zur Infektion mit rekombinante Viren

Effizienz der Transfektion mit rekombinante DNA im vergleich zur Infektion mit rekombinante Viren

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Transfection

Infection

cells exposed to1'000'000 particles/cell12 hours

cells exposed to 3 particle/cell30 min

Ergo das gentransfer mittels rekombinante Viren ist ueber 1'000'000-fach effizienter

als jene nicht-viral transfer methode

kleine Parade von Genransfervektorenkleine Parade von GenransfervektorenUNIFR

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Adenovirus

Adeno-associated V.

Retrovirus (incl. HIV)

Naked DNA

Liposomes & Co.

Oligonucleotides

rekombinante Adenovirenrekombinante AdenovirenUNIFR

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Approaches

Generation I

Generation III

Hybrid adenos: Adeno-RV Adeno-AAV Adeno-Transposase

Examples OTC deficiency (clin, ---) Cystic Fibrosis (clin, --- ) Oncolytic viruses (clin, +++)

Advantages / Limitations

8 Kb capacity Generation I >30 Kb capacity Generation IIIAdeno can be grown at very high titers,However Do not integrate

Can contain RCAs

Are toxic /immunogenic

r4ekombinante Adeno-associated-virus (AAV)r4ekombinante Adeno-associated-virus (AAV)UNIFR

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Examples Hemophilia A (clin, animal, +++) Gaucher (clin, animal, +++) Brain Ischemia (animal, +++) Cystic fibrosis (animal, +/-)

Advantages / Limitations

Persistence in the genome permits long-term expression, high titers are easilyobtained, 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.

Approaches

Helper-dependent production

Helper independent production

Cis-complementing vectors

Co-infection

Rekombinante Retroviren (inkl. HIV)Rekombinante Retroviren (inkl. HIV)UUNIFR

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UUNIFR

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Approaches

Murine Retroviruses

VSV-pseudotyped RV

Lentiviruses !

Self-inactivating RV

Combination viruses

Examples SCID (IL2R defect, Paris) (clin, +++) Adenosine Deaminase deficiency (clin, +++!!!) Parkinson (preclin, +++) Anti cancer (clin +/-)

Advantages / Limitations

9 Kb capacity + integration throughtransposition also in quiescent cells(HIV), permit in principle long-termtreatments, however disturbed by: Insertional mutagenesis

Gene silencing

High mutation rate

Low titer of production

Reine oder komplexierte DNSReine oder komplexierte DNSUNIFR

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Approaches

Naked DNA injection /biolistic

Naked DNA + pressure

Naked DNA + electroporation

Liposomal formulations

Combinations

Advantages / Limitations

Unlimited size capacity + lowerimmunogenicity and lower bio-riskof non viral formulations isdisturbed by

Low efficiency of gene transfer

Even lower stable integration

Examples Critical limb Ischemia (clin, +++) Cardiac Ischemia (clin, +/-) Vaccination (clin, +/-) Anti restenosis (preclin. +/-)

OligonuklotideOligonuklotideUNIFR

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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, +++)

Recap: Limitierungen der heutigen GenvektorenRecap: Limitierungen der heutigen GenvektorenUNIFR

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Adenovirus- no persistence- limited packaging- toxicity, immunogenicity

Biolistic bombardmentor local direct injection- limited area

Retrovirus (incl. HIV) & AAV- limited packaging- random insertion- unstable genome

General- antibody response- limited packaging- gene silencing- random insertion

Solutions:- synthetic viruses (“Virosomes”)

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”)

*klinische Versuche in der SGT**klinische Versuche in der SGT*

UNIFRRusconi2003

UNIFRRusconi2003

Der klassische klinische Weg: viel Zeit und GeldDer klassische klinische Weg: viel Zeit und GeldUNIFR

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0 Idea 0

2 Cell culture assays 0.5 Mio

5 Pre-clinical testsanimal models 2 Mio

7 Clinical phase I5-20 patientsverify side effects 6 Mio

10 Clinical phase II30-100 patientsdosis escalation 12 Mio

15 Clinical Phase III>300- 1000 patientsmulticentricdouble blind 80 Mio

16>> Registration / Availability

year event costs U$D

This means:assuming 20% of new developmentsmakes it to final registration,the average investment is 300-500 Mio U$Dfor each approved drug/procedure

Vernichten wir mindestens vier Mythen bei der Gentherapie

Vernichten wir mindestens vier Mythen bei der Gentherapie

UNIFRRusconi2003

UNIFRRusconi2003

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...)

Trends bei der klinischen SGT experimentierungTrends bei der klinischen SGT experimentierungUNIFRRusconi2003

UNIFRRusconi2003

cancer

hered.

Infect.vasc.

40

60

100

20

80

trials

500

1500

1000

patients

1992 1994 1996 19981990 2000

21% overall still pending or not yet Initiated !www.wiley.com/genetherapy

66% phase I21% phase I-II11% phase II0.8% phase II-III0.7% phase III

As of August 2003:660 registered protocols3672 treated patients

Ergo en dépit de son age la

TGS peut compter couramment seulement 1% d'essais en phase III

II-II

II

Einige MeilensteineEinige MeilensteineUNIFRRusconi2003

UNIFRRusconi2003

1990, 1993, 2000 // ADA deficiencyF Anderson, M Blaese // C Bordignon

Anderson, 1990

Bordignon, 2000 (ESGT, Stockholm)2002, science 296, 2410 ff)

1997, 2000, Critical limb ischemiaJ Isner († 4.11.2001), I Baumgartner, Circulation 1998

Isner, 1998

1998, RestenosisV Dzau, HGT 1998

Dzau, 1999

1999, Crigler Njiar (animal)C Steer, PNAS 1999

Kmiec, 1999

2000, HemophiliaM Kay, K High

2000, SCIDA Fischer, Science April 2000

Fischer, 20002002

2000, correction Apo E4 (animal model)G. Dickson, 2000 esgt, 2002 BBA

Dickson, 2000

2000, correction Parkinson (animal model)P Aebischer, Science, Nov 2000

Aebischer, 2000

2001, ONYX oncolytic VirusesD Kirn (Cancer Gene Ther 9, p 979-86)

Kirn, 2000,20012002

Intravascular adenoviral agents in cancer patients:

Lessons from clinical trials(review)

*Die hoch- und tief-punkte ... **Die hoch- und tief-punkte ... *

UNIFRRusconi2003

UNIFRRusconi2003

Zwei besonders frustrierende Faelle:Muskelschwund und Mucoviszidose

Zwei besonders frustrierende Faelle:Muskelschwund und Mucoviszidose

UNIFRRusconi2003

UNIFRRusconi2003

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

Die mesit befuerchtete Nebeneffekte der GentherapieDie mesit befuerchtete Nebeneffekte der GentherapieUNIFRRusconi2003

UNIFRRusconi2003

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

Random integration in genome

-> insertional mutagenesis (-> cancer risk)

Ergo die Nebeneffekte waren nicht so scvheinbar wenn

SGT ineffizient war Heute muessen wir diese Nebeneffekte seriös

betrachten

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

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.

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).

4 bittere Feststellungen aber nur einen Patient bis jetzt direkt an SGT gestorben

4 bittere Feststellungen aber nur einen Patient bis jetzt direkt an SGT gestorben

UNIFRRusconi2003

UNIFRRusconi2003

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.

Der klinische Versuch in Paris X-SCID (A. Fischer, Hôpital Necker)

Der klinische Versuch in Paris X-SCID (A. Fischer, Hôpital Necker)

UNIFRRusconi2003

UNIFRRusconi2003

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

Gene Therapeutical approach explant BM (3-6 month old) select CD34+ transduce with retroviral vector encoding gamma(c) re-infusion, follow-up

Conventional treatments maintenance under sterile condition treatment with antibiotics transplant of matching bone marrow

Die Odyssee des klinischen Versuchs in ParisDie Odyssee des klinischen Versuchs in ParisUNIFRRusconi2003

UNIFRRusconi2003

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

Die FragenDie FragenUNIFRRusconi2003

UNIFRRusconi2003

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 goodgood bad not goodgood bad not goodbad 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?

Anhäufung von hoch- und tief-Punkte: ein Rollercoaster!Anhäufung von hoch- und tief-Punkte: ein Rollercoaster!UNIFRRusconi2003

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high

Low

moo

d

NIHMotulskireport

Lentivectorsin pre-clinic

Adeno III

J. Isner

ADA

R. Crystal

Adeno I

90 91 92 93 94 95 96 97 98 99 00 01 02

AAV germline in mice?

V.Dzau

A. FischerM. Kay

lentivectorsin clinics?

NFP37

C Bordignon

Ergo whenever a reasonable cruise

speed was achieved, a major adverse event has brought us back square one

03

Adverseevents inParis

J. WilsonJ. Gelsinger

Weitere Faktoren die zum schlechten 'Image' der Gentherapie beigetragen haben

Weitere Faktoren die zum schlechten 'Image' der Gentherapie beigetragen haben

UNIFRRusconi2003

UNIFRRusconi2003

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...

*Conclusions & Perspectives**Conclusions & Perspectives*

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SchlussfolgerungenSchlussfolgerungenUNIFRRusconi2003

UNIFRRusconi2003

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 décompresseurSorenson Video 3 sont requis pour visualiser

cette image.

Perspectiven: SGT wird weiter fortschreitentrotz schwieruigkeiten und unvermeidbare Unfälle

Perspectiven: SGT wird weiter fortschreitentrotz schwieruigkeiten und unvermeidbare Unfälle

UNIFRRusconi2003

UNIFRRusconi2003

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 fuer die aufmerksamkeit und fuer spezielle Fragen, bitte schreiben sie an:sandro.rusconi@unifr.choder besuchen sie die WEB seite:www.unifr.ch/nfp37

...Danke ... und bleiben wir optimistisch...Danke ... und bleiben wir optimistischUNIFRRusconi2003

UNIFRRusconi2003

Swiss National Research Foundation

My collaborators at UNIFR

Biovalley ProgramGymnasium Liestal

END, let's open the DiscussionEND, let's open the DiscussionUNIFRRusconi2003

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***Diskussions-slides...***Diskussions-slides...UNIFRRusconi2002

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text ttt

Gene therapy in Switzerland: the 30 projects financed by the NFP37 programme (1996-2001)

Gene therapy in Switzerland: the 30 projects financed by the NFP37 programme (1996-2001)

UNIFRRusconi2003

UNIFRRusconi2003

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

NationalesForschungsprogramm 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

The SGT acrobatics: matching vectors / delivery system / disease

The SGT acrobatics: matching vectors / delivery system / disease

UNIFRRusconi2003

UNIFRRusconi2003

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)

Die Hauptkrankheit des 21. Jahrhunderts: VeralterungDie Hauptkrankheit des 21. Jahrhunderts: VeralterungUNIFR

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60

70

80

50

1920 1940 1960 1980 1991900

Life

exp

ecta

ncy

(CH

)

4

20 40 60 80

100

10

1

canc

er in

cide

nce

1900 200020 40 60 80

100%

M

E2/E

E3/E4

E4/E4

Alz

heim

er’s

fre

e %

1900 2000

aa getting oldcomp2.mov

Pas toutes les stratégies de transfert se basent sur un ancrage au hasard

Pas toutes les stratégies de transfert se basent sur un ancrage au hasard

UNIFRRusconi2003

UNIFRRusconi2003

Random integrating vectors r-retroviruses r-lentiviruses r-AAV plasmids (low frequency) plasmids + transposase (eg 'sleeping beauty')

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)

Ergo genotoxic non-genotoxic

Specifically integrating vectors hybrid vectors (HSV-AAV) Phage 31 integrase-based designer integrase

Mais un virus c'est quoi?Une machine auto-réplicative extrèmement efficace

Mais un virus c'est quoi?Une machine auto-réplicative extrèmement efficace

UUNIFR

Rusconi

2003ß

UUNIFR

Rusconi

2003ß

E L1 L2

standard viral genome

100 nm

replication

entry disassemblydocking genome replication

late genes exp

assembly

capsid

E L1 L2

Spread

Etc...

early genes exp

Comment peut-on construire des virus récombinants?Comment peut-on construire des virus récombinants?UNIFR

Rusconi

2002

UNIFR

Rusconi

2002

E L1 L2 rprp

Wild type genome Normal target cells Virions

Recombinant genome R-Virions

E E E

EE

EE

Packaging cells

Normal target cells

X

PackagingPackagingPackaging

D'autres tecnolgies émergentes entrent en compétition brutale avec la thérapie génique

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?

UNIFRRusconi2003

UNIFRRusconi2003

V

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

La génétique est practiquée depuis de millénaires, la biologie moléculaire seulement depuis 30 ans

La génétique est practiquée depuis de millénaires, la biologie moléculaire seulement depuis 30 ans

UNIFRRusconi

2003

UNIFRRusconi

2003

100’000 b.C. Empirical genetics

10’000 b.C.Biotechnology

2000 a.d.Molecular biology

2001 a.d, Genomics

When/where/ may be SGT indicated?When/where/ may be SGT indicated?UNIFRRusconi2003

UNIFRRusconi2003

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

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)

Ergo: there are many indications

for SGT as stand-alone or as complementary therapy

Rapid and transient action required

Adeno II, Plasmid, modulatory oligonucleotides

Trauma or infection(Ischemia, fracture, burn, wound, acute infection, anaphyllaxis)

rapid & transient expression of cytotoxic or immunomodulators

Adeno II, Plasmid, oncolytic recombinant viruses

Solid tumors +/- metastat.(cervical, breast, brain, skin)

No rapid expression necessary, persistence required, low toxicity

AAV, nonviral, LentiLocal chronic or progressive (ex. CNS, joints, eyes)

Justifications /IssuesMost suitable vector

persistence of expression of the transferred gene, minimize readministration

Chronic Metabolic (ex. OTC, Gaucher, Haemophilia, hematopoietic)

AAV, Lenti, Adeno III, r-retroviruses, repair oligo

Which vector for which disease categoryWhich vector for which disease categoryUNIFRRusconi2003

UNIFRRusconi2003

Disease Type

Comparing relevant issues in the two main 'vectorology' sectors (viral versus nonviral)

Comparing relevant issues in the two main 'vectorology' sectors (viral versus nonviral)

UNIFRRusconi2003

UNIFRRusconi2003

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)

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

The THREE missions of medicineThe THREE missions of medicineUNIFR

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2003

UNIFR

Rusconi

2003

Prevention

Diagnosis

Therapy

'Molecular Medicine'Application of the

know-how in molecular genetics

to medicine

+

+

+

Ideal properties of a systemically delivered non-viral formulation

Ideal properties of a systemically delivered non-viral formulation

UNIFRRusconi2003

UNIFRRusconi2003

Stability particle should resist serum inactivation particle should be inert to immune inactivation

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

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

Public perception problemsPublic perception problemsUNIFRRusconi2003

UNIFRRusconi2003

Negative perception of manipulative genetics general aversion of genetic manipulation fear of catastrophic scenarios

Deception after excessive promises hopes reinforced by media spectacularisation and

over-simplification deception after non-complied deadline

Confusion with other gene-based and non-gene-based technologies

stem cell technology human cloning procedures genetically modified food

*d Why so many cancer trials?*d Why so many cancer trials?UNIFRRusconi2002

UNIFRRusconi2002

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

*d Ethical dilemmas can sometimes slow down progress and hamper objective appreciations

UNIFRRusconi2002

UNIFRRusconi2002

ADA gene therapy 1990-1999« it does not work »

Feeling:It ’s time we face reality, my friends…We ’re not exactly rocket scientists

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 )

vect

or

*d Cancer molecular treatment Example:Oncolytic viruses on the example of ONYX-015

*d Cancer molecular treatment Example:Oncolytic viruses on the example of ONYX-015

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 immuno-

compromised patients (chemotherapy)

UNIFRRusconi2002

UNIFRRusconi2002

*d Recap: what is a virus ? -> A superbly efficient replicating machine

*d Recap: what is a virus ? -> A superbly efficient replicating machine

UUNIFR

Rusconi

2002

UUNIFR

Rusconi

2002

E L1 L2

standard viral genome

100 nm

replication

entry disassemblydocking genome replication

late genes exp

assembly

capsid

E L1 L2

Spread

Etc...

early genes exp

*d Examples of gene transfer treatments against cancer*d Examples of gene transfer treatments against cancerUNIFRRusconi2002

UNIFRRusconi2002

percentage of trials(therapeutic potential)

Directly cytotoxic Prodrug activation Tumor-specific cytotoxic expression

38% of protocols(strong complementary effect, possible relapse)

Immunostimulatory Transfer of Immuno-attracting functions Instruction of immune cells

31% of protocols(very strong potential, low relapse chances)

Immunoprotective Transfer chemoresistance genes in

immune cells

5 % of protocols(very limited application, laborious, not widely explored)

Tumor modulation Restore tumor-suppressor functions down-regulate pro-oncogenic functions

26 % of protocols(good effect but low bystander and likely relapse)

Type of treatment examples

*d Gene correction strategies (independent of delivery vectors)

*d Gene correction strategies (independent of delivery vectors)

UUNIFR

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2002

UUNIFR

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2002

Approaches

Trans-splicing mRNA

Ribozymic splicing of RNA

Chimeroplasts-induced repair

Triple-helix-guided 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

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…)*d Gene therapy ‘ FAM ’ (Frequently Asserted Myths…)UNIFRRusconi2002

UNIFRRusconi2002

...is withdrawing huge public funding

Gene therapy ...

Wrong: 1998: USA < 2% of NIH budget, CH less than 1% of NF budget

...will be invariably expensive Wrong: example DNA based vaccination

Isner, 1998

...is solely targeting hereditary diseases Wrong: Cancer, Cardiovascular, Restenosis, Ischemia, ...

Fischer, 2000

...has not yet proven its therapeutic value

Kmiec, 1999

Wrong: J. Isner/ I. Baumgartner (ischemia), A. Fischer (SCID), C Bordignon (ADA); M. Kay (Haemophilia, F McCormick (cancer), …...is based only on a ‘shotgun insertion' Wrong: Gene correction 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