Sandro Rusconi (09.03.52)Sandro Rusconi (09.03.52)UNIFRRusconi

2005

UNIFRRusconi

2005

'Therapeutische' Gentherapie: Stand 2005

Sept 15, 2005Uniklinik Balgrist1972-75 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 andUniv. 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 Cultureand University Affairs of Canton Ticino

Eigentlich gemeint war:Therapeutischer

Gentransfer: Stand 2005

Gene therapy: A 15-years hailstorm of highly emotionalised good and bad news

Gene therapy: A 15-years hailstorm of highly emotionalised good and bad news

UNIFRRusconi

2005

UNIFRRusconi

2005

BBC, NBC, CNN,...

New York TimesWashington PostTimesLe MondeFrankfurter Allgemeine...

Feb 1990 First trial ADA deficiency

Dec 1988 IL-2 cancer treatment trial

Mar 1994 SAE cystic fibrosis

NatureScienceNEJM...

Jun 1995 Motulsky NIH report

Feb 1996 r-lentiviruses

Oct 1998 VEGF ischemia

Jess

e Gelsi

nger Oct

1999

A Fischer, E Thrasher Paris & UK Dec 2000

AAV germline Sept 2000

C Bordignon, Milano trial May 2002

First SAE Paris Sep 2002 second SAE Paris Feb 2003

Internet

Autoimmunity monkeys May 2004

SiRNA preclinical 2004

third SAE Paris Jan 2005Selten hat eine medizinische Technik so viel Rauch und so

wenig Feuer produziert

How many of you have heard mostly bad news... ?mostly good news...?

1 Gen -> 1 oder mehrere Funktionen1 Gen -> 1 oder mehrere Funktionen

RNA(s)DNA

GENE

Protein(s)

2-5 FUNCTIONS

Gene expression

Transcription / translation

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

100 ’000 genes(50 ’000 genes?)

UNIFRRusconi

2005

UNIFRRusconi

2005

Multifunctional character of genes implies: cross talk with different pathways unclarified hyerarchical position unclarified side-effects potential

Ergo 'ein Gen -> eine Function'

ist so falsch wie'eine Krakheit -> ein Medikament'

Recap: was ist ein Gen?:ein grosses Molekuel mit informativem Inhalt

Recap: was ist ein Gen?:ein grosses Molekuel mit informativem Inhalt

RNA(s)DNA Protein(s)

GENE FUNCTIONTranscription / translation

codingspacer spacerregulatoryDNA

RNA

Therefore, to fullfil its role, a transferred gene segment must include:

regulatory sequences for Transcription proper signals for RNA Maturation/transport proper signals for mRNA Translation proper signals for mRNA Degradation

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2005

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2005

1 Organismus -> mehr als 105 entwicklungsgenetisch kontrollierte Funktionen

1 Organismus -> mehr als 105 entwicklungsgenetisch kontrollierte Funktionen

2m 2 mm 0.2mm

0.02mm

DNA RNA Protein

0.001mm Zur Erinnerung1 Cm3 Gewebe 1'000'000'000 Zellen!

UNIFRRusconi

2005

UNIFRRusconi

2005

Gentherapie als logische Folge: die dritte AeraGentherapie als logische Folge: die dritte AeraUNIFRRusconi

2003

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

Ergo gene transfer is a logical

development of molecular biology

Somatische Gentherapie (SGT): DefinitionSomatische Gentherapie (SGT): Definition

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

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2005

UNIFRRusconi

2005

Wieso 'somatisch'?Wieso 'somatisch'?

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 tranformierung von

keimbahnzellen ist zur Zeitvermieden (technische und ethische Probleme)

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2005

UNIFRRusconi

2005

Wann gibt heute eine Indikation fuer SGT ?Wann gibt heute eine Indikation fuer SGT ?

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: viele Indikationen

Perverse deviation dreams (even with current technologyI:

gene-based sports doping performance amelioration cosmetics

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2005

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UNIFRRusconi

2005Pharmacologische BetrachtungenPharmacologische Betrachtungen

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

Ergo: Therapy with nucleic acids Spezielle Formulierung Viel komplexer als konventionelle Medikament-Therapie Geringere Reversibilitaet

VIER grundlegende Fragen der SGTVIER grundlegende Fragen der SGT

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?

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2005

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2005

DREI Kategorien von anatomische Gen-LieferungDREI Kategorien von anatomische Gen-Lieferung

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

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2005

UNIFRRusconi

2005

Ergo ex vivo or local delivery are

currently preferred over systemic delivery

ZWEI Vektor-Typen: non-viral & viral

ZWEI Vektor-Typen: non-viral & viral

a

b

Non-viral transfer(transfection of plasmids)

Viral gene transfer(Infection by r-vectors)

Nuclear envelope barrier! see, Nature BiotechDecember 2001

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2005

Ergo viral transfer is much more efficient

nonviral transfer must solve a number of hurdles- serum protection/stability- target docking- endosomal escape- nuclear trafficking- genomic integration- anti apoptotic functions- immunological camouflage - ...

Transfection versus InfectionTransfection versus Infection

Transfection

Infection

exposed to106 particles/cell12 hours

exposed to 1 particle/cell30 min

Ergo virally mediated gene transfer is millions of times more efficent than nonviral

transfer (when calculated in terms of transfer/particle)

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2005

UNIFRRusconi

2005

Kurze Liste von VektorenKurze Liste von Vektoren

r-Adenovirus

r-Adeno-Associated V.

r-Retrovirus (incl. HIV)

Naked DNA

Liposomes & Co.

Oligonucleotides

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UNIFRRusconi

2005

Recap: Limitierungen heutiger VektorenRecap: Limitierungen heutiger Vektoren

r-Adenovirus- no persistence- limited packaging- toxicity, immunogenicity

Biolistic bombardmentor local direct injection- limited area

Electroporation- limited organ access

Liposomes, gene correction & Co.- rather inefficient transfer

General- low transfer efficiency- no or little genomic integration

Solutions:- improved liposomes with viral properties (“Virosomes”)

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2004

UNIFRRusconi

2004

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”)

Ergo the future will probably see an increasing

interest in viral-like, but artificial particles

Gentherapie in der Klinik: Trials Worldwide (cumulative)Gentherapie in der Klinik: Trials Worldwide (cumulative)

cancer

hered.

Infect.vasc.

40

60

100

20

80

trials

500

1500

1000

patients

1992 1994 1996 19981990 2000

20% overall still pending or not yet Initiated !www.wiley.com/genetherapy

66% phase I19% phase I-II13% phase II0.8% phase II-III1.7% phase III

As of January 2005:938 cumulative protocols (90-2005)4700 treated /enrolled 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' paradigm

II-II

II

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2005

UNIFRRusconi

2005

! As of Jan 1, 2004:1 approved product in China (Gendicine, by Sibiono Inc. 2004)2600 Patients treated in 2004

Klinische Meilensteine der GentherapieKlinische Meilensteine der Gentherapie

Anderson, 1990

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

1990, 1993, 2000, // ADA deficiencyF Anderson, M Blaese 90/93/ C Bordignon 2000/2004 Isner, 1998

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

2000, HemophiliaM Kay, K High

Fischer, 20002002

2000, 2002, X-SCIDA Fischer, 2000/2002, Thrasher 2003

Kirn, 2000,200120022003

Intravascular adenoviral agents in cancer patients:

Lessons from clinical trials(review)

dropped in 2004?licensed China 2005?

2001, 2003 ONYX oncolytic VirusesD Kirn (Cancer Gene Ther 9, p 979-86)

Manuel GrezHans Peter HossleReinhard Seger2004/2005

very encouraging data from just initiated clinical trial,prospected >10 patients

2004, Chronic Granulomatous DiseaseM Grez Frankfurt; R Seger Zürich

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2005

Approved commercialisation of Gendicine (Jan 2004) for cancer treatment in China.-> ! Hum Gene Ther 16, 1016 ff.

SibionoShenzen

2004/2005 Gendicine (adeno-p53 vector)L Peng, Sibiono Inc, Shenzen, China

25 lives were so far documentedly saved by GT in european trials (x-SCID, ADA, CGD) (France, UK, Italy) (all in phase I)

~200 lives quality-improved in several other phase I and II trials

~nnn lives saved or quality-improved ?by Gendicine (50'000 patients prospected for 2006)

Zwei persistierende FrustrationsfälleZwei persistierende Frustrationsfälle

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'

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2005

UNIFRRusconi

2005

Die meist befürchtete Nebeneffekte der GentherapieDie meist befürchtete Nebeneffekte der Gentherapie

Immune response to vector

immune response or long term side effects from

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«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

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2005

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2005

immune response or long term side effects from

new or foreign gene product (-> autoimmunity)

Paris, Jan 14, 2003, A Fischer: retrovirus X-SCID (bone marrow) same cohorta second patient developed a similar leukemia 30 trials in USA were temporarily suspended

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.

UPenn, Sept. 19, 1999, J. Wilson: adenovirus , OTC deficiency (liver) one patient (Jesse Gelsinger) died of a severe septic shock. Many trials were put on hold for several months (years).

SAEs1: Vom Gelsingers' Tod bis zu den Paris' Leukaemias

SAEs1: Vom Gelsingers' Tod bis zu den Paris' Leukaemias

NY May 5, 1995, R. Crystal: adenovirus, cystic fibrosis (lung) one patient mild pneumonia-like conditionTrial interrupted and many others on hold.

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2005

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2005

Most Recent Paris' Trial Newsdiscussed under:

www.unifr.ch/nfp37/adverse03.html

Tomorrow (16.09.05) A Fischer will talk at the Kontderspital

(Workshop organised by R Seger)

Paris, Jan 24, 2005, A Fischer:

retrovirus X-SCID (bone marrow) same cohort

a third patient developed a similar leukemia

what will happen?

Ergogene therapy can produce both short-term and long-term severe side effects through acute immunogenicity or insertional mutagenesis (cancer risk)

SAEs2: Recent Autoimmunity ReportsSAEs2: Recent Autoimmunity Reports

Blood, 1 May 2004, Vol. 103, No. 9, comment: pp. 3248-3249Autoimmunity 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

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2005

UNIFRRusconi

2005

K High, ASGT June meeting 2004[Abstract1002] Immune Responses to AAV and to Factor IX in a Phase I Study of AAV-Mediated, Liver-DirectedGene Transfer for Hemophilia B

Ergosomatic gene transfer and ectopic transgene expression can generate mid-term auto- immunity

SAEs3: Nicht-wissenschaftliche Faktoren die Fortschritt und Image von GT negativ beeinflusst haben

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

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

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2005

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2005

1625

Gentherapie Hoehe und Tiefe: a true roller-coaster ride!

Gentherapie Hoehe und Tiefe: a true roller-coaster ride!

>90

high

Low

moo

d

NIHMotulskireport

Lentivectors

Adeno III

J. Isner

F Anderson

R. Crystal

Adeno I

A. FischerM. Kay

AAV germline in mice?

Ergo whenever a reasonable cruise

speed was achieved, a major adverse event has brought us back «square one» or even below

V.Dzau

Paris I and IILeukaemias

J. Gelsinger

90 91 92 93 94 95 96 97 98 99 00 01 02 03 04

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UNIFRRusconi

2005

05

C Bordignon

5

lentivectorshopes

Auto-immunity

gendicine

Paris III

?

?

?

4 companies

Schlussfolgerungen: GT hat Konzepte bewiesen aber bleibt immer noch im Pionierzustand

Schlussfolgerungen: GT hat Konzepte bewiesen aber bleibt immer noch im Pionierzustand

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 !

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2005

UNIFRRusconi

2005

Aussichte: GT wird fortschreiten trotz den gängigen und zhukuenftiken Zwischenfälle

Aussichte: GT wird fortschreiten trotz den gängigen und zhukuenftiken Zwischenfälle

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

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2005

UNIFRRusconi

2005

Meinen 'Proust's questionnaire' bezüglich GentherapieMeinen 'Proust's questionnaire' bezüglich GentherapieUNIFRRusconi

2005

UNIFRRusconi

2005

will GT ever make it into routine clinical practice ? yes

The most worrying adverse-effect? immunity

Which will bloom: viral or non viral transfer? combination thereof

Is insertional mutagenesis an important hurdle? No

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

when will GT widely established ? not tomorrow

Thank you all for the patience and attention,

sandro.rusconi@unifr.chor visit:

www.unifr.ch/nfp37/

...Danke, und ... let's remain optimistic...Danke, und ... let's remain optimistic

My UNIFR and TI collaborators

Ch. Gerber, B. Fuchs

Orthopedics Update

UNIFRRusconi

2005

UNIFRRusconi

2005

Ergo let's look forward

to a safe landing

That's all, folks!That's all, folks!UNIFRRusconi

2005

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2005

www.unifr.ch/nfp37

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2004

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2004