Sandro Rusconi (9.3.52)Sandro Rusconi (9.3.52)UNIFRRusconi

2005

UNIFRRusconi

2005

'Gene doping':Is it coming? Is it there?

Lausanne 21.09.2005AISTS 'genes and sport'

1972-75 School teacher (Locarno, Switzerland)

1975-79 Graduation in Biology UNI Zuerich, Switzerland

1979-82 PhD curriculum UNI Zuerich, molecular biology

1982-84 Research assistant UNI Zuerich

1984-86 Postdoc UCSF, K Yamamoto, (San Francisco)

1987-93 Principal Investigator, UNI Zuerich, PD

1994-today Professor Biochemistry UNI Fribourg

1996-2002 Director Swiss National Research Program 37

'Somatic Gene Therapy'

2002-03 Sabbatical, Tufts Med. School Boston 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

What is therapeutic gene transfer (gene therapy)?functions transfer, objectiives, somatic

Which possibilities exist gor Gene-based Doping ?doping in general, gene doping perspectives, obstacles, detectability, side effects, risk-benefits

How far has gene therapy progressed ?principles, goals, obstacles, clinical achievements

Issues that will be addressedIssues that will be addressed

What is a 'gene'?1 gene -> several fiunctions, genes language, gene expression, gene manipulation

Conclusionstechnically too early but recklessness and money make it moving anyway

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UNIFRRusconi

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Quintessence

read it in this orange box

Doping in top sports has some deadly aspects...Doping in top sports has some deadly aspects...UNIFRRusconi2005

UNIFRRusconi2005

Name Sport Age Date CauseDenis Zanette (Ita) cyclist 33 10.01.2003 cardiac arrest dentist roomFabrice Salanson (Fra) cyclist 23 03.06.2003 cardiac arrest hotel roomVivien Foe (Cam) football 28 26.06.2003 cardiac arrest on the fieldJose Maria Jimenez(Spa) ciclist 32 8.12.2003 cardiac arrest hospitalMiklos Feher (Hun) football 24 25.01.2004 cardiac arrest on the fieldRaymond Junikis basket 23 10.02.2004 cardiac arrest at the matchJohan Sermon cyclist 21 13.02.1004 cardiac arrest at homeMarco Pantani cyclist 28 14,02.2004 cardiac arrest hotel room??? ... ... ... ...

Questions what is the perception of risk/benefit in sports? Can all this worsen when gene transfer enters the scene?

Mythos 'Gene': in the good and in the bad ...Mythos 'Gene': in the good and in the bad ...

MedicineNeue Medikamente, neue Heilungschancen, neue Diagostik, ...

AgricultureNeue Eigenschaften von Nutzpflanzen/ Nutztieren ...

AgricultureOekologische Katastrophen, Gesundheitspropleme, «GMO=Giftig»

MedicineBio-Waffen, Monster-Generation, Designer-babies, ...

Ergo:we must first eliminate these myths

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UNIFRRusconi

2005

Myths 'Gene therapy':- against hereditary diseases- transmissible modification

Myths 'Gene doping':- better than conventional- hereditary genetic modification- pre-natal designer athletes

1 Organism -> 1013 Cells, distributed in specialised organs and tissues

1 Organism -> 1013 Cells, distributed in specialised organs and tissues

2m 2 mm 0.2mm

0.02mm

DNA RNA Protein

0.001mm

Ergo at each cell division the genetic

marterial is copied in 1 cm3 -> 1'000'000'000 cells

UNIFRRusconi

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UNIFRRusconi

2005

The old motto: 'one gene one function' is obsolete...The old motto: 'one gene one function' is obsolete...

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?)

Ergo:side effects in gene transfer might becaused also by supplementary andyet undiscovered functions of a given gene

UNIFRRusconi

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UNIFRRusconi

2005

But ...what is actually 'a gene'?:...a regulated nano-machine for the production of RNA

But ...what is actually 'a gene'?:...a regulated nano-machine for the production of RNA

RNADNA Protein

GENE FUNCTIONTranscription / translation

codingspacer spacerregulatoryDNA

RNA

UNIFRRusconi

2005

UNIFRRusconi

2005

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

The reductionist Paradigm of molecular biologistsThe reductionist Paradigm of molecular biologists

GENE transfer FUNCTION transfer

GENE KO FUNCTION KO

GENE OK FUNCTION OK

DNA

GENE

Protein

FUNCTION(s)Gene transfer can imply: Transfer of a new Function, or Transfer of a compensatory F., or Transfer einer interfering Function

UNIFRRusconi

2005

UNIFRRusconi

2005

Gene transfer as logical consequence: the third era of molecular biology

Gene transfer as logical consequence: the third era of molecular biology

EightiesGenes 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

UNIFRRusconi

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UNIFRRusconi

2005

Gene transfer (Gene therapy): logical consequence of development in molecular biology

Somatic Gene therapy (SGT) Definition and applications

Somatic Gene therapy (SGT) Definition and applications

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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UNIFRRusconi

2005

Pharmacological considerations, differences with conventional medication therapy

Pharmacological considerations, differences with conventional medication therapy

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

Theray with nucleic acids (DNA) Need special formulation (vectors) more complex less reversible

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UNIFRRusconi2003

UNIFRRusconi2003

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

Somatic Cells: all the other cells of the body

Ergo:somatic gene transfer is NOT aiming at GERM LINE cellsThe modification is therefore NOT HERITABLE

Why 'somatic'?Why 'somatic'?UNIFRRusconi

2005

UNIFRRusconi

2005

The four technical basic questions in SGTThe four technical basic questions in SGTUNIFRRusconi2003

UNIFRRusconi2003

Efficiency of gene transfer

Specificity of gene transfer

Persistence of gene transfer

Toxicity of gene transfer

Remember!

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UNIFRRusconi

2005

The variables which disease? which gene? which vector? which target organ? which type of delivery?

Three anatomical delivery methods in SGT:Three anatomical delivery methods in SGT:

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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UNIFRRusconi

2005

Ergo ex vivo or local delivery are

currently preferred over systemic delivery

Two classes of vectors:viral / non viral

Two classes of vectors:viral / non viral

A

B

Transfert non viral(transfection)

viral transfer(Infection)

Nuclear envelope barrier!

direct nuclear shuttling!

UNIFRRusconi

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UNIFRRusconi

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Why are viruses 'better'? 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 - ...

Example: transfection (non viral) versus Infection (viral) transfer of a reporter gene

Example: transfection (non viral) versus Infection (viral) transfer of a reporter gene

Transfection

Infection

cells exposed to1'000'000 particles/cell12 hours

cells exposed to 3 particle/cell30 min

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UNIFRRusconi

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

transfer (when calculated in terms of transfer/particle)

Mini- List von current gene transfer methodsMini- List von current gene transfer methods

r-Adenovirus

r- Adeno-associated V.

r- Retrovirus

r- Lentivirus

Naked DNA

Liposomes & Co.

Oligonucleotides

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UNIFRRusconi

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Recap: Limitations of current vectorsRecap: Limitations of current vectors

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”)

UNIFRRusconi

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UNIFRRusconi

2005

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

The traditional clinical path: lots of time/money

The traditional clinical path: lots of time/money

UNIFR

Rusconi

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UNIFR

Rusconi

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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

Fazit:on average only 1out of 5 drugs makes it to approval->

500 Mio U$D investment per successful drug !!!

Gene therapy in the clinics: Trials Worldwide (cumulative)

Gene therapy in the clinics: 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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UNIFRRusconi

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

clinical milestones in gene therapyclinical milestones in gene therapy

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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UNIFRRusconi

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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)

Die most feared side effects of Gene TherapyDie most feared side effects of Gene Therapy

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

UNIFRRusconi

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UNIFRRusconi

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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: from Pennsylvania to ParisSAEs1: from Pennsylvania to Paris

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

UNIFRRusconi

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UNIFRRusconi

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Most Recent Paris' Trial Newsdiscussed under:

www.unifr.ch/nfp37/adverse03.html

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 Reports in gene transfer...should open the eyes of potential dopers

SAEs2: Recent Autoimmunity Reports in gene transfer...should open the eyes of potential dopers

Blood (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

UNIFRRusconi

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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

Ergo somatic gene transfer and ectopic

transgene expression is detectable and can generate mid-term auto- immunity sounds quite risky for Epo doping

Now, lets dedicate completely to gene doping:.. is ist thinkable?

Now, lets dedicate completely to gene doping:.. is ist thinkable?

Ergo:Realistically speaking, efforts in SGT should be currently restricted to severe diseases, and we should close the talk here,however...

UNIFRRusconi

2005

UNIFRRusconi

2005Gene therapy (features summary)- treatment not hereditary- principle works - not yet clinically established- high risk with todays vectorology- applicable to virtually all disease status- pioneer status

The three levels of Doping...The three levels of Doping...

Before thecompetition

(anabolic enhancers)

During the competition(performance enhancers)

After thecompetition

(repair enhancers)

'Molecular treatmentsApplication of the

know-how in molecular genetics

to doping

+

+

+

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UNIFRRusconi

2005

Which gene-transfer-Strategies could be concretely conceivable for doping?

Which gene-transfer-Strategies could be concretely conceivable for doping?

ex vivo, hematopoietic tissue:pro hematopoietic (Epo receptor, oxygen transport...)

in vivo local (example muscle):metabolic enhancers, growth factors, muscular fiber changers, cardio-modulators (glucose/oxygen, MGF, IGF-1, anti-myostatin, Epo)

in vivo local (example joints):pain reducers, inflammation inhibitors, recovery and repair factors (anti-TNF, BMPs, ...)

in vivo systemic:anabolic enhancers, endocrine factors, pain killers, vascular controllers, (hormone metabolising enzymes, proenkephalins, ...)

UNIFRRusconi

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UNIFRRusconi

2005

Ergo:Doping with gene transfer is conceivable at many different levels

The features IGF-1 -> growth factor for muscles AAV Vector, intra muscular Rat model , + or - training

Resultsmuscle force and muscle mass increased beyond levels obtained in training

The experiments of Lee Sweeney (2004) have raised further smoke...

The experiments of Lee Sweeney (2004) have raised further smoke...

Gene transfer of IGF-1 (J. App Physiol 96, 1097 ff (2004))

Ergook, Dr. Sweeney, transfer of IGF-1 in rats significantly increases muscle performance,, but...

- + - +

- - ++training:

IGF-1:

mus

cle

forc

e

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UNIFRRusconi

2005

questions- can it be extrapolated to humans?- kg muscle to tranduce ?- how to manufacture sufficient vector?- symmetry of effects?- adverse side effects?

Which side effects should we expect if gene transfer would be currently applied in doping

Which side effects should we expect if gene transfer would be currently applied in doping

Short -mid term Autoimmunity Hyperimmunity Toxic shock

Long term Fibrosis Cancer conventional side- effects of

administered factors Inaccessibility to future gene

therapy interventions (immunity to vectors)

Specially dangerous: Recklessness... Improper technology

(unsuitable vector, low competence of doctors)

Improper Material

(contaminated with pathogens or pyrogens)

Insufficient follow-up

UNIFRRusconi

2005

UNIFRRusconi

2005

Which would be the objective limitations of gene doping?

Which would be the objective limitations of gene doping?

Viral gene transfer immune problems limited readministration possibilities general toxicity, genotoxicity

Nonviral gene transfer generally inefficient lack of persistence, requires readministration

Strategy-independent problems laborious, not readily available long term gene expression difficult to control irreversible effects or permanent tagging asymmetry of effects

Ergo:risks seem today currently higher than benefits

UNIFRRusconi

2005

UNIFRRusconi

2005

R

N

Detection possibilities of gene doping

Detection possibilities of gene doping

Antibody detection (viral antigens)

r-nucleic acids detection (PCR)

recombinant protein / post-translational

modification detection (MALDI-TOF )

Anatomically difficult to detect

(if locally administered)

-> but leaves permanent genetic marking

Detection of nucleic acids cannot be performed in body fluids

(except in early phase after systemic administration)

-> might require specific tissue biopsy

Ergo foreign genes detectable only short-

term in blood or body fluids, but - foreign genes detectable long term in tissue biopsies, and- abnormal gene products detectable (example GT erythropoietin in monkeys)

UNIFRRusconi

2005

UNIFRRusconi

2005

Comparison of advantages/disadvantages: with respect to conventional Doping

Comparison of advantages/disadvantages: with respect to conventional Doping

Category Drug/protein Gene-based

Rapidity of effects rapid slow

Reversibility rapid slow

Complexity of treatm. simple complex

Associated risks depends high

Concealability possible difficult

/impossible

Dosage straightforward difficult

Ergo:the odds would speak actually against Gene doping

but:awareness of the above needs common sense, a property that is rare in the doping field

and:...there are several borderline sports lacking doping control

UNIFRRusconi

2005

UNIFRRusconi

2005

Question:...isnt it the high publicity over gene doping just a sort of psychologically intimidating strategy by some nations (this has happened before in doping)

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cette image.

Doping with Gene transfer: Proust's questionnaire

Doping with Gene transfer: Proust's questionnaire

UNIFRRusconi

2005

UNIFRRusconi

2005

Is someone in the field currently 'thinking GT' for doping ? yes

Which country may produce the first human cases? ...China?

Shall it be effective at all ? probably little

Will gene doping be more effective than conventional? No

Will some athlete suffer or even die from GT attempts? yes

Which gene will be first transferred for doping purposes ? Epo

Shall gene doping remain difficult to detect ? No

M Proust 1871-1922

Which sports will produce the first case ? horse racing?

Why is then gene doping still so attractive ? ignorance

for correspondence:sandro.rusconi@unifr.ch

for info :www.unifr.ch/nfp37

... Thank you, and let's hope that sports can continue producing genuine emotions and fairplay

... Thank you, and let's hope that sports can continue producing genuine emotions and fairplay

my collaborators at UNIFR

AISTS, Prof. Bengt Kayser

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2005

UNIFRRusconi

2005

«That's all folks» ...looking forward to your questions

«That's all folks» ...looking forward to your questions

www.unifr.ch/nfp37

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2005

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2005

UNIFRRusconi2002

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