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Sandro Rusconi
Sandro Rusconi
Schedule
Schedule
1 Gene -> 1 or more functions
1 Gene -> 1 or more functions
What is in fact a gene
What is in fact a gene
1 Organism -> more than 105 genetically-controlled Functions
1 Organism -> more than 105 genetically-controlled Functions
Reductionistic molecular biology paradigm (gene defects and gene
Reductionistic molecular biology paradigm (gene defects and gene
Not only the genome determines the health status
Not only the genome determines the health status
Gene amplification / manipulation techniques (genetic engineering,
Gene amplification / manipulation techniques (genetic engineering,
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The FOUR eras of molecular medicine
The FOUR eras of molecular medicine
The major disease and medical challenge of the 21st century: Ageing
The major disease and medical challenge of the 21st century: Ageing
Now, let's talk about Somatic Gene Therapy (somatic gene transfer)
Now, let's talk about Somatic Gene Therapy (somatic gene transfer)
Why 'somatic'
Why 'somatic'
Somatic gene therapy’s (gene transfer) four fundamental questions
Somatic gene therapy’s (gene transfer) four fundamental questions
Pharmacological considerations for DNA transfer
Pharmacological considerations for DNA transfer
THREE classes of physiological gene delivery
THREE classes of physiological gene delivery
TWO classes of gene transfer vehicles: non-viral & viral
TWO classes of gene transfer vehicles: non-viral & viral
Transfection with recombinant DNA Vs Infection with recombinant
Transfection with recombinant DNA Vs Infection with recombinant
but remember
but remember
Recap: current limitations of popular gene transfer vectors
Recap: current limitations of popular gene transfer vectors
Gene Therapy in the clinic: Trials Wordldwide
Gene Therapy in the clinic: Trials Wordldwide
Gene Therapy Milestones
Gene Therapy Milestones
The most feared potential side-effects of gene transfer
The most feared potential side-effects of gene transfer
Four bitter lessons, but only one treatment-related death so far
Four bitter lessons, but only one treatment-related death so far
Ups and Downs and current status of Gene Therapy: a true roller
Ups and Downs and current status of Gene Therapy: a true roller
The THREE levels of doping
The THREE levels of doping
Which gene transfer approaches would be compatible with doping
Which gene transfer approaches would be compatible with doping
Which would be the objective current limitations in gene-based doping
Which would be the objective current limitations in gene-based doping
Which side effects could be feared in gene-based doping strategies
Which side effects could be feared in gene-based doping strategies
Putative detection methods for gene-transfer-based doping strategies
Putative detection methods for gene-transfer-based doping strategies
Final side-by-side comparison: gene-based doping versus drug- or
Final side-by-side comparison: gene-based doping versus drug- or
Somatic gene transfer: conclusions
Somatic gene transfer: conclusions
...Thanks, and let's hope that fair sports will continue to rise
...Thanks, and let's hope that fair sports will continue to rise
UNIFR Rusconi 2002
UNIFR Rusconi 2002

Презентация на тему: «Sandro Rusconi». Автор: . Файл: «Sandro Rusconi.ppt». Размер zip-архива: 2631 КБ.

Sandro Rusconi

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1 Sandro Rusconi

Sandro Rusconi

UNIFR Rusconi 2003

Gene transfer: limits and potential as doping vehicle

Geneva 30.09.03 AISTS 'genes & sport' workshop

1972-75 Primary 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) 1987-94 Group leader, UNI Zuerich (mol. bio., PD) 1994-today Professor Biochemistry UNI Fribourg 1996-02 Director Swiss National Research Program 37 'Somatic Gene Therapy' 2001-today Swiss Natl. Res. Program 50 'Endocrine disruptors' 2002-03 Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department 2002-05 President Union of Swiss Societies for Experimental Biology (USGEB)

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2 Schedule

Schedule

UNIFR Rusconi 2003

Basic understanding of 'genes': what is a gene, how many genes, molecular biology dogma genetic diseases, environmental factors, ageing

Essential concepts on 'molecular medicine' & molecular doping: applications and problems,

Techniques of gene transfer (Gene Therapy) problems and solutions, vectors, clinical achievements

Gene-based doping applications, comparison with other doping, detection

Conclusions plausibility table

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3 1 Gene -> 1 or more functions

1 Gene -> 1 or more functions

UNIFR Rusconi 2003

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4 What is in fact a gene

What is in fact a gene

: a segment of DNA acting as a regulated machine for RNA production

UNIFR Rusconi 2003

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regulatory

coding

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5 1 Organism -> more than 105 genetically-controlled Functions

1 Organism -> more than 105 genetically-controlled Functions

UNIFR Rusconi 2003

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6 Reductionistic molecular biology paradigm (gene defects and gene

Reductionistic molecular biology paradigm (gene defects and gene

transfer)

UNIFR Rusconi 2003

sport boxe 01.mov

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7 Not only the genome determines the health status

Not only the genome determines the health status

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UNIFR Rusconi 2003

also acquired conditions may have a genetic component that modulates their healing trauma fractures burns infections

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8 Gene amplification / manipulation techniques (genetic engineering,

Gene amplification / manipulation techniques (genetic engineering,

recombinant DNA) are simple

UNIFR Rusconi 2003

Science-grade material can be essentially prepared in your cellar ...not so clinical-grade material!

segments of genomic DNA can be specifically cut and isolated

isolated segment can be recombined with a plasmid vector

plasmid vector is transferred into bacteria where it can multiply

isolated recombinant DNA can be further recombined to obtain the final desired molecule

Final molecule is transferred into cells or organisms

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nucleus & DNA.mov

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The THREE missions of medicine, impact of molecular techniques

UNIFR Rusconi 2003

Prevention

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

Diagnosis

Therapy

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10 The FOUR eras of molecular medicine

The FOUR eras of molecular medicine

UNIFR Rusconi 2003

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genomeABC.mov

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11 The major disease and medical challenge of the 21st century: Ageing

The major disease and medical challenge of the 21st century: Ageing

UNIFR Rusconi 2003

NB: many treatments that slow down ageing or age-related degenerative diseases are also potential doping treatments

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12 Now, let's talk about Somatic Gene Therapy (somatic gene transfer)

Now, let's talk about Somatic Gene Therapy (somatic gene transfer)

UNIFR Rusconi 2003

Chronic treatment

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

Acute treatment

Preventive treatment

Hereditary disorders

Acquired disorders

molecular_therapycardio1.mov

Loss-of-function

Gain-of-function

NFP37 somatic gene therapy www.unifr.ch/nfp37

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13 Why 'somatic'

Why 'somatic'

UNIFR Rusconi 2003

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

Ergo: somatic gene transfer is a post-natal treat- ment aiming at somatic cells and consequently does not lead to a hereditary transmission of the genetic alteration --> is NOT a GENETIC SELECTION!

Somatic Cells: all the other cells of the body

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14 Somatic gene therapy’s (gene transfer) four fundamental questions

Somatic gene therapy’s (gene transfer) four fundamental questions

UNIFR Rusconi 2003

Efficiency of gene transfer

Specificity of gene transfer

Persistence of gene transfer

Toxicity of gene transfer

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15 Pharmacological considerations for DNA transfer

Pharmacological considerations for DNA transfer

UNIFR Rusconi 2003

Classical Drugs

Protein Drugs

Nucleic Acids

Therapy with nucleic acids requires particulated formulation is much more complex than previous drug deliveries has a different degree of reversibility (dosage problem)

Mw 20 ’000- 100 ’000 Da Biologically prepared Slower diffusion/action Oral delivery not possible Cellular delivery: - act extracellularly Can be delivered as soluble molecules nm size rapidly reversible treatment

Mw 50- 500 Daltons Synthetically prepared Rapid diffusion/action Oral delivery possible Cellular delivery: - act at cell surface - permeate cell membrane - imported through channels Can be delivered as soluble molecules ?ngstrom/nm size rapidly reversible treatment

Mw N x 1’000’000 Da Biologically prepared Slow diffusion Oral delivery inconceivable Cellular delivery: - no membrane translocation - no nuclear translocation - no biological import Must be delivered as complex carrier particles 50-200 nm size slowly or not reversible

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16 THREE classes of physiological gene delivery

THREE classes of physiological gene delivery

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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17 TWO classes of gene transfer vehicles: non-viral & viral

TWO classes of gene transfer vehicles: non-viral & viral

UNIFR Rusconi 2003

Non-viral transfer (transfection)

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Nuclear envelope barrier! see, Nature Biotech December 2001

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18 Transfection with recombinant DNA Vs Infection with recombinant

Transfection with recombinant DNA Vs Infection with recombinant

viruses

UNIFR Rusconi 2003

Transfection

exposed to 106 particles/cell 12 hours

Infection

exposed to 3 particle/cell 30 min

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19 but remember

but remember

.. "Nobody's perfect "!

List of popular vectors/methods

UNIFR Rusconi 2003

Naked DNA Liposomes & Co. Oligonucleotides

Adenovirus Adeno-associated V. Retrovirus (incl. HIV)

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20 Recap: current limitations of popular gene transfer vectors

Recap: current limitations of popular gene transfer vectors

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) - limited package - random insertion - unstable genome

Liposomes, gene correction & Co. - very inefficient transfer

General - antibody response - limited packaging - gene silencing

General - low transfer efficiency 1/10’000 of viruses’ in vivo

Solutions: - synthetic viruses (“Virosomes”)

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

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21 Gene Therapy in the clinic: Trials Wordldwide

Gene Therapy in the clinic: Trials Wordldwide

UNIFR Rusconi 2003

As of Sept. 2002: 599 registered protocols 4000 treated patients

86% phase I 13% phase II 1 % phase III

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

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22 Gene Therapy Milestones

Gene Therapy Milestones

UNIFR Rusconi 2003

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, ESGT congress, 7.10.2000 Stockholm

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

2001, ONYX oncolytic Viruses D Kirn (Gene Ther 8, p 89-98)

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23 The most feared potential side-effects of gene transfer

The most feared potential side-effects of gene transfer

UNIFR Rusconi 2003

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) side effects of newly acquired gene product Contamination of germ line cells

Random integration in genome -> insertional mutagenesis (-> cancer risk)

Ergo many effects are due to 'primitiveness' of the today's protocols for the moment side effects would (should) ethically limit GT to serious diseases without valid alternatives

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24 Four bitter lessons, but only one treatment-related death so far

Four bitter lessons, but only one treatment-related death so far

UNIFR Rusconi 2003

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.

!! Most Recent Paris' Trial News www.unifr.ch/nfp37/adverse.html

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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25 Ups and Downs and current status of Gene Therapy: a true roller

Ups and Downs and current status of Gene Therapy: a true roller

coaster ride!

UNIFR Rusconi 2003

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Ergo whenever a reasonable cruise speed was achieved, a major adverse event has brought us back square one

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

Lentivectors in pre-clinic

Adverse events in Paris

J. Wilson J. Gelsinger

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26 The THREE levels of doping

The THREE levels of doping

UNIFR Rusconi 2003

Before the competition (anabolic enhancers)

'Molecular treatments Application of the know-how in molecular genetics to doping

During the competition (performance enhancers)

After the competition (repair enhancers)

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27 Which gene transfer approaches would be compatible with doping

Which gene transfer approaches would be compatible with doping

strategies

UNIFR Rusconi 2003

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, anti-myostatin,...)

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, ...)

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28 Which would be the objective current limitations in gene-based doping

Which would be the objective current limitations in gene-based doping

strategies

UNIFR Rusconi 2003

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

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29 Which side effects could be feared in gene-based doping strategies

Which side effects could be feared in gene-based doping strategies

UNIFR Rusconi 2003

Short -mid term Autoimmunity Hyperimmunity Toxic shock

sport hunter jones.mov

Long term Fibrosis Cancer conventional side- effects of administered factors Inaccessibility to future gene therapy interventions (immunity)

Intrinsic to reckless application (probably the biggest danger) malpractice (unsuitable vector/administration route) non-clinical grade material (pathogens or allergens) lack of follow-up

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30 Putative detection methods for gene-transfer-based doping strategies

Putative detection methods for gene-transfer-based doping strategies

and their linked problems

UNIFR Rusconi 2003

sport weigh women 2.mov

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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

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31 Final side-by-side comparison: gene-based doping versus drug- or

Final side-by-side comparison: gene-based doping versus drug- or

protein-based doping

UNIFR Rusconi 2003

Category Drug/protein Gene-based

Rapidity of effects rapid slow

Ergo: The odds would speak currently rather against the adoption of gene-based doping,

Reversibility rapid slow

Dosage straightforward difficult

but: this applies to common-sense clinical practice, and this aspect is not guaranteed in the doping field

Complexity of treatm. simple complex

Associated risks depends high

and: ... there are several sporting disciplines where doping is not rigourously (or not at all) verified.

Concealability possible difficult /impossible

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32 Somatic gene transfer: conclusions

Somatic gene transfer: conclusions

UNIFR Rusconi 2003

somatic gene transfer has been originally developed for the treatment of diseases (genetical or acquired) must be distinguished from genetic selection has the potential to be applied for pre- during- and post-performance enhancement currently still experimental and not technically mature for applications in non-lethal conditions has already raised the interest of doping field major risk linked with premature application single gene transfer for enhancement will create more problems than it could solve

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33 ...Thanks, and let's hope that fair sports will continue to rise

...Thanks, and let's hope that fair sports will continue to rise

genuine emotions: yesterday, today and tomorrow!

AISTS, MSA program

UNIFR Rusconi 2003

if you are too shy to ask send an e-mail to: sandro.rusconi@unifr.ch or visit: www.unifr.ch/nfp37

My collaborators at UNIFR

Swiss National Research Foundation

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34 UNIFR Rusconi 2002

UNIFR Rusconi 2002

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