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					                                                                                    UNIFR
Sandro Rusconi (9.3.52)                             Lausanne 21.09.2005             Rusconi
                                                   AISTS 'genes and sport'
                                                                                    2005
1972-75      School teacher (Locarno, Switzerland)
1975-79      Graduation in Biology UNI Zuerich, Switzerland            'Gene doping':
1979-82      PhD curriculum UNI Zuerich, molecular biology
                                                                        Is it coming?
1982-84      Research assistant UNI Zuerich
1984-86      Postdoc UCSF, K Yamamoto, (San Francisco)
                                                                          Is it there?
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 and
             Univ. 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 Culture
               and University Affairs of Canton Ticino
                                                                          UNIFR
Issues that will be addressed                                             Rusconi
                                                                          2005

 What is a 'gene'?
 1 gene -> several fiunctions, genes language, gene expression,
 gene manipulation
 What is therapeutic gene transfer (gene therapy)?
 functions transfer, objectiives, somatic                   Quintessence
 How far has gene therapy progressed ?
 principles, goals, obstacles, clinical achievements         read it in this
                                                              orange box
 Which possibilities exist gor Gene-based Doping ?
 doping in general, gene doping perspectives, obstacles,
 detectability, side effects, risk-benefits
 Conclusions
 technically too early but recklessness and money
 make it moving anyway
                                                                                       UNIFR
Doping in top sports has some deadly aspects...                                        Rusconi
                                                                                       2005

 Name                      Sport      Age   Date         Cause
 Denis Zanette (Ita)       cyclist    33    10.01.2003   cardiac arrest dentist room
 Fabrice Salanson (Fra)    cyclist    23    03.06.2003   cardiac arrest hotel room
 Vivien Foe (Cam)          football   28    26.06.2003   cardiac arrest on the field
 Jose Maria Jimenez(Spa)   ciclist    32    8.12.2003    cardiac arrest hospital
 Miklos Feher (Hun)        football   24    25.01.2004   cardiac arrest on the field
 Raymond Junikis           basket     23    10.02.2004   cardiac arrest at the match
 Johan Sermon              cyclist    21    13.02.1004   cardiac arrest at home
 Marco 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?
                                                                      UNIFR
Mythos 'Gene': in the good and in the bad ...                         Rusconi
                                                                      2005

Medicine
Neue Medikamente, neue Heilungschancen,
neue Diagostik, ...
                     Myths 'Gene therapy':
                                        Medicine
                     - against hereditary diseases
                                        Bio-Waffen, Monster-Generation,
                     - transmissible modification
                                        Designer-babies, ...
 Agriculture
 Neue Eigenschaften von Nutzpflanzen/
 Nutztieren ...      Myths 'Gene doping':
                     - better than conventional
                                        Agriculture
                     - hereditary genetic modification
                                        Oekologische Katastrophen,
                     - pre-natal designer athletes
                                        Gesundheitspropleme, «GMO=Giftig»

 Ergo:
 we must first eliminate these myths
                                                                           UNIFR
1 Organism ->  1013  Cells,                                                Rusconi
distributed in specialised organs and tissues                              2005

       2m                     2 mm                          0.2mm




                                              0.02mm
                              0.001mm

DNA     RNA     Protein
                                         Ergo
                                            at each cell division the genetic
                                             marterial is copied
                                            in 1 cm3 -> 1'000'000'000 cells
                                                                           UNIFR
The old motto: 'one gene one function' is obsolete...                      Rusconi
                                                                           2005


      DNA                  RNA(s)                    Protein(s)
                   Transcription / translation




                     Gene expression
     GENE                     Ergo:               2-5 FUNCTIONS
                              side effects in gene transfer might be
                              caused also by supplementary and
                              yet undiscovered functions of a given gene


100 ’000 genes                                   >300 ’000 functions
(50 ’000 genes?)                                 (>150 ’000 functions)
                                                                               UNIFR
 But ...what is actually 'a gene'?:                                            Rusconi
 ...a regulated nano-machine for the production of RNA                         2005


         DNA                 RNA                      Protein



         GENE                                        role,
                           Therefore, to fullfil its FUNCTION
                   Transcription / translation
                                 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
                                                    RNA


                                                    DNA
spacer          regulatory                coding                   spacer
                                                                    UNIFR
The reductionist Paradigm of molecular biologists                   Rusconi
                                                                    2005



   DNA                                  Protein




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

   GENE OK                        Transfer einer interfering Function
                                      FUNCTION OK

   GENE KO                           FUNCTION KO

   GENE transfer                     FUNCTION transfer
                                                                            UNIFR
Gene transfer as logical consequence: the third era of                      Rusconi
molecular biology                                                           2005

 Eighties
 Genes as probes           Nineties
                           Genes as factories
                                                      Y2K
                                                      Genes as drugs


           1 2 3 4 5
          ok ** ok ** **

                              50

                                                      3000
                              10
                                   80 85 90 95 99     1000

                 Gene transfer (Gene therapy):             80 85 90 95 00

                logical consequence of development in molecular biology
   Somatic Gene therapy (SGT)                                             UNIFR
                                                                          Rusconi
   Definition and applications                                            2005

    Definition of SGT:                               Chronic treatment
    'Use genes as drugs':                               Acute treatment
    Correcting disorders by
    somatic gene transfer                                 Preventive treatment

NFP37 somatic gene therapy
    www.unifr.ch/nfp37
                             Hereditary disorders
                               Acquired disorders

                                  Loss-of-function
                                    Gain-of-function
Pharmacological considerations,                                                           UNIFR
                                                                                          Rusconi
differences with conventional medication therapy                                          2005
Classical Drugs                    Protein Drugs                    Nucleic Acids
   Mw 50- 500 Daltons                Mw 20 ’000- 100 ’000 Da      Mw N x 1’000’000 Da
                                                                    
   Synthetically prepared            Biologically prepared       Biologically prepared

   Rapid diffusion/action            Slower diffusion/action     Slow diffusion
   Oral delivery possible            Oral delivery not possible  Oral delivery inconceivable

   Cellular delivery:                Cellular delivery:          Cellular delivery:

    - act at cell surface              - act extracellularly        - no membrane translocation
    - permeate cell membrane                                        - no nuclear translocation
    - imported through channels                                     - no biological import
   Can be delivered as               Can be delivered as         Must be delivered as

    soluble molecules                  soluble molecules            complex carrier particles
    Ångstrom/nm size                   nm size                      50-200 nm size
   rapidly reversible treatment      rapidly reversible treatment slowly or not reversible
                                                                  



 Theray with nucleic acids (DNA)
    Need special formulation (vectors)
    more complex
    less reversible
                                                                                     UNIFR
                                                                                     UNIFR
Why 'somatic'?                                                                       Rusconi
                                                                                     Rusconi
                                                                                     2003
                                                                                     2005


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




                            Ergo:
                            somatic gene transfer is
                            NOT aiming at GERM LINE cells
    Somatic Cells: all the The cells of the body is therefore
                            other modification
                            NOT HERITABLE
                                                               UNIFR
                                                               UNIFR
The four technical basic questions in SGT                      Rusconi
                                                               Rusconi
                                                               2003
                                                               2005

                                                   Remember!
     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?
                                                                                UNIFR
Three anatomical delivery methods in SGT:                                       Rusconi
                                                                                2005


  Ex-vivo                In-vivo                          In-vivo
                         topical delivery                 systemic delivery
                                            Ergo
                                               ex vivo or local delivery are
                                                currently preferred over
                                               systemic delivery
                    V




         Examples:                   Examples:                             Examples:
         - bone marrow               - brain                               - intravenous
         - liver cells               - muscle                              - intra-arterial
         - skin cells                - eye                                 - intra-peritoneal
                                     - joints
                                     - tumors
Two classes of vectors:                                                               UNIFR
                                                                                      Rusconi
viral / non viral
                                                                                      2004

   Transfert non viral                 A
   (transfection)
                                                Why are viruses 'better'?
                                                   viral transfer is much more efficient

                                                   nonviral transfer must solve a
                                                     B
                                                    number of hurdles
                                                    - serum protection/stability
                                                    - target docking
                                                            Nuclear envelope barrier!
                                                    - endosomal escape
                                                    - nuclear trafficking
   viral transfer
                                                    - genomic integration
   (Infection)                                      - anti apoptotic functions
                                                    - immunological camouflage
                                                    - ...


                    direct nuclear shuttling!
Example: transfection (non viral) versus Infection (viral)                     UNIFR
                                                                               Rusconi
transfer of a reporter gene                                                    2005

                               Transfection
                       cells exposed to
                       1'000'000 particles/cell
                       12 hours
                                                              Infection
                                                          cells exposed to
                                                          3 particle/cell
                                                          30 min




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

   transfer (when calculated in terms of transfer/particle)
                                                       UNIFR
Mini- List von current gene transfer methods           Rusconi
                                                       2005


       r-Adenovirus                 Naked DNA


       r- Adeno-associated V.       Liposomes & Co.


       r- Retrovirus                Oligonucleotides

       r- Lentivirus
                                                                                                  UNIFR
Recap: Limitations of current vectors                                                             Rusconi

 r-Adenovirus
                                                                                                  2005
 - no persistence                            Biolistic bombardment
 - limited packaging                         or local direct injection
 - toxicity, immunogenicity                  - limited area

      r-AAV                                       Electroporation
      - no integration in host g.                 - limited organ access
      - very limited packaging
      - autoimmunity?
                                                      Liposomes, gene correction & Co.
         r-Retrovirus (incl. HIV)                     - rather inefficient transfer
         - limited packaging
         - random insertion
         - unstable genome
                                                            General
                                                            - low transfer efficiency
             General
                                                            - no or little genomic integration
             - antibody response
             - limited packaging
             - gene silencing              Ergo
             - Manufacturing limitations                      Solutions:
                                            the future will probably see an increasing
                                                              - improved liposomes
                 Solutions:                  interest in viral-like, but artificial particles
                                                                with viral properties (“Virosomes”)
                 - synthetic viruses
                   (“Virosomes”)
The traditional clinical path:                                                      UNIFR
                                                                                    Rusconi
lots of time/money                                                                  2005


         year   event                         costs U$D

         0      Idea                          0

         2      Cell culture assays           0.5 Mio     Fazit:
                                                          on average only 1out of 5 drugs
         5      Pre-clinical tests                        makes it to approval
                animal models                 2 Mio
                                                          ->
         7      Clinical phase I
                5-20 patients                             500 Mio U$D investment
                verify side effects           6 Mio       per successful drug !!!
         10     Clinical phase II
                30-100 patients
                dosis escalation              12 Mio

         15     Clinical Phase III
                >300- 1000 patients
                multicentric
                double blind                  80 Mio

         16>>   Registration / Availability
Gene therapy in the clinics:                                                         UNIFR
                                                                                     Rusconi
Trials Worldwide (cumulative)
                                                                                     2005
   trials                                                                   patients
            Ergo
               in spite of 13 year- research only
                less than 2% of the trials has          As of January 2005:
                reached phase III
   100         not necessarily due to the «novel»                            1500
                                                        938 cumulative protocols (90-2005)
                                                        4700 treated /enrolled patients
                'fail early, fail fast'cancer
                                       paradigm
    80                                                                          II
                                                        66% phase I
    60      ! As of Jan 1, 2004:                        19% phase I-II         1000
            1 approved product in China                 13% phase II         I-II
                                        hered.
            (Gendicine, by Sibiono Inc. 2004)           0.8% phase II-III                 I
    40      2600 Patients treated in 2004               1.7% phase III
                                                                                    500
    20                                                vasc.overall still pending
                                                       20%
                                                                   Infect.
                                                       or not yet Initiated !
                                                        www.wiley.com/genetherapy

     1990 1992            1994         1996          1998     2000
                                                                                          UNIFR
clinical milestones in gene therapy                                                       Rusconi
                                                                                          2005
1990, 1993, 2000, // ADA deficiency                                   Anderson, 1990
F Anderson, M Blaese 90/93/ C Bordignon 2000/2004                           Isner, 1998
                                                                                Fischer,
                                                                         Kirn, 2000
1997, 2000, Critical limb ischemia                                       2000, 2002
J Isner († 4.11.2001), I Baumgartner, 1998
            25 lives                                                     2001 Grez
                                                                          Manuel
            were so
2000, Hemophilia far documentedly saved by GT in                         2002 Peter Hossle
                                                                          Hans Sibiono
            european trials (x-SCID, ADA, CGD)
M Kay, K High                                                            2003 Shenzen
                                                                          Reinhard Seger
            (France, UK, Italy) (all in phase I)          Intravascular adenoviral agents
                                                                          2004/2005
2000, 2002, X-SCID                                                in cancer patients:
            ~200 lives quality-improved
A Fischer, 2000/2002, Thrasher 2003                          Lessons from clinical from
                                                         very encouraging data trials
            in several other phase I and II trials                     (review)
                                                         just initiated clinical trial,
2001, 2003 ONYX oncolytic Viruses                                  dropped in 2004?
                                                         prospected >10 patients
            ~nnn lives saved or quality-improved ?              licensed China 2005?
D Kirn (Cancer Gene Ther 9, p 979-86)
            by Gendicine (50'000 patients prospected 2000 (ESGT, Stockholm)
                                              Bordignon,      Approved commercialisation of
            for 2006)
2004, Chronic Granulomatous Disease
                                                              Gendicine (Jan
                                              2002, science 296, 2410 ff) 2004) for cancer
                                                            treatment in China.
M Grez Frankfurt; R Seger Zürich                            -> ! Hum Gene Ther 16, 1016 ff.

2004/2005 Gendicine (adeno-p53 vector)
L Peng, Sibiono Inc, Shenzen, China
                                                       UNIFR
Die most feared side effects of Gene Therapy           Rusconi
                                                       2005
   Immune response to vector
   immune response or long term side effects from
    new or foreign gene product (-> autoimmunity)
   General toxicity of viral vectors
   Adventitious contaminants in recombinant viruses
   Random integration in genome
    -> insertional mutagenesis (-> cancer risk)
   Contamination of germ line cells

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
                                                                                                   UNIFR
SAEs1: from Pennsylvania to Paris                                                                  Rusconi
                                                                                                   2005
NY May 5, 1995, R. Crystal:
adenovirus, cystic fibrosis (lung)
one patient mild pneumonia-like condition
Trial interrupted and many others on hold.
                                                                          Most Recent Paris' Trial News
UPenn, Sept. 19, 1999, J. Wilson:                                              discussed under:
adenovirus , OTC deficiency (liver)                                     www.unifr.ch/nfp37/adverse03.html
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:
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.

Paris, Jan 14, 2003, A Fischer:                                 Ergo
retrovirus X-SCID (bone marrow) same cohort
a second patient developed a similar leukemia                   gene therapy can produce both short-
30 trials in USA were temporarily suspended                     term and long-term severe side effects
                                                                through acute immunogenicity or
                                                                insertional mutagenesis (cancer risk)
SAEs2: Recent Autoimmunity Reports in gene transfer...                                      UNIFR
                                                                                            Rusconi
should open the eyes of potential dopers
                                                                                            2005
Blood (2004), Vol. 103, No. 9, comment: pp. 3248-3249
Autoimmunity 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
                                                           Ergo
K High, ASGT June meeting 2004                              somatic gene transfer and ectopic
[Abstract1002] Immune Responses to AAV and to
Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed
                                                             transgene expression
Gene Transfer for Hemophilia B                              is detectable and
                                                            can generate mid-term auto- immunity
                                                            sounds quite risky for Epo doping
Now, lets dedicate completely to gene doping:   UNIFR
                                                Rusconi
.. is ist thinkable?
                                                2005
Gene 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

 Ergo:
 Realistically speaking, efforts in SGT
 should be currently restricted to severe
 diseases, and we should close the talk
 here,
 however...
                                                                 UNIFR
The three levels of Doping...                                    Rusconi
                                                                 2005

         Before the
                      +
        competition
    (anabolic enhancers)
                                        'Molecular treatments
                                            Application of the
                           +                  know-how in
   During the competition                   molecular genetics
                                                to doping
  (performance enhancers)


                                       +
                                     After the
                                    competition
                                (repair enhancers)
Which gene-transfer-Strategies                                      UNIFR
                                                                    Rusconi
could be concretely conceivable for doping?
                                                                    2005
   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):            Ergo:
    pain reducers, inflammation inhibitors, recovery and gene transfer is
                                               Doping with
    repair factors (anti-TNF, BMPs, ...)       conceivable at many
                                                different levels
   in vivo systemic:
    anabolic enhancers, endocrine factors, pain killers,
    vascular controllers, (hormone metabolising
    enzymes, proenkephalins, ...)
The experiments of Lee Sweeney (2004)                                     UNIFR
                                                                          Rusconi
have raised further smoke...
                                                                          2005
Gene transfer of IGF-1 (J. App Physiol 96, 1097 ff (2004))


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




                                            muscle force
Results
muscle force and muscle mass increased
beyond levels obtained in training          questions
                                            - can it be extrapolated to humans?
                                            - kg muscle to tranduce ?
                                            - how to manufacture sufficient vector?
Ergo
                                            - symmetry of effects?
ok, Dr. Sweeney, transfer of IGF-1 in
rats significantly increases muscle
                                            - adverse      -
                                                           +
                                          training: side effects?   -      +
performance,, but...                         IGF-1:        - -      +    +
Which side effects should we expect if gene transfer                      UNIFR
                                                                          Rusconi
would be currently applied in doping
                                                                          2005

Short -mid term
   Autoimmunity
   Hyperimmunity
   Toxic shock

Long term                        Specially dangerous: Recklessness...
 Fibrosis                        Improper technology

 Cancer                           (unsuitable vector, low competence of doctors)
 conventional side- effects of   Improper Material
  administered factors             (contaminated with pathogens or pyrogens)
 Inaccessibility to future gene
                                  Insufficient follow-up
  therapy interventions (immunity
  to vectors)
Which would be the objective limitations                    UNIFR
                                                            Rusconi
of gene doping?
                                                            2005
Viral gene transfer
                                 Ergo:
 immune problems                risks seem today
                                 currently
 limited readministration possibilities
                                 higher than benefits
 general toxicity, genotoxicity

 Nonviral gene transfer
  generally inefficient

  lack of persistence, requires readministration

  Strategy-independent problems                                    N
   laborious, not readily available

   long term gene expression difficult to control
                                                        R
   irreversible effects or permanent tagging

   asymmetry of effects
Detection possibilities                                                     UNIFR
                                                                            Rusconi
of gene doping
                                                                            2005

   Antibody detection (viral antigens)
   r-nucleic acids detection (PCR)
   recombinant protein / post-translational
    modification detection (MALDI-TOF )


   Anatomically difficult to detect
    (if locally administered)             Ergo
                                           foreign genes detectable only short-
    -> but leaves permanent genetic marking
                                            term in blood or body fluids, but
                                            - foreign genes detectable long term

                                            in tissue body fluids
    Detection of nucleic acids cannot be performed inbiopsies, and
    (except in early phase after systemic administration)
                                            - abnormal gene products detectable
    -> might require specific tissue biopsy (example GT erythropoietin in
                                            monkeys)
Comparison of advantages/disadvantages:                                 UNIFR
                                                                        Rusconi
with respect to conventional Doping
                                                                        2005

Category
    Ergo:               Drug/protein         Gene-based
   the odds would speak actually against Gene doping
Rapidity of effects rapid           slow

   but:
Reversibility           rapid                  slow
   awareness of the above needs common sense,
   a property that is rare in the doping field
Dosage                  straightforward difficult
   and:
Complexity of treatm. simple                  complex
   ...there are several borderline sports lacking doping control

Associated risks
    Question:           depends              high
   ...isnt it the high publicity over gene doping just a sort of
                         possible               difficult /impossible
   psychologically intimidating strategy by some nations (this has
Concealability
   happened before in doping)
 Doping with Gene transfer:                                                      UNIFR
                                                                                 Rusconi
 Proust's questionnaire
                                                                                 2005

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

Which sports will produce the first case ?                     horse racing?

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

Will gene doping be more effective than conventional?          No

Shall it be effective at all ?                                 probably little

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

Why is then gene doping still so attractive ?                  M Proust 1871-1922
                                                               ignorance
                                                   UNIFR
... Thank you, and let's hope that sports can      Rusconi
continue producing genuine emotions and fairplay   2005

 AISTS,
 Prof. Bengt Kayser

 my collaborators at UNIFR


 for correspondence:
 sandro.rusconi@unifr.ch

 for info :
 www.unifr.ch/nfp37
                                       UNIFR
«That's all folks»                     Rusconi
...looking forward to your questions   2005




       www.unifr.ch/nfp37
UNIFR
Rusconi
2002