DIMENSIONS by jennyyingdi

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									                                                                                     UNIFR
Technical Remarks                                                                    Rusconi
                                                                                     2002
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                                                                 S. Rusconi
                                                                               UNIFR
Sandro Rusconi                                                                 Rusconi
                                                                               2002

1972-75      Primary school teacher (Locarno, Switzerland)      Bern, Nov 22, 2002
1975-79      Graduation in Biology UNI Zuerich, Switzerland       Swiss Olympic
1979-82      PhD curriculum UNI Zuerich, molecular biology     gene doping workshop
1982-84      Research assistant UNI Zuerich
1984-86      Postdoc UCSF, K Yamamoto, (San Francisco)           Sports doping:
1987-91      Principal Investigator, UNI Zuerich (mol. bio.)   Is there a realistic
1994-today   Professor Biochemistry UNI Fribourg                 application for
1996-today   Director Swiss National Research Program 37
                                                                 gene transfer?
             'Somatic Gene Therapy'
2001         Participant Swiss Natl. Res Program 50
             'Endocrine disruptors'
2002         Sabbatical, Tufts Med. School Boston and
             Univ. Milano, Pharmacology Department
2002         President Union of Swiss Societies for
             Experimental Biology (USGEB)
                                                                 UNIFR
Schedule                                                         Rusconi
                                                                 2002

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
                                                           UNIFR
Genetics has been used since millennia,                    Rusconi
Molecular Biology, only since 30 years                     2001



                                      100’000 b.C.
                                      Empirical genetics



                                      10’000 b.C.
                                      Biotechnology


                                      2000 a.d.
                                      Molecular biology

                                      2001 a.d, Genomics
                                                                         UNIFR
1 Gene -> 1 or more functions                                            Rusconi
                                                                         2001

     DNA                   RNA                       Protein
                   Transcription / translation




                     Gene expression
     GENE                                          2-5 FUNCTIONS




100 ’000 genes                                   >300 ’000 functions
(50 ’000 genes?)                                 (>150 ’000 functions)
                                                                      UNIFR
 What is in fact a gene?: a segment of DNA acting as a                Rusconi
 regulated machine for RNA production                                 2002

         DNA                 RNA                   Protein



         GENE      Transcription / translation    FUNCTION




                                                  RNA


                                                  DNA
spacer          regulatory               coding              spacer
                                                        UNIFR
1 Organism -> more than 105                             Rusconi
genetically-controlled Functions                        2002
      2m                     2 mm               0.2mm




                                       0.02mm
                             0.001mm

DNA     RNA    Protein
                                                     UNIFR
Reductionistic molecular biology paradigm            Rusconi
(gene defects and gene transfer)                     2002


   DNA                            Protein




  GENE                           FUNCTION(s)

  GENE OK                        FUNCTION OK

  GENE KO                        FUNCTION KO

  GENE transfer                  FUNCTION transfer
                                                                     UNIFR
Gene amplification / manipulation techniques                         Rusconi
(genetic engineering, recombinant DNA)                               2002

 segments of genomic DNA can be specifically cut and isolated



 isolated segment can be recombined with a plasmid vector
             Science-grade material
             can be essentially prepared in your cellar
 plasmid vector is transferred into bacteria where it can multiply
             ...not so clinical-grade material!
 isolated recombinant DNA can be further recombined to obtain
 the final desired molecule

 Final molecule is transferred into cells or organisms
                                                                       UNIFR
The FOUR eras of molecular medicine                                    Rusconi
                                                                       2002
 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
  Y2K+n Post-genomic improvements 99 former technologies
                                     of  1000

                                                      80 85 90 95 00
                                                                                                                          UNIFR
      The major disease of the 21st century: Ageing                                                                       Rusconi
                                                                                                                          2002



                                                                               100




                                                            cancer incidence
                                                                                                          1 0 0%




                                                                                     Alzheimer’s free %
                                                                               10




                                                                               1                                                 E2 /E
                                            many treatments that slow down ageing
Life expectancy (CH)




                       80
                                                                                                                                 M
                       70                   or age-related degenerative diseases
                                                                               4
                                                                                                                            E3 /E4
                                                                      treatments
                                            are also potential doping 4 0 6 0 8 0
                                                                  20                                                       E4 /E4
                       60
                       50                                                                                    1900 2000
                                                                                                              20 40 60    80

                       1900   1920   1940    1960   1980   199                                                     1900   2000
   Now, let's talk about Somatic Gene Therapy                         UNIFR
                                                                      Rusconi
   (somatic gene transfer)                                            2002
    Definition of GT:                    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
Somatic gene therapy’s (gene transfer)                     UNIFR
                                                           Rusconi
four fundamental questions                                 2002
                                                   Remember!
       Efficiency of gene transfer
            Specificity of gene transfer

                  Persistence of gene transfer


                       Toxicity of gene transfer
                                                                                    UNIFR
Why 'somatic'?                                                                      Rusconi
                                                                                    2001

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



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


   Somatic Cells: all the other cells of the body
                                                                  UNIFR
Pharmacological considerations                                    Rusconi
                                                                  2001

Classical Drugs     Protein Drugs       Nucleic Acids

                                           Mw N
  Mw 50- 500 Daltons 20 ’000- 100 ’000 Da x 1’000’000 Da
                      Mw
                      Biologically prepared
 Synthetically prepared                    Biologically prepared

                      Slower diffusion/action
 Rapid diffusion/action                    Slow diffusion

                      Oral delivery not possible
 Oral delivery 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

                    as
    Can be delivered  Can be delivered asMust be delivered as
                                         
    soluble molecules soluble molecules complex carrier particles
    Ångstrom/nm size nm size               50-200 nm size
                                                                  UNIFR
THREE classes of physiological gene delivery                      Rusconi
                                                                  2001



  Ex-vivo                In-vivo                 In-vivo
                         topical delivery        systemic delivery



                    V




         Examples:                   Examples:               Examples:
         - bone marrow               - brain                 - intravenous
         - liver cells               - muscle                - intra-arterial
         - skin cells                - eye                   - intra-peritoneal
                                     - joints
                                     - tumors
                                                                UNIFR
TWO classes of gene transfer vehicles: non-viral & viral        Rusconi
                                                                2001


          Non-viral transfer    a
          (transfection)


          Viral gene transfer
          (Infection)                 b

                                          Nuclear envelope barrier!
                                          see, Nature Biotech
                                          December 2001
Transfection with recombinant DNA                            UNIFR
                                                             Rusconi
Vs Infection with recombinant viruses                        2001




                      Transfection
                      exposed to
                      106 particles/cell
                      12 hours
                                            Infection
                                           exposed to
                                           3 particle/cell
                                           30 min
                                                      UNIFR
Quick parade of popular vectors/methods               Rusconi
                                                      2002



       Adenovirus                  Naked DNA


       Adeno-associated V.         Liposomes & Co.


       Retrovirus (incl. HIV)
                                   Oligonucleotides
                                                                      UNIFR
Recombinant Adenoviruses                                              Rusconi
                                                                      2002

    Approaches              Advantages / Limitations

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

                               Can contain RCAs
    Hybrid adenos:             Are toxic /immunogenic
       Adeno-RV
       Adeno-AAV           Examples
       Adeno-Transposase    OTC deficiency (clin, ---)

                             Cystic Fibrosis (clin, --- )

                             Oncolytic viruses (clin, +++)
                                                                       UNIFR
Recombinant AAV (adeno-associated-virus)                               Rusconi
                                                                       2002

  Approaches                      Advantages / Limitations

  Helper-dependent production     Persistence in the genome permits long-
                                  term expression, high titers are easily
                                  obtained, immunogenicity is very low,
  Helper independent production   However the major problem is:

                                     Small capacity (<4.5 kb) which does
                                      not allow to accommodate large genes
  Cis-complementing vectors
                                      or gene clusters.


  Co-infection                    Examples
                                   Hemophilia A (clin, animal, +++)

                                   Gaucher (clin, animal, +++)

                                   Brain Ischemia (animal, +++)
                                   Cystic fibrosis (animal, +/-)
                                                                   UUNIFR
Recombinant Retroviruses (includes HIV-based)                      Rusconi
                                                                   2002

  Approaches               Advantages / Limitations

  Murine Retroviruses      9 Kb capacity + integration through
                           transposition also in quiescent cells
                           (HIV), permit in principle long-term
  VSV-pseudotyped RV       treatments, however disturbed by:
                            Insertional mutagenesis

                              Gene silencing
  Lentiviruses !              High mutation rate
                              Low titer of production
  Self-inactivating RV
                           Examples
                            SCID (IL2R defect, Paris) (clin, +++)

  Combination viruses       Adenosine Deaminase deficiency (clin, +++!!!)

                            Parkinson (preclin, +++)

                            Anti cancer (clin +/-)
                                                                         UNIFR
Naked / complexed DNA                                                    Rusconi
                                                                         2002

 Approaches                       Advantages / Limitations

 Naked DNA injection /biolistic   Unlimited size capacity + lower
                                  immunogenicity and lower bio-risk
                                  of non viral formulations is
 Naked DNA + pressure             disturbed by

                                     Low efficiency of gene transfer
 Naked DNA + electroporation         Even lower stable integration

                                  Examples
 Liposomal formulations            Critical limb Ischemia (clin, +++)
                                   Cardiac Ischemia (clin, +/-)

                                   Vaccination (clin, +/-)
 Combinations                      Anti restenosis (preclin. +/-)
                                                             UNIFR
Oligo-nucleotides                                            Rusconi
                                                             2002

 Approaches               Advantages / Limitations

 Antisense                these procedures may be suitable for :

                             handling dominant defects
 Ribozymes/DNAzymes          transient treatments (gene modulation)
                             permanent treatments (gene correction)
 Triple helix

                          Examples
 Decoy / competitors       Anti cancer (clin,preclin., +/-)
                           Restenosis (clin, +++)

                           Muscular Distrophy (animal, +++)
 Gene-correcting oligos   √!
Recap: current limitations of popular                                            UNIFR
                                                                                 Rusconi
gene transfer vectors                                                            2002

 Adenovirus
                                   Biolistic bombardment
 - no persistence
                                   or local direct injection
 - limited packaging
                                   - limited area
 - toxicity
 - immunogenicity
                                      Electroporation
     Retrovirus (incl. HIV)           - limited organ access
     - limited package
     - random insertion                   Liposomes, gene correction & Co.
     - unstable genome                    - very inefficient transfer
          General
          - antibody response                  General
          - limited packaging                  - low transfer efficiency
          - gene silencing                       1/10’000 of viruses’ in vivo

             Solutions:                            Solutions:
             - synthetic viruses                   - improved liposomes
               (“Virosomes”)                         with viral properties (“Virosomes”)
                                                          UNIFR
The most feared potential side-effects of gene transfer   Rusconi
                                                          2002

   Immune response to vector
   immune response to new or foreign gene product
   General toxicity of viral vectors
   Adventitious contaminants in recombinant viruses
   Random integration in genome
    -> insertional mutagenesis (-> cancer risk)
   Contamination of germ line cells
                                                                    UNIFR
Gene Therapy in the clinic: Trials Wordldwide                       Rusconi
                                                                    2002
   trials                                                patients

                                         As of Sept. 2002:
   100                                                         1500
                                         599 registered protocols
                                         4000 treated patients
                        cancer
     80
     60                                  86% phase I         1000
                                         13% phase II
                                         1 % phase III
     40                       hered.
                                                               500
     20                                vasc.overall still pending
                                        21%
                                                    Infect.
                                        or not yet Initiated !
                                         www.wiley.com

      1990 1992     1994    1996    1998        2000
                                                                                     UNIFR
Gene Therapy Milestones                                                              Rusconi
                                                                                     2002
1990, 1993, 2000 // ADA deficiency                             Anderson, 1990
                                                                      Isner, 1998
F Anderson, M Blaese // C Bordignon                                  Dzau, 1999
                                                                           Kmiec, 1999
1997, 2000, Critical limb ischemia                                          Fischer, 2000
                                                                                Dickson, 2000
J Isner († 4.11.2001), I Baumgartner, Circulation 1998                      Aebischer, 2000
                                                                              Kirn, 2001
1998, Restenosis
V Dzau, HGT 1998
1999, Crigler Njiar (animal)
C Steer, PNAS 1999
                                                               Clinical trials with ONYX-015,
2000, Hemophilia
                                                                     what we learned?
M Kay, K High
2000, SCID                                                                 (Review)
A Fischer, Science April 2000
                                          Bordignon, 2000 (ESGT, Stockholm)
2000, correction Apo E4 (animal model) proves efficacy of the same protocol
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)
Gene Therapy Adverse events:                                      UNIFR
                                                                  Rusconi
NY 1995 // UPenn 1999 // Paris 2002                               2002

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 was interrupted and many others were put on hold.
UPenn, Sept. 19, 1999, J. Wilson: in a trial with adenovirus
mediated gene transfer to treat OTC deficiency (liver) one patient
(Jesse Gelsinger) died of a severe septic shock. Many trials were
put on hold for several months (years).

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.
       Ups and Downs of Gene Therapy:                                 UNIFR
                                                                      Rusconi
       a true roller coaster ride!                                    2002
high                                                    A. Fischer lentivectors
                                                        M. Kay       in clinics?
                          R. Crystal
         Adeno I                                   V.Dzau          C Bordignon
                                            J. Isner
    ADA                                                         AAV
                            NIH        Adeno III
                                                                germline
mood




                            Motulski
                            report Lentivectors                 in mice?
        Ergo:                       in pre-clinic                        Paris
        in spite of its respectable age,             J. Wilson
Low     gene transfer is still in its infancy        J. Gelsinger
        and still produces more controversies
        than clinical results                   NFP37

       90   91   92   93   94   95   96   97   98   99   00    01     02
                                                              UNIFR
The THREE levels of doping                                    Rusconi
                                                              2002



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




                                    +
                                  After the
                                 competition
                             (repair enhancers)
Which gene transfer approaches would be                    UNIFR
                                                           Rusconi
compatible with doping strategies                          2002

   ex vivo, hematopoietic tissue:
    erythropoietin?

   in vivo local (example muscle):
    metabolic enhancers, growth factors,
    muscular fiber changers

   in vivo local (example joints):
    pain reducers, inflammation inhibitors, recovery and
    repair factors

   in vivo systemic:
    anabolic factors, endocrine factors, pain killers
Which are the objective current limitations in         UNIFR
                                                       Rusconi
gene-based doping strategies                           2002
Viral gene transfer
 immune problems

 limited readministration

 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
Which side effects could be feared in                            UNIFR
                                                                 Rusconi
gene-based doping strategies                                     2002

Short -mid term
   Autoimmunity
   Hyperimmunity
   Toxic shock

Long term                          Intrinsic to reckless application
 Fibrosis                         (probably the biggest danger)
 Cancer                            malpractice (unsuitable
 Conventional effects of            vector/administration route)
  administered factors              non-clinical grade material
 Inaccessibility to future gene
                                     (adventitious pathogens
  therapy interventions (immunity)
                                     or allergens)
                                    lack of follow-up
Which detection methods would be (or not) evisageable                        UNIFR
                                                                             Rusconi
for gene-based doping strategies                                             2002

   Antibody detection (viral antigens or other epitopes)
   recombinant-nucleic acids detection (PCR)
   recombinant protein detection
     (MALDI-TOF / proteomics)


           Gene transfer may be anatomically difficult to
            detect (if locally administered) but leaves
            permanent genetic marking

                        the detection of nucleic acids cannot be
                         performed in body fluids (except for systemically
                         administered treatments) and might require
                         specific tissue biopsy
Final side-by-side comparison:                                   UNIFR
                                                                 Rusconi
gene-based doping versus drug- or protein-based doping           2002

Category              Drug/protein    Gene-based

Rapidity of effects   rapid           slow

Reversibility         rapid           slow/none

Dosage        Ergo: straightforward difficult
              The odds speak currently rather against the adoption of
Complexity of treatm. simple
              gene-based doping,       complex
              but this applies to common-sense clinical practice,
                                       high
Associated risks thisdepends not guaranteed in the doping field
              and       aspect is

Detectability         arduous         'straightforward'
                                                                UNIFR
...Thanks !                                                     Rusconi
                                                                2002

 Swissolympics
                                      Our own project/goal may indeed
                                      appear very small and harmless...
 My collaborators at UNIFR
                                       This does not necessarily apply
                                            to its consequences...
 Swiss National Research Foundation



 Thank you all for the attention,
 and... if you are too shy to ask
 send an e-mail to:
 sandro.rusconi@unifr.ch
 or visit:
 www.unifr.ch/nfp37
                    UNIFR
discussion slides   Rusconi
                    2002
                                                                                                            UNIFR
Examples of inheritable gene defects                                                                        Rusconi
                                                                                                            2002
Polygenic defects               Type     estimated                   genetics            behaviour   environment
(‘ frequent ’)                          min - max

Diabetes                         poly      1      - 4%
Hyperurikemia                    Multi     2
                         Monogenic defects- 15 %                     estimated
Glaucoma                         poly      1      - 2%
                         (‘ rare ’)                                   min - max
Displasia                        Multi     1      - 3%
                         Cystic fibrosis, muscular dystrophy
Hypercolesterolemia              Multi     1      - 5%
                         immodeficiencies, metabolic diseases, all together
Syn-& Polydactyly                poly      0.1    - 1%
                         Hemophilia...                               0.4  - 0.7%
Congenital cardiac defects       Multi     0.5    - 0.8 %
Manic-depressive psychosis       Multi     0.4    - 3%
Miopy                            poly     Predispositions
                                           3      - 4%                    Type      estimated
Polycystic kidney                poly      0.1    - 1%                             min - max
Psoriasis                        Multi (*) Alzheimer%
                                           2      - 3                     Multi    7       - 27 %
Schizofrenia                     Multi (*) Parkinson%
                                           0.5    - 1                     Multi    1       - 3%
Scoliosis                        Multi (*) Breast cancer
                                           3      - 5%                    Multi    4       - 8%
                                          (*) Colon Carcinoma             Multi    0.1     - 1%
                                          (*) Obesity                     Multi    0.5     - 2%
                                          (*) Alcolholism/ drug addiction Multi    0.5     -  3%

                                                     Sum of incidences                          min - max
                                                     (all defects)                              32  - 83%
The long way to drug/procedure registration is the                             UNIFR
                                                                               Rusconi
principal cause of financial burden, but we cannot avoid it                    2002


        year   event                         costs U$D

        0      Idea                          0

        2      Cell culture assays           0.5 Mio     This means:
                                                         assuming 20% of new developments
        5      Pre-clinical tests                        makes it to final registration,
               animal models                 2 Mio
                                                         the average investment is
        7      Clinical phase I                          300-500 Mio U$D
               5-20 patients                             for each approved drug/procedure
               verify side effects           6 Mio

        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
                                                                           UNIFR
Not only the genome determines the health status...                        Rusconi
                                                                           2002

                                      genetics   behaviour   environment



             Muscle distrophy
             Familial Breast Cancer
             Sporadic Breast Cancer
             Lung Cancer


             Obesity
             Artherosclerosis


             Alzheimer
             Parkinson ’s


             Drug Abuse
             Homosexuality
Recap: what is a virus ? ->                                        UUNIFR
                                                                   Rusconi
A superbly efficient replicating machine                           2002

        100 nm


                     docking   entry        disassembly    genome replication
                                         early genes exp



            capsid
  replication
     E L1 L2                            assembly                  late genes
     E L1 L2                                                          exp



standard viral genome                      Spread

                               Etc...
Engineering of replication-defective, recombinant viruses      UNIFR
                                                               Rusconi
(Principle)                                                    2002


rp   E   L1 L2   rp



 Wild type genome         Normal target cells        Virions



     X                      E       E           E

                                E           E
                            E
                                        E




 Recombinant genome       Packaging cells            R-Virions



                          Normal target cells
                                                                             UNIFR
'Classical' GT models and strategies                                         Rusconi
                                                                             2002

   Disease                 transferred function        Clinical Results

   ADA deficiency          ADA normal gene             1990 F. Anderson,
    (Immunodeficiency)       (enzyme)                     2002 C. Bordignon

   Cystic Fibrosis         CFTR gene                   no significant results
    (Lung, Pancreas)         (chlorine transporter)       in spite of several trials

   Haemophilia B           Factor IX gene              1999-2000 M. Kay and K.
    (Blood)                  (blood clotting factor       Horwitz
   SCID                    IL2R gene                   2000 A. Fischer
    (Immunodeficiency)       (gamma-C receptor)
   Limb ischaemia          VEGF gene                   1998 J. Isner
    (Hands, Feet)            (vascular promoter)
   Cardiac ischaemia       VEGF gene                   2000 J. Isner
    (Heart)                  (vascular promoter)

								
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