Sandro Rusconi (09.03.52)

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					                                                                                   UNIFR
Sandro Rusconi (09.03.52)                                                          Rusconi
                                                                                   2005
                                                                   Sept 15, 2005
1972-75      School teacher (Locarno, Switzerland)                Uniklinik Balgrist
1975-79      Graduation in Biology UNI Zuerich, Switzerland
1979-82      PhD curriculum UNI Zuerich, molecular biology       'Therapeutische'
1982-84      Research assistant UNI Zuerich                        Gentherapie:
1984-86      Postdoc UCSF, K Yamamoto, (San Francisco)              Stand 2005
1987-93      Principal Investigator, UNI Zuerich, PD
1994-today   Professor Biochemistry UNI Fribourg               Eigentlich gemeint war:
1996-2002    Director Swiss National Research Program 37          Therapeutischer
             'Somatic Gene Therapy'                            Gentransfer: Stand 2005
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
Gene therapy: A 15-years hailstorm of                           UNIFR
                                                                Rusconi
highly emotionalised good and bad news
                                                                2005
                 BBC, NBC, CNN,...
                    New York Times
                    Washington Post
                    Times
              Selten hat eine medizinische
                    Le viel Rauch
              Technik soMonde und so
                    Frankfurter Allgemeine
                 wenig Feuer produziertC Bordignon, Milano trial May 2002
                    ...
              How many of you have heard
                  mostlyNature
                         bad news... ?
                  mostlyScience
                         good news...?
                        NEJM          Internet
                        ...
                                                                             UNIFR
1 Gen -> 1 oder mehrere Funktionen                                           Rusconi
                                                                             2005

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




                     Gene expression
                           Ergo
    GENE                                             Function'
                                   'ein Gen -> eine 2-5 FUNCTIONS
                                    ist so falsch wie
                                   'eine Krakheit -> ein Medikament'



                               Multifunctional character of genes implies:
100 ’000 genes                                  >300 ’000 functions
                                cross talk with different pathways
(50 ’000 genes?)                                (>150 ’000 functions)
                                unclarified hyerarchical position

                                unclarified side-effects potential
                                                                             UNIFR
 Recap: was ist ein Gen?:                                                    Rusconi
 ein grosses Molekuel mit informativem Inhalt                                2005

         DNA                 RNA(s)                Protein(s)


                                     Therefore, to fullfil its role,
         GENE      Transcription / translation
                                       a transferred gene segment must include:
                                                   FUNCTION
                                      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
1 Organismus -> mehr als 105                                    Rusconi
entwicklungsgenetisch kontrollierte Funktionen                  2005
      2m                    2 mm                       0.2mm




                                            0.02mm
                                 0.001mm         Zur Erinnerung
                                                 1 Cm3 Gewebe
                                                  1'000'000'000 Zellen!
DNA     RNA    Protein
                                                                                   UNIFR
Gentherapie als logische Folge: die dritte Aera                                    Rusconi
                                                                                   2003
 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
                                            Ergo
                                               gene transfer is a80 85 90 95 00
                                                                   logical
                                                development of molecular biology
                                                                      UNIFR
   Somatische Gentherapie (SGT): Definition                           Rusconi
                                                                      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
                                                                                UNIFR
Wieso 'somatisch'?                                                              Rusconi
                                                                                2005

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


Ergo
   tranformierung von                                         i.e. somatic gene therapy
    keimbahnzellen ist                                         is a treatment aiming at
    zur Zeit                                                   somatic cells and conse-
    vermieden (technische und
    ethische Probleme)                                         quently does not lead to
                                                               a hereditary transmission
                                                               of the genetic alteration


   Somatic Cells: all the other cells of the body
                                                                                     UNIFR
Wann gibt heute eine Indikation fuer SGT ?                                           Rusconi
                                                                                     2005

 No existing cure or treatment
    most monogenic diseases

          Side effects and limitations of protein injection
             interleukin 12 (cancer)
              -> toxic effects and rapid degradation
             VEGF (ischemias)                             Ergo:
              -> angiomas                                   viele Indikationen
             Factor VIII or IV (hemophilia)
              -> insufficient basal level

                       Complement to conventional
                                                              Perverse deviation dreams
                           increases specificity of conventional therapy (cancer)
                                                                 (even with current
                           increases efficacy of conventional therapy (hemophilia)
                                                                 technologyI:
                                                                gene-based sports doping
                                  Life quality burden of patient
                                   costs of enzyme therapy  performance amelioration
                                                               (ex. ADA)
                                                                (ex. Insulin)
                                   burden of daily injections  cosmetics
                                                                                             UNIFR
Pharmacologische Betrachtungen                                                               Rusconi
                                                                                             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

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

 Geringere Reversibilitaet
                                                      UNIFR
VIER grundlegende Fragen der SGT                      Rusconi
                                                      2005

       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
DREI Kategorien von anatomische Gen-Lieferung                                  Rusconi
                                                                               2005

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




        Examples:                    Examples:                         Examples:
        - bone marrow                - brain                           - intravenous
        - liver cells                - muscle                          - intra-arterial
        - skin cells                 - eye                             - intra-peritoneal
                                     - joints
                                     - tumors
ZWEI Vektor-Typen:                                                           UNIFR
                                                                             Rusconi
non-viral & viral
                                                                             2005
    Non-viral transfer
    (transfection of plasmids)
                                 a


Viral gene transfer              Ergo
(Infection by r-vectors)            viral transfer is much more efficient

                                 
                                                b
                                     nonviral transfer must solve a
                                     number of hurdles
                                                       Nuclear envelope barrier!
                                     - serum protection/stability
                                     - target docking see, Nature Biotech
                                     - endosomal escapeDecember 2001
                                     - nuclear trafficking
                                     - genomic integration
                                     - anti apoptotic functions
                                     - immunological camouflage
                                     - ...
                                                                               UNIFR
Transfection versus Infection                                                  Rusconi
                                                                               2005
                             Transfection
                             exposed to
                             106 particles/cell
                             12 hours
                                                              Infection
                                                             exposed to
                                                             1 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
Kurze Liste von Vektoren                             Rusconi
                                                     2005

       r-Adenovirus               Naked DNA


       r-Adeno-Associated V.      Liposomes & Co.


       r-Retrovirus (incl. HIV)
                                  Oligonucleotides
                                                                                                 UNIFR
Recap: Limitierungen heutiger Vektoren                                                           Rusconi

r-Adenovirus
                                                                                                 2004
- 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”)
                                                                                     UNIFR
Gentherapie in der Klinik: Trials Worldwide (cumulative)                             Rusconi
                                                                                     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
Klinische Meilensteine der Gentherapie                                                    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
Zwei persistierende Frustrationsfälle                                            Rusconi
                                                                                 2005

 Muscular dystrophy
  (incidence 1: 3000 newborn males)
    requires persistence of expression
    extremely large gene (14 kb transcript, 2 megaBP gene
    unclear whether regulation necessary
    unclear at which point disease is irreversible


 Cystic fibrosis
  (incidence 1: 2500 newborns)
    most luminal attempts failed because of anatomical /    In spite of genes discovered
     biochemical barrier: no receptors, mucus layer            in the 90ties:
    large gene that requires probably regulation             lacking suitable vector
    requires long term regulation                            no satisfactory delivery
    unclear at which point disease becomes irreversible
                                                               method
                                                              no persistence

                                                              treatment 'too late'
                                                       UNIFR
Die meist befürchtete Nebeneffekte der Gentherapie     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
SAEs1: Vom Gelsingers' Tod bis zu den Paris'                                                       UNIFR
                                                                                                   Rusconi
Leukaemias                                                                                         2005
NY May 5, 1995, R. Crystal:                                               Most Recent Paris' Trial News
adenovirus, cystic fibrosis (lung)                                             discussed under:
one patient mild pneumonia-like condition                               www.unifr.ch/nfp37/adverse03.html
Trial interrupted and many others on hold.

UPenn, Sept. 19, 1999, J. Wilson:                                           Tomorrow (16.09.05)
adenovirus , OTC deficiency (liver)                                 A Fischer will talk at the Kontderspital
one patient (Jesse Gelsinger) died of a severe septic shock.         (Workshop organised by R Seger)
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)
                                                                                      UNIFR
SAEs2: Recent Autoimmunity Reports                                                    Rusconi
                                                                                      2005
Blood, 1 May 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


K High, ASGT June meeting 2004
[Abstract1002] Immune Responses to AAV and to            Ergo
Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed
Gene Transfer for Hemophilia B                           somatic gene transfer and ectopic
                                                         transgene expression can
                                                         generate mid-term auto- immunity
SAEs3: Nicht-wissenschaftliche Faktoren die Fortschritt                              UNIFR
                                                                                     Rusconi
und Image von GT negativ beeinflusst haben
                                                                                     2005
   'Naive' statements in the early 90ties
   Excess of speculative financing in mid-late 90ties.
   Concomitance with stock-market euphoria
   Reckless statements/promises or misreporting in late 90ties
   Tendency by the media to spectacularise good and/or bad news




                                Ergo
                                 too much money, too much time pressure, too much media

                                  exposure among the image killer factors.
                                 The fundamental error: we pretended making a business issue

                                  out of a scientific issue
       Gentherapie Hoehe und Tiefe:                                                          UNIFR
                                                                                             Rusconi
       a true roller-coaster ride!
                                                              >90    A. Fischer              2005
                                                                     M. Kay
high
                            R. Crystal
            Ergo                                         V.Dzau
                                                                       C Bordignon
            whenever a reasonable cruise
            
                Adeno I
            speed was achieved, a major                J. Isner
            adverse event has brought us
        F Anderson                                                                         lentivectors
            back «square one» or even                                  AAV
                                NIH               Adeno III                                hopes
            below                                                      germline
mood




                                Motulski
                                                                       in mice?
                                  report

                                         25
                                              Lentivectors                                           ?
                                                                                                  gendi
                                                                                                  cine
                                                                            16               Auto-     ?
                                                                                             immunity
Low
                                                      J. Gelsinger
                                                                          Paris I and II                  ?
                companies                                                 Leukaemias          5
        4
                                                                                                  Paris III
       90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05
Schlussfolgerungen: GT hat Konzepte bewiesen aber                                         UNIFR
                                                                                          Rusconi
bleibt immer noch im Pionierzustand
                                                                                          2005
Fundamentally
   many new potentially therapeutic genes identified
   All types of diseases can be virtually treated by gene
    transfer
   We start to manage efficiency, specificity, persistence and
    toxicity



    Vectors and models
       Choice of among a number of viral and non viral vectors
       NonViral vectors lower toxicity/danger
        BUT -> inefficient
       Viral vectors limited packaging and high toxicity
        BUT -> efficient



        Clinically                                                Ergo
           Over 1000 trials and >4000 patients in 15 years        we are somewhat ahead but still
           Only a handful phase III                                in the pioneering phase !
           Periodical pitfalls
           Gendicine approved in China (2004)
Aussichte: GT wird fortschreiten trotz den gängigen und                                              UNIFR
                                                                                                     Rusconi
zhukuenftiken Zwischenfälle
                                                                                                     2005
Fundamental level & vectorology

   Better understanding of gene interactions and networking
   Gene inhibition through Si RNA, designed Zn finger
   specifically integrating gene constructs
   artificial chromosomes become more realistic
   novel, semi-artificial particles


    Preclinically
       scaling up to larger animal models (dog and monkey)
       new transgenic models may give improved similarities to
        human diseases



        Clinically
           Use of recombinant lentiviruses
                                                                      Ergo
           Increase of Phase III procedures over the next 5 years       Accidents typical of prototypic status
           therapeutical applications may be registered within 3-5      hurdles can be overcome
            years                                                        the genuine potential of SGT is
           challenge by other emerging therapies                         intact
                                                                               UNIFR
 Meinen 'Proust's questionnaire' bezüglich Gentherapie                         Rusconi
                                                                               2005

will GT ever make it into routine clinical practice ?           yes

when will GT widely established ?                               not tomorrow

The most worrying adverse-effect?                               immunity

Is insertional mutagenesis an important hurdle?                 No

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

Who shall 'win' the race: gene transfer or cell therapy?        both or neither

Will GT be applicable also for non-severe conditions?           yes

Which will be the best inhibitor function: antisense,           ...whatever
 intrabodies, aptamers, ribozymes, DNAzymes, SiRNA,
 designer Zn Fingers, triple helix, small drugs, ...whatever?   M Proust 1871-1922
                                                           UNIFR
 ...Danke, und ... let's remain optimistic                 Rusconi
                                                           2005
Orthopedics Update
Ch. Gerber, B. Fuchs

My UNIFR and TI collaborators
                                  Ergo
Thank you all for                    let's look forward
the patience                          to a safe landing
and attention,

sandro.rusconi@unifr.ch
or visit:
www.unifr.ch/nfp37/
                                          UNIFR
That's all, folks!                        Rusconi
                                          2005




                     www.unifr.ch/nfp37
UNIFR
Rusconi
2004

				
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