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Sandro Rusconi (09.03.52)

UNIFR Rusconi 2003. 20 Oktober 2003 Liestal Biovalley. Sandro Rusconi (09.03.52). 2003: wohin führt uns Gentherapie?. 1972-75 School teacher (Locarno, Switzerland) 1975-79 Graduation in Biology UNI Zuerich, Switzerland 1979-82 PhD curriculum UNI Zuerich, molecular biology

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Sandro Rusconi (09.03.52)

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  1. UNIFR Rusconi 2003 20 Oktober 2003 Liestal Biovalley Sandro Rusconi (09.03.52) 2003: wohin führt uns Gentherapie? 1972-75 School teacher (Locarno, Switzerland) 1975-79 Graduation in Biology UNI Zuerich, Switzerland 1979-82 PhD curriculum UNI Zuerich, molecular biology 1982-84 Research assistant UNI Zuerich 1984-86 Postdoc UCSF, K Yamamoto, (San Francisco) 1987-93 Principal Investigator, UNI Zuerich, PD 1994-todayProfessor Biochemistry UNI Fribourg 1995-today Director Swiss National Research Program 37 'Somatic Gene Therapy' 2002-03Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department 2002-05 President Union of Swiss Societies for Experimental Biology (USGEB) *essentielle wiederholung in Genetik* *grundkonzepte der Gentherapie* *Klinische experimentiering in SGT* *Hohe und Tiefe in der SGT**Schlussfolgerungen und Perspektiven* Courmayeur, March 2003 a a a a a a

  2. UNIFR Rusconi2003 *essentielle Wiederholungen in Genetik* a a a a a a

  3. DNA RNA Protein Transcription / translation GENE 2-5 FUNCTIONS Gene expression 100 ’000 genes (50 ’000 genes?) >300 ’000 functions (>150 ’000 functions) UNIFR Rusconi 2003 1 Gen -> 1 oder mehrere Funktionen1 Genom -> über Nx100'000 Funktionen a a a a a a

  4. 2 mm 0.2mm 2m 0.02mm 0.001mm DNA RNA Protein UNIFR Rusconi 2003 1 Organismus -> 1013 Zellen, verteilt und spezialisiert in Organe und Gewebe • 1 Cm3 Gewebe • 1'000'000'000 Zellen a a a a a a

  5. DNA RNA Protein GENE Transcription / translation FUNCTION RNA DNA UNIFR Rusconi 2003 Aber was istr eigentlich 'ein Gen'?:eine regulierbare Nano-machine zur Herstellung von RNA • Um wirksam zu sein sollte ein transferierter Gen beinhalten: • Sequenzen fuer Genregulation • Signale fuer reifung/transport der RNA • Signale fuer Uebersetzung in proteinen spacer regulatory coding spacer a a a a a a

  6. DNA Protein GENE FUNCTION(s) GENE OK FUNCTION OK GENE KO FUNCTION KO GENE transfer FUNCTION transfer UNIFR Rusconi 2003 Das reduktionistische Paradigma des Molekularbiologes • Gentransfer kann beinhalte: • transfer einer neuen Funktion, oder • transfer einer kompensierenden F., oder • transfer einer interferierenden Funktion a a a a a a a a a a a a

  7. genetics behaviour environment UNIFR Rusconi 2003 Beispiele von Vererbbare Defekte Polygenic defects Type estimated (‘ frequent ’) min - max Diabetes poly 1 - 4 % Hyperurikemia Multi 2 - 15 % Glaucoma poly 1 - 2 % Displasia Multi 1 - 3 % Hypercolesterolemia Multi 1 - 5 % Syn-& Polydactyly poly 0.1 - 1 % Congenital cardiac defects Multi 0.5 - 0.8 % Manic-depressive psychosis Multi 0.4 - 3 % Miopy poly 3 - 4 % Polycystic kidney poly 0.1 - 1 % Psoriasis Multi 2 - 3 % Schizofrenia Multi 0.5 - 1 % Scoliosis Multi 3 - 5 % Monogenic defects estimated (‘ rare ’)min - max Cystic fibrosis, muscular dystrophy immodeficiencies, metabolic diseases, all together Hemophilia... 0.4 - 0.7% Predispositions Type estimated min - max (*) Alzheimer Multi 7 - 27 % (*) Parkinson Multi 1 - 3 % (*) Breast cancer Multi 4 - 8 % (*) Colon Carcinoma Multi 0.1 - 1 % (*) Obesity Multi 0.5 - 2 % (*) Alcolholism/ drug addiction Multi 0.5 - 3% • Ergo: • Jedermann ist Träger von mindestens einen Defekt • Viele Defekte manifestieren sich erst spät im Leben (Anfälligkeiten) • Einige Anfälligkeiten sind positiv (langlebigkeit, Infektionsresistenz etc...) Sum of incidences min - max (all defects) 32 - 83% a a a a a a

  8. genetics behaviour environment Muscle distrophy Familial Breast Cancer Sporadic Breast Cancer Lung Cancer Obesity Artherosclerosis Alzheimer Parkinson ’s Drug Abuse Homosexuality UNIFR Rusconi 2003 Das genom ist nicht das einzige determinant des gesund-krang gleichgewichtes • Sogar die heilung von 'erworbene' krankheiten kann genetisch bedingt sein: • Trauma, Wunde • Brüche • Verbrennungen, Infektionen • Vergiftungen a a a a a a

  9. UNIFR Rusconi2003 *grundkonzepte in der Somatischen Gentherapie (SGT)* a a a a a a

  10. Eighties Genes as probes Nineties Genes as factories Y2K Genes as drugs 50 1 2 3 4 5 Y2K+n Post-genomic improvements of former technologies 3000 10 80 85 90 95 99 1000 ok ok ** ** ** 80 85 90 95 00 UNIFR Rusconi 2003 Die 4 Aeren der molekularen Medizin genomeABC.mov a a a a a a

  11. UNIFR Rusconi2003 Somatische Gentherapie (SGT) Definierung und Anwendungsbereich Chronic treatment Definition of SGT: 'Use genes as drugs': Correcting disorders by somatic gene transfer Acute treatment Preventive treatment NFP37 somatic gene therapy www.unifr.ch/nfp37 Hereditary disorders Acquired disorders Loss-of-function Gain-of-function a a a a a a

  12. UNIFR Rusconi2003 Das Prinzip darf einfach sein, aber der Teufel liegt häufig in den Details... There are many things that are simple in principle, like... getting a train ticket... ! try this 5 min before departureand with a group of Chinese tourists in front parking your car... ! try this at noon, any given day in Zuerich or Paris ... counting votes... ! ask Florida's officials ... gene therapy... look at progress in 13 years... a a a a a a

  13. UNIFR Rusconi 2003 Pharmakologische Betrachtungen Classical Drugs Protein Drugs Nucleic Acids • Mw 20 ’000- 100 ’000 Da • Biologically prepared • Slower diffusion/action • Oral delivery not possible • Cellular delivery: - act extracellularly • Can be delivered as soluble moleculesnm size • rapidly reversible treatment • Mw 50- 500 Daltons • Synthetically prepared • Rapid diffusion/action • Oral delivery possible • Cellular delivery: - act at cell surface- permeate cell membrane- imported through channels • Can be delivered as soluble moleculesÅngstrom/nm size • rapidly reversible treatment • Mw N x 1’000’000 Da • Biologically prepared • Slow diffusion • Oral delivery inconceivable • Cellular delivery:- no membrane translocation - no nuclear translocation- no biological import • Must be delivered as complex carrier particles50-200 nm size • slowly or not reversible O H O H O O O H O H O O O H O H • Thérapies avec acides nucléiques • nécessitent de formulation en micro-particules • bien plus complexes que la pharmacologie conventionnelle • différent niveau de reversibilité (problème de dosage et de maitrise des effets indésirables O a a a a a a

  14. UNIFR Rusconi2003 Wieso 'somatisch'? • Germ Line Cells: the cells (spermatocytes and oocytes and their precursors) that upon fertilisation can give rise to a descendant organism • Ergo • transformation of germ line cells is avoided, to exclude risk of erratic mutations due to insertional mutagenesis i.e. somatic gene therapy 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 a a a a a a

  15. Remember! Efficiency Specificity Persistence Toxicity UNIFR Rusconi2003 Die vier Grundfragen bei der SGT Efficiency of gene transfer Specificity of gene transfer Persistence of gene transfer Toxicity of gene transfer • Le variables • welche Krankheit? • Welches Gen? • Welches Vektor? • Welches Organ / Gewebe? • Welche Transfermethode? a a a a a a a a a a a a

  16. V UNIFR Rusconi 2003 Die drei Transfer-wege bei der SGT: Ex-vivo In-vivo topical delivery In-vivo systemic delivery Examples: - bone marrow - liver cells - skin cells Examples: - brain - muscle - eye - joints - tumors Examples: - intravenous - intra-arterial - intra-peritoneal a a a a a a

  17. A B UNIFR Rusconi 2003 Die zwei klassen von 'Vektoren':virale / nicht-virale Transfert non viral (transfection) Nuclear envelope barrier! viral transfer (Infection) direct nuclear shuttling! a a a a a a

  18. UNIFR Rusconi 2003 Effizienz der Transfektion mit rekombinante DNA im vergleich zur Infektion mit rekombinante Viren Transfection cells exposed to 1'000'000 particles/cell 12 hours Infection cells exposed to 3 particle/cell 30 min • Ergo • das gentransfer mittels rekombinante Viren ist ueber 1'000'000-fach effizienter als jene nicht-viral transfer methode a a a a a a

  19. UNIFR Rusconi 2003 kleine Parade von Genransfervektoren Naked DNA Liposomes & Co. Oligonucleotides Adenovirus Adeno-associated V. Retrovirus (incl. HIV) but remember... "Nobody's perfect "! a a a a a a

  20. Efficiency +++ Specificity Persistence Toxicity ++ UNIFR Rusconi 2003 rekombinante Adenoviren • Approaches • Generation I • Generation III • Hybrid adenos: • Adeno-RV • Adeno-AAV • Adeno-Transposase • Advantages / Limitations • 8 Kb capacity Generation I >30 Kb capacity Generation IIIAdeno can be grown at very high titers,However • Do not integrate • Can contain RCAs • Are toxic /immunogenic • Examples • OTC deficiency (clin, ---) • Cystic Fibrosis (clin, --- ) • Oncolytic viruses (clin, +++) a a a a a a

  21. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2003 r4ekombinante Adeno-associated-virus (AAV) Approaches Helper-dependent production Helper independent production Cis-complementing vectors Co-infection • Advantages / Limitations • Persistence in the genome permits long- • term expression, high titers are easily • obtained, immunogenicity is very low, • However the major problems are: • insertional mutagenesis • Small capacity (<4.5 kb) which does not allow to accommodate large genes or gene clusters. • Examples • Hemophilia A (clin, animal, +++) • Gaucher (clin, animal, +++) • Brain Ischemia (animal, +++) • Cystic fibrosis (animal, +/-) a a a a a a

  22. Efficiency Specificity Persistence Toxicity UUNIFR Rusconi 2003 Rekombinante Retroviren (inkl. HIV) Approaches Murine Retroviruses VSV-pseudotyped RV Lentiviruses ! Self-inactivating RV Combination viruses • Advantages / Limitations • 9 Kb capacity + integration through • transposition also in quiescent cells • (HIV), permit in principle long-term • treatments, however disturbed by: • Insertional mutagenesis • Gene silencing • High mutation rate • Low titer of production • Examples • SCID (IL2R defect, Paris) (clin, +++) • Adenosine Deaminase deficiency (clin, +++!!!) • Parkinson (preclin, +++) • Anti cancer (clin +/-) a a a a a a

  23. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2003 Reine oder komplexierte DNS Approaches Naked DNA injection /biolistic Naked DNA + pressure Naked DNA + electroporation Liposomal formulations Combinations • Advantages / Limitations • Unlimited size capacity + lower • immunogenicity and lower bio-risk • of non viral formulations is • disturbed by • Low efficiency of gene transfer • Even lower stable integration • Examples • Critical limb Ischemia (clin, +++) • Cardiac Ischemia (clin, +/-) • Vaccination (clin, +/-) • Anti restenosis (preclin. +/-) a a a a a a

  24. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2003 Oligonuklotide Approaches Antisense Ribozymes/DNAzymes Triple helix Decoy / competitors Gene-correcting oligos • Advantages / Limitations • these procedures may be suitable for : • handling dominant defects • transient treatments (gene modulation) • permanent treatments (gene correction) • Examples • Anti cancer (clin,preclin., +/-) • Restenosis (clin, +++) • Muscular Distrophy (animal, +++) √ ! a a a a a a

  25. UNIFR Rusconi 2003 Recap: Limitierungen der heutigen Genvektoren Adenovirus - no persistence - limited packaging - toxicity, immunogenicity Biolistic bombardment or local direct injection - limited area Electroporation - limited organ access Retrovirus (incl. HIV) & AAV - limited packaging - random insertion - unstable genome Liposomes, gene correction & Co. - very inefficient transfer General - antibody response - limited packaging - gene silencing- random insertion General - low transfer efficiency - no or little genomic integration Solutions: - improved liposomes with viral properties (“Virosomes”) Solutions: - synthetic viruses (“Virosomes”) a a a a a a

  26. UNIFR Rusconi2003 *klinische Versuche in der SGT* a a a a a a

  27. UNIFR Rusconi 2003 Der klassische klinische Weg: viel Zeit und Geld yearevent costs U$D 0Idea 0 2Cell culture assays 0.5 Mio 5Pre-clinical tests animal models 2 Mio 7Clinical phase I 5-20 patients verify side effects 6 Mio 10Clinical phase II 30-100 patients dosis escalation 12 Mio 15Clinical Phase III >300- 1000 patients multicentric double blind 80 Mio 16>>Registration /Availability This means: assuming 20% of new developments makes it to final registration, the average investment is 300-500 Mio U$D for each approved drug/procedure Molecular therapy is NO EXCEPTION ! a a a a a a

  28. UNIFR Rusconi2003 Vernichten wir mindestens vier Mythen bei der Gentherapie • Classical Gene Therapy Image • Hereditary disease • culprit gene must be known • requires 100% efficiency of transfer/expr. • gene transfer/expression must persist • Reality • Many acquired diseases can be treated(ex. infections, traumatic lesions, tumors,...) • 'Short circuit' or symptomatic treatments (ex. neurodeg. conditions with trophic factors) • Few % sufficient for many diseases(ex. hemophilia, limb ischaemia ...) • No persistence required in many cases (ex. vaccination, cytotoxic antitumoral factors, restenosis prevention, acute rejection prevention...) a a a a a a

  29. trials patients 100 1500 cancer 80 II 1000 60 I-II I hered. 40 500 vasc. 20 Infect. 1990 1992 1994 1996 1998 2000 UNIFR Rusconi2003 Trends bei der klinischen SGT experimentierung • Ergo • en dépit de son age la TGS peut compter couramment seulement 1% d'essais en phase III As of August 2003:660 registered protocols 3672 treated patients 66% phase I 21% phase I-II 11% phase II 0.8% phase II-III 0.7% phase III 21% overall still pending or not yet Initiated ! www.wiley.com/genetherapy a a a a a a

  30. Isner, 1998 Anderson, 1990 Dzau, 1999 Dickson, 2000 Kmiec, 1999 Aebischer, 2000 Fischer, 2000 2002 Kirn, 2000, 2001 2002 Intravascular adenoviral agents in cancer patients: Lessons from clinical trials (review) Bordignon, 2000 (ESGT, Stockholm)2002, science 296, 2410 ff) UNIFR Rusconi2003 Einige Meilensteine 1990, 1993, 2000 // ADA deficiency F Anderson, M Blaese // C Bordignon 1997, 2000, Critical limb ischemia J Isner († 4.11.2001), I Baumgartner, Circulation 1998 1998, Restenosis V Dzau, HGT 1998 1999, Crigler Njiar (animal) C Steer, PNAS 1999 2000, Hemophilia M Kay, K High 2000, SCID A Fischer, Science April 2000 2000, correction Apo E4 (animal model) G. Dickson, 2000 esgt, 2002 BBA 2000, correction Parkinson (animal model) P Aebischer, Science, Nov 2000 2001, ONYX oncolytic Viruses D Kirn (Cancer Gene Ther 9, p 979-86) a a a a a a

  31. UNIFR Rusconi2003 *Die hoch- und tief-punkte ... * a a a a a a

  32. UNIFR Rusconi2003 Zwei besonders frustrierende Faelle:Muskelschwund und Mucoviszidose • 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) • luminal attempts failed because of anatomical / biochemical barrier: no receptors, mucus layer • large gene that requires probably regulation • requires long term regulation • unclear at which point disease becomes irreversible • Trotz Isolierung der entspre-chenden Genen in 1984 • kein geeignetes Vektor • keine geeignete Lieferungsmethode a a a a a a

  33. UNIFR Rusconi2003 Die mesit befuerchtete Nebeneffekte der Gentherapie • 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 • Random integration in genome-> insertional mutagenesis (-> cancer risk) • Ergo • die Nebeneffekte waren nicht so scvheinbar wenn SGT ineffizient war • Heute muessen wir diese Nebeneffekte seriös betrachten a a a a a a

  34. UNIFR Rusconi2003 4 bittere Feststellungen aber nur einen Patient bis jetzt direkt an SGT gestorben NY May 5, 1995, R. Crystal: in a trial with adenovirus mediated gene transfer to treat cystic fibrosis (lung) one patient developed a mild pneumonia-like condition and recovered in two weeks. The trial interrupted and many others on hold. UPenn, Sept. 19, 1999, J. Wilson: in a trial with adenovirus mediated gene transfer to treat OTC deficiency (liver) one patient (Jesse Gelsinger) died of a severe septic shock. Many trials were put on hold for several months (years). Paris, Oct 2, 2002, A Fischer: in a trial with retrovirus mediated gene transfer to treat SCID (bone marrow) one patient developed a leukemia-like condition.The trial has been suspended to clarify the issue of insertional mutagenesis, and some trials in US and Germany have been put on hold. Paris, Jan 14, 2003, A Fischer: a second patient of the cohort of 9 comes up with a similar disease than the one reported in october 2002. 30 trials in USA are temporarily suspended a a a a a a

  35. UNIFR Rusconi2003 Der klinische Versuch in Paris X-SCID (A. Fischer, Hôpital Necker) Disease • deficiency of the receptor gamma(c) • incapacity of maturing lymphocytes • severe combined immunodeficiency • lethal at 4 months if untreated • survival 10 years under sterile conditions Conventional treatments • maintenance under sterile condition • treatment with antibiotics • transplant of matching bone marrow • Gene Therapeutical approach • explant BM (3-6 month old) • select CD34+ • transduce with retroviral vector encoding gamma(c) • re-infusion, follow-up a a a a a a

  36. UNIFR Rusconi2003 Die Odyssee des klinischen Versuchs in Paris Chronology • 1998 start treatment of patients • 2000 publication results first 2 patients • 2001/2002 publication further 8 patients • 9 out of 10 responded well, back home, normal life Adverse 1 • summer 2002, high WBC in a 36 months patient • september 2002, hyper-proliferatory cells with insertion in proximity of LMO2 gene, notification authorities • October 2003, public disclosure, chemotherapy, good response, report at ESGT congress. Adverse 2 • december 2002, T cell hyper-proliferation in a second, 36 months patient • hyper-proliferatory cells also contain insertion of transgene close to LMO2 gene • January 2003, notification to authorities, public disclosure, treatment chemotherapy a a a a a a

  37. UNIFR Rusconi2003 Die Fragen Facts • in both patients insertion of the transgene in proximity of LMO2 • this type of insertion not found in CD34+ cells in these patients • LMO2 expression is apparently increased in these patients • LMO2 gene already known as proto-oncogene involved in some chromosomal-translocations found in some leukaemias • gamma(c) receptor can respond to IL-2, IL-5, IL-7, IL-9, IL-15, Il-21 and ... • gamma(c) receptor is therefore itself a pro-proliferatory and anti-apoptotic signaling molecule • Questions/hypotheses • is this adverse event specific for the disease status? • is the transgene contributing to the hyper-proliferatory potential? • is the gamma(c) synergising with LMO2? • Has there been such an adverse event in the over 20 retrovirally transduced patients treated so far for other diseases? Perspectives if the answers are 'YES' 'NO' 'UNK' goodbadnot good goodbadnot good goodbadnot good badgoodnot good a a a a a a

  38. high mood Low NFP37 UNIFR Rusconi2003 A. Fischer M. Kay Anhäufung von hoch- und tief-Punkte: ein Rollercoaster! lentivectors in clinics? R. Crystal V.Dzau Adeno I C Bordignon J. Isner ADA AAV germline in mice? NIH Motulski report Adeno III • Ergo • whenever a reasonable cruise speed was achieved, a major adverse event has brought us back square one Lentivectors in pre-clinic Adverse events in Paris J. Wilson J. Gelsinger 90 91 92 93 94 95 96 97 98 99 00 01 02 03 a a a a a a

  39. UNIFR Rusconi2003 Weitere Faktoren die zum schlechten 'Image' der Gentherapie beigetragen haben • Naive statements by some good-willing scientists in the early 90ties • Not-so-naive statements by not-so-naive scientists in search of fame • Huge amount of money that flowed into the research and development that attracted many incompetent researchers. • Concomitance with stock-market euphoria (little attention to realism) • Reckless statements or misreporting by greedy scientists or company managers to increase the value of their stock options (memorandum by the ASGT on conflict of interest 2000, www.asgt..org) • Tendency by the media to spectacularise good news and/or bad news • Ergo • zuviel geld und spekulation: ein explosiver cocktail, wie beim Sport oder Kunst... a a a a a a

  40. UNIFR Rusconi2003 *Conclusions & Perspectives* a a a a a a

  41. UNIFR Rusconi2003 Schlussfolgerungen • Grundkonzepte • The therapeutic gene transfer in somatic cells must cope with: efficiency, specificity, persistence and toxicity • many genes with potential therapeutic value have been identified, and essentially all types of diseases can be treated by gene transfer • Vektoren und Modelle • There is the choice of a certain number of viral and non viral vectors, none of them being generally applicable • Viral vectors have the advantage of efficiency and nonviral vector the advantage of lower toxicity/danger. • Viral vectors have the disadvantage of limited packaging and some toxicity, while nonviral vector have the major disadvantage of low efficiency of transfer • Klinische Versuche • over 600 trials and 3500 patients in 12 years • only a handful of trials is now reaching phase III • Progress further slowed down by periodical pitfalls a a a a a a

  42. UNIFR Rusconi2003 Perspectiven: SGT wird weiter fortschreitentrotz schwieruigkeiten und unvermeidbare Unfälle • Grundlage Forschung und 'Vektorologie' • the better understanding of gene interactions and networking (functional genomics) could improve the utilisation of gene-based or gene targeted strategies • novel paradigms can become available (Si RNA, PNA triplex etc...) • specifically integrating gene constructs or artificial chromosomes become more realistic • Praeklinische Forschung • scaling up to larger animal models (dog and monkey) permits better appreciation of dosage requirements • new transgenic models may give improved similarities to human diseases • Klinische Forschung • Use of recombinant lentiviruses may be imminent • Increase of Phase III procedures over the next 5 years • First therapeutical applications may be registered within 3-5 years • challenge by other emerging therapies • Ergo • der grösste teil der fehler waren menschliche Fehler • Die Huerde koennen bewältigt werden. a a a a a a

  43. UNIFR Rusconi2003 ...Danke ... und bleiben wir optimistisch Biovalley Program Gymnasium Liestal My collaborators at UNIFR Swiss National Research Foundation Danke fuer die aufmerksamkeit und fuer spezielle Fragen, bitte schreiben sie an: sandro.rusconi@unifr.ch oder besuchen sie die WEB seite: www.unifr.ch/nfp37 a a a a a a

  44. UNIFR Rusconi2003 END, let's open the Discussion a a a a a a

  45. UNIFR Rusconi2002 ***Diskussions-slides... • text • ttt a a a a a a

  46. UNIFR Rusconi2003 Gene therapy in Switzerland: the 30 projects financed by the NFP37 programme (1996-2001) NFP37phase Aphase B (96-99) (99-01) Submissions 30 26 Granted 19 18 Total requested 32 Mio 9 Mio Granted 7.6 Mio 6 Mio DISEASE ORIENTATION Cancer 8 10 Acquired disorders 2 7 Vector development 5 3 Hereditary disorders 2 4 Infectious diseases 1 2 RESEARCH LEVEL Fundamental 10 7 Preclinical (animal models) 5 9 Clinical phase I 2 3 Clinical Phase II 0 1 Clinical Phase III 0 0 Ethical/social aspects 1 1 Nationales Forschungsprogramm 37 NFP37 « somatic gene therapy »www.unifr.ch/nfp37 • Please Note • the NFP37 represented at most 30% of the Swiss-based experimentation in SGT during 1996-2001 a a a a a a

  47. UNIFR Rusconi2003 The SGT acrobatics: matching vectors / delivery system / disease • Chronic Conditions • Slow onset of expression acceptable • Initiation of the treatment weeks/months/years before 'point of no return' (ex. cystic fibrosis) • persisting expression of the transgene or re-administration required (example hemophilia) • Usually based on compensation of 'genetic loss-of-function' (permanent re-gain of function; ex. ADA) • Regulation of gene expression often necessary (because of persistence) • For some diseases even a small % of tissue transformation is already therapeutic • Acute Conditions • Rapid onset of expression necessary • Initiation of the treatment minutes/hours/days before 'point of no return' (ex. brain ischemia) • persisting expression of the transgene not required, occasional re-administration (example ischemia) • Usually based on augmentation of resident function (transient gain of function; ex. VEGF) • Regulation of gene expression not necessary (because of transiency) • For most diseases even a small % of transformation is already therapeutic • Ergo • many divergent variables must be matched for each case • an advantage for one purpose becomes a disadvantage for another (viceversa) a a a a a a

  48. 1 0 0 8 0 1 0 0 % 7 0 cancer incidence 1 0 Life expectancy (CH) 6 0 Alzheimer’s free % E 2 / E 5 0 1 M E 3 / E 4 1 9 0 0 1 9 2 0 1 9 4 0 1 9 6 0 1 9 8 0 1 9 9 4 E 4 / E 4 2 0 4 0 6 0 8 0 2 0 4 0 6 0 8 0 1900 2000 1900 2000 UNIFR Rusconi 2003 Die Hauptkrankheit des 21. Jahrhunderts: Veralterung aa getting oldcomp2.mov a a a a a a

  49. UNIFR Rusconi2003 Pas toutes les stratégies de transfert se basent sur un ancrage au hasard • Ergo • genotoxic • non-genotoxic • Random integrating vectors • r-retroviruses • r-lentiviruses • r-AAV • plasmids (low frequency) • plasmids + transposase (eg 'sleeping beauty') • Specifically integrating vectors • hybrid vectors (HSV-AAV) • Phage 31 integrase-based • designer integrase • Transient, non integrating vectors • adenovirus • plasmid • RNA virus based • oligonucleotides (SiRNA, antisense, ribozymes) • artificial chromosomes • Gene correction vectors • chimeroplasts (RNA-DNA chimeric oligos) • single stranded DNA (homologous recom) a a a a a a

  50. 100 nm L1 L1 L2 L2 capsid E E UUNIFR Rusconi 2003ß Mais un virus c'est quoi?Une machine auto-réplicative extrèmement efficace docking entry disassembly genome replication early genes exp replication late genes exp assembly standard viral genome Spread Etc... a a a a a a

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