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Sandro Rusconi (09.03.52)

UNIFR Rusconi 2005. Sandro Rusconi (09.03.52). Sept 15, 2005 Uniklinik Balgrist. 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

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Sandro Rusconi (09.03.52)

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  1. UNIFR Rusconi 2005 Sandro Rusconi (09.03.52) Sept 15, 2005 Uniklinik Balgrist 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-today Professor Biochemistry UNI Fribourg 1996-2002 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) 2002-06Euregenethy Network (EU-harmonsiation of biosafety and ethical aspects in gene therapy) 'Therapeutische' Gentherapie: Stand 2005 Eigentlich gemeint war: Therapeutischer Gentransfer: Stand 2005 2005-xx Director of Governmental Division for Culture and University Affairs of Canton Ticino a a a a a a

  2. UNIFR Rusconi 2005 Gene therapy: A 15-years hailstorm of highly emotionalised good and bad news BBC, NBC, CNN,... Jesse Gelsinger Oct 1999 New York Times Washington Post Times Le Monde Frankfurter Allgemeine ... Feb 1990 First trial ADA deficiency A Fischer, E Thrasher Paris & UK Dec 2000 Dec 1988 IL-2 cancer treatment trial Selten hat eine medizinische Technik so viel Rauch und so wenig Feuer produziert How many of you have heard mostly bad news... ?mostly good news...? AAV germline Sept 2000 Mar 1994 SAE cystic fibrosis C Bordignon, Milano trial May 2002 Jun 1995 Motulsky NIH report First SAE Paris Sep 2002 Feb 1996 r-lentiviruses Nature Science NEJM ... second SAE Paris Feb 2003 SiRNA preclinical 2004 Autoimmunity monkeys May 2004 Oct 1998 VEGF ischemia third SAE Paris Jan 2005 Internet a a a a a a

  3. DNA RNA(s) Protein(s) Transcription / translation GENE 2-5 FUNCTIONS Gene expression 100 ’000 genes (50 ’000 genes?) >300 ’000 functions (>150 ’000 functions) UNIFR Rusconi 2005 1 Gen -> 1 oder mehrere Funktionen • Ergo • 'ein Gen -> eine Function' ist so falsch wie'eine Krakheit -> ein Medikament' • Multifunctional character of genes implies: • cross talk with different pathways • unclarified hyerarchical position • unclarified side-effects potential a a a a a a

  4. DNA RNA(s) Protein(s) GENE Transcription / translation FUNCTION RNA DNA UNIFR Rusconi 2005 Recap: was ist ein Gen?:ein grosses Molekuel mit informativem Inhalt • Therefore, to fullfil its role, a transferred gene segment must include: • regulatory sequences for Transcription • proper signals for RNA Maturation/transport • proper signals for mRNA Translation • proper signals for mRNA Degradation spacer regulatory coding spacer a a a a a a

  5. 2 mm 0.2mm 2m 0.02mm 0.001mm DNA RNA Protein UNIFR Rusconi 2005 1 Organismus -> mehr als 105entwicklungsgenetisch kontrollierte Funktionen • Zur Erinnerung • 1 Cm3 Gewebe • 1'000'000'000 Zellen! a a a a a a

  6. Eighties Genes as probes Nineties Genes as factories Y2K Genes as drugs 50 1 2 3 4 5 3000 10 80 85 90 95 99 1000 ok ok ** ** ** 80 85 90 95 00 UNIFR Rusconi 2003 Gentherapie als logische Folge: die dritte Aera • Ergo • gene transfer is a logical development of molecular biology a a a a a a

  7. UNIFR Rusconi 2005 Somatische Gentherapie (SGT): Definition 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

  8. UNIFR Rusconi 2005 Wieso 'somatisch'? • Germ Line Cells: the cells (spermatocytes and oocytes and their precursors) that upon fertilisation can give rise to a descendant organism • Ergo • tranformierung von keimbahnzellen ist zur Zeitvermieden (technische und ethische Probleme) 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

  9. UNIFR Rusconi 2005 Wann gibt heute eine Indikation fuer SGT ? • 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)-> angiomas • Factor VIII or IV (hemophilia)-> insufficient basal level • Ergo: • viele Indikationen • Complement to conventional • increases specificity of conventional therapy (cancer) • increases efficacy of conventional therapy (hemophilia) • Perverse deviation dreams (even with current technologyI: • gene-based sports doping • performance amelioration • cosmetics • Life quality burden of patient • costs of enzyme therapy (ex. ADA) • burden of daily injections (ex. Insulin) a a a a a a

  10. UNIFR Rusconi 2005 Pharmacologische 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 • Ergo: Therapy with nucleic acids • Spezielle Formulierung • Viel komplexer als konventionelle Medikament-Therapie • Geringere Reversibilitaet O H O a a a a a a

  11. UNIFR Rusconi 2005 VIER grundlegende Fragen der SGT 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? a a a a a a

  12. V UNIFR Rusconi 2005 DREI Kategorien von anatomische Gen-Lieferung Ex-vivo In-vivo topical delivery In-vivo systemic delivery • Ergo • ex vivo or local delivery are currently preferred over 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

  13. UNIFR Rusconi 2005 ZWEI Vektor-Typen: non-viral & viral Non-viral transfer (transfection of plasmids) a Viral gene transfer (Infection by r-vectors) • Ergo • viral transfer is much more efficient • nonviral transfer must solve a number of hurdles- serum protection/stability- target docking- endosomal escape- nuclear trafficking- genomic integration- anti apoptotic functions- immunological camouflage - ... b Nuclear envelope barrier! see, Nature Biotech December 2001 a a a a a a

  14. UNIFR Rusconi 2005 Transfection versus Infection Transfection exposed to 106 particles/cell 12 hours Infection exposed to 1 particle/cell 30 min • Ergo • virally mediated gene transfer is millions of timesmore efficent than nonviral transfer (when calculated in terms of transfer/particle) a a a a a a

  15. UNIFR Rusconi 2005 Kurze Liste von Vektoren Naked DNA Liposomes & Co. Oligonucleotides r-Adenovirus r-Adeno-Associated V. r-Retrovirus (incl. HIV) but remember... "Nobody's perfect "! a a a a a a

  16. UNIFR Rusconi 2004 Recap: Limitierungen heutiger Vektoren r-Adenovirus - no persistence - limited packaging - toxicity, immunogenicity Biolistic bombardment or local direct injection - limited area r-AAV - no integration in host g. - very limited packaging - autoimmunity? Electroporation - limited organ access Liposomes, gene correction & Co. - rather inefficient transfer r-Retrovirus (incl. HIV) - limited packaging - random insertion - unstable genome General - low transfer efficiency - no or little genomic integration General - antibody response - limited packaging - gene silencing - Manufacturing limitations • Ergo • the future will probably see an increasing interest in viral-like, but artificial particles Solutions: - improved liposomes with viral properties (“Virosomes”) Solutions: - synthetic viruses (“Virosomes”) a a a a a a

  17. 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 Rusconi 2005 Gentherapie in der Klinik: Trials Worldwide (cumulative) • Ergo • in spite of 13 year- research only less than 2% of the trials has reached phase III • not necessarily due to the «novel»'fail early, fail fast' paradigm As of January 2005:938 cumulative protocols (90-2005) 4700 treated /enrolled patients 66% phase I 19% phase I-II 13% phase II 0.8% phase II-III 1.7% phase III ! As of Jan 1, 2004: 1 approved product in China (Gendicine, by Sibiono Inc. 2004) 2600 Patients treated in 2004 20% overall still pending or not yet Initiated ! www.wiley.com/genetherapy a a a a a a

  18. Isner, 1998 Anderson, 1990 Fischer, 2000 2002 Kirn, 2000, 2001 2002 2003 Manuel Grez Hans Peter Hossle Reinhard Seger 2004/2005 Intravascular adenoviral agents in cancer patients: Lessons from clinical trials (review) dropped in 2004? licensed China 2005? very encouraging data from just initiated clinical trial, prospected >10 patients Bordignon, 2000 (ESGT, Stockholm)2002, science 296, 2410 ff) SibionoShenzen Approved commercialisation of Gendicine (Jan 2004) for cancer treatment in China. -> ! Hum Gene Ther 16, 1016 ff. UNIFR Rusconi 2005 Klinische Meilensteine der Gentherapie 1990, 1993, 2000, // ADA deficiency F Anderson, M Blaese 90/93/ C Bordignon 2000/2004 1997, 2000, Critical limb ischemia J Isner († 4.11.2001), I Baumgartner, 1998 25 lives were so far documentedly saved by GT in european trials (x-SCID, ADA, CGD) (France, UK, Italy) (all in phase I) ~200 lives quality-improvedin several other phase I and II trials ~nnn lives saved or quality-improved?by Gendicine (50'000 patients prospected for 2006) 2000, Hemophilia M Kay, K High 2000, 2002, X-SCID A Fischer, 2000/2002, Thrasher 2003 2001, 2003 ONYX oncolytic Viruses D Kirn (Cancer Gene Ther 9, p 979-86) 2004, Chronic Granulomatous Disease M Grez Frankfurt; R Seger Zürich 2004/2005 Gendicine (adeno-p53 vector) L Peng, Sibiono Inc, Shenzen, China a a a a a a

  19. UNIFR Rusconi 2005 Zwei persistierende Frustrationsfälle • 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 / biochemical barrier: no receptors, mucus layer • large gene that requires probably regulation • requires long term regulation • unclear at which point disease becomes irreversible • In spite of genes discovered in the 90ties: • lacking suitable vector • no satisfactory delivery method • no persistence • treatment 'too late' a a a a a a

  20. UNIFR Rusconi 2005 Die meist befürchtete Nebeneffekte der Gentherapie • Immune response to vector • immune response or long term side effects from 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 • immune response or long term side effects from new or foreign gene product (-> autoimmunity) • Random integration in genome-> insertional mutagenesis (-> cancer risk) • 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 a a a a a a

  21. UNIFR Rusconi 2005 SAEs1: Vom Gelsingers' Tod bis zu den Paris' Leukaemias NY May 5, 1995, R. Crystal: adenovirus, cystic fibrosis (lung) one patient mild pneumonia-like condition Trial interrupted and many others on hold. Most Recent Paris' Trial News discussed under: www.unifr.ch/nfp37/adverse03.html Tomorrow (16.09.05) A Fischer will talk at the Kontderspital(Workshop organised by R Seger) UPenn, Sept. 19, 1999, J. Wilson: adenovirus , 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: 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: retrovirus X-SCID (bone marrow) same cohort a second patient developed a similar leukemia 30 trials in USA were temporarily suspended Ergo gene therapy can produce both short-term and long-term severe side effects through acute immunogenicity or insertional mutagenesis (cancer risk) Paris, Jan 24, 2005, A Fischer: retrovirus X-SCID (bone marrow) same cohort a third patient developed a similar leukemia what will happen? a a a a a a

  22. UNIFR Rusconi 2005 SAEs2: Recent Autoimmunity Reports 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 Factor IX in a Phase I Study of AAV-Mediated, Liver-Directed Gene Transfer for Hemophilia B Ergo somatic gene transfer and ectopic transgene expression can generate mid-term auto- immunity a a a a a a

  23. UNIFR Rusconi 2005 SAEs3: Nicht-wissenschaftliche Faktoren die Fortschritt und Image von GT negativ beeinflusst haben • '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 a a a a a a

  24. >90 ? 25 ? 16 ? 5 4 UNIFR Rusconi 2005 A. Fischer M. Kay Gentherapie Hoehe und Tiefe: a true roller-coaster ride! high R. Crystal • Ergo • whenever a reasonable cruise speed was achieved, a major adverse event has brought us back «square one» or even below V.Dzau C Bordignon Adeno I J. Isner F Anderson lentivectors hopes AAV germline in mice? NIH Motulski report Adeno III mood Lentivectors gendi cine Auto-immunity Low Paris I and II Leukaemias J. Gelsinger companies Paris III 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 a a a a a a

  25. UNIFR Rusconi 2005 Schlussfolgerungen: GT hat Konzepte bewiesen aber bleibt immer noch im Pionierzustand • 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/dangerBUT -> inefficient • Viral vectors limited packaging and high toxicityBUT -> efficient • Ergo • we are somewhat ahead but still in the pioneering phase ! • Clinically • Over 1000 trials and >4000 patients in 15 years • Only a handful phase III • Periodical pitfalls • Gendicine approved in China (2004) a a a a a a

  26. UNIFR Rusconi 2005 Aussichte: GT wird fortschreiten trotz den gängigen und zhukuenftiken Zwischenfälle • 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 • Increase of Phase III procedures over the next 5 years • therapeutical applications may be registered within 3-5 years • challenge by other emerging therapies • Ergo • Accidents typical of prototypic status • hurdles can be overcome • the genuine potential of SGT is intact a a a a a a

  27. UNIFR Rusconi 2005 Meinen 'Proust's questionnaire' bezüglich Gentherapie 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, intrabodies, aptamers, ribozymes, DNAzymes, SiRNA, designer Zn Fingers, triple helix, small drugs, ...whatever? ...whatever M Proust 1871-1922 a a a a a a

  28. UNIFR Rusconi 2005 ...Danke, und ... let's remain optimistic Orthopedics Update Ch. Gerber, B. Fuchs My UNIFR and TI collaborators • Ergo • let's look forward to a safe landing Thank you all for the patience and attention, sandro.rusconi@unifr.ch or visit: www.unifr.ch/nfp37/ a a a a a a

  29. UNIFR Rusconi 2005 That's all, folks! www.unifr.ch/nfp37 a a a a a a

  30. UNIFR Rusconi 2004 a a a a a a

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