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Sandro Rusconi ( ) UNIFR 2005

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1 Sandro Rusconi (09.03.52) UNIFR 2005
Sept 15, 2005 Uniklinik Balgrist School teacher (Locarno, Switzerland) Graduation in Biology UNI Zuerich, Switzerland PhD curriculum UNI Zuerich, molecular biology Research assistant UNI Zuerich Postdoc UCSF, K Yamamoto, (San Francisco) Principal Investigator, UNI Zuerich, PD 1994-today Professor Biochemistry UNI Fribourg Director Swiss National Research Program 37 'Somatic Gene Therapy' Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department President Union of Swiss Societies for Experimental Biology (USGEB) Euregenethy 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 How many of you have heard mostly bad news... ? mostly good news...?
Gene therapy: A 15-years hailstorm of highly emotionalised good and bad news UNIFR Rusconi 2005 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 1 Gen -> 1 oder mehrere Funktionen
UNIFR Rusconi 2005 DNA GENE RNA(s) Protein(s) 2-5 FUNCTIONS Gene expression Transcription / translation Ergo 'ein Gen -> eine Function' ist so falsch wie 'eine Krakheit -> ein Medikament' >300 ’000 functions (>150 ’000 functions) 100 ’000 genes (50 ’000 genes?) Multifunctional character of genes implies: cross talk with different pathways unclarified hyerarchical position unclarified side-effects potential a a a a a a

4 Recap: was ist ein Gen?: ein grosses Molekuel mit informativem Inhalt
UNIFR Rusconi 2005 RNA(s) DNA Protein(s) GENE FUNCTION Transcription / translation 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 DNA RNA spacer regulatory coding spacer a a a a a a

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

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

7 Somatische Gentherapie (SGT): Definition
UNIFR Rusconi 2005 Definition of SGT: 'Use genes as drugs': Correcting disorders by somatic gene transfer Chronic treatment Acute treatment Preventive treatment NFP37 somatic gene therapy Hereditary disorders Acquired disorders Loss-of-function Gain-of-function a a a a a a

8 Wieso 'somatisch'? UNIFR 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 keimbahnzellen ist zur Zeit vermieden (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 Wann gibt heute eine Indikation fuer SGT ?
UNIFR 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) -> 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 Pharmacologische Betrachtungen
UNIFR Rusconi 2005 Classical Drugs Protein Drugs Nucleic Acids Mw 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 molecules Ångstrom/nm size rapidly reversible treatment Mw 20 ’  ’000 Da Biologically prepared Slower diffusion/action Oral delivery not possible Cellular delivery: - act extracellularly Can be delivered as soluble molecules 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 particles nm size slowly or not reversible O H O H O O H O O H O O H O 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 VIER grundlegende Fragen der SGT
UNIFR 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? a a a a a a

12 DREI Kategorien von anatomische Gen-Lieferung
UNIFR Rusconi 2005 Ex-vivo In-vivo topical delivery In-vivo systemic delivery Ergo ex vivo or local delivery are currently preferred over systemic delivery V 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 ZWEI Vektor-Typen: non-viral & viral
UNIFR Rusconi 2005 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 Transfection versus Infection
UNIFR 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) a a a a a a

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

16 Recap: Limitierungen heutiger Vektoren
UNIFR Rusconi 2004 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 Gentherapie in der Klinik: Trials Worldwide (cumulative)
UNIFR Rusconi 2005 40 60 100 20 80 trials 500 1500 1000 patients 1992 1994 1996 1998 1990 2000 cancer 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 ( ) 4700 treated /enrolled patients I I-II II 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 hered. vasc. 20% overall still pending or not yet Initiated ! Infect. a a a a a a

18 Klinische Meilensteine der Gentherapie
UNIFR Rusconi 2005 1990, 1993, 2000, // ADA deficiency F Anderson, M Blaese 90/93/ C Bordignon 2000/2004 Anderson, 1990 Bordignon, 2000 (ESGT, Stockholm) 2002, science 296, 2410 ff) Isner, 1998 Fischer, 2000 2002 Kirn, 2000, 2001 2002 2003 Intravascular adenoviral agents in cancer patients: Lessons from clinical trials (review) dropped in 2004? licensed China 2005? 1997, 2000, Critical limb ischemia J Isner († ), I Baumgartner, 1998 Manuel Grez Hans Peter Hossle Reinhard Seger 2004/2005 very encouraging data from just initiated clinical trial, prospected >10 patients 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-improved in several other phase I and II trials ~nnn lives saved or quality-improved ? by Gendicine (50'000 patients prospected for 2006) Approved commercialisation of Gendicine (Jan 2004) for cancer treatment in China. -> ! Hum Gene Ther 16, 1016 ff. Sibiono Shenzen 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 ) 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 Zwei persistierende Frustrationsfälle
UNIFR 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 / 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 Die meist befürchtete Nebeneffekte der Gentherapie
UNIFR Rusconi 2005 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 SAEs1: Vom Gelsingers' Tod bis zu den Paris' Leukaemias
UNIFR Rusconi 2005 NY May 5, 1995, R. Crystal: adenovirus, cystic fibrosis (lung) one patient mild pneumonia-like condition Trial interrupted and many others on hold. Most Recent Paris' Trial News discussed under: Tomorrow ( ) 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 SAEs2: Recent Autoimmunity Reports
UNIFR Rusconi 2005 Blood, 1 May 2004, Vol. 103, No. 9, comment: pp 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 SAEs3: Nicht-wissenschaftliche Faktoren die Fortschritt und Image von GT negativ beeinflusst haben
UNIFR Rusconi 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 a a a a a a

24 Gentherapie Hoehe und Tiefe: a true roller-coaster ride!
UNIFR Rusconi 2005 >90 A. Fischer M. Kay 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 AAV germline in mice? lentivectors hopes NIH Motulski report Adeno III mood Lentivectors ? gendi cine 25 16 Auto- immunity Low Paris I and II Leukaemias J. Gelsinger companies 5 4 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 Schlussfolgerungen: GT hat Konzepte bewiesen aber bleibt immer noch im Pionierzustand
UNIFR Rusconi 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 Over 1000 trials and >4000 patients in 15 years Only a handful phase III Periodical pitfalls Gendicine approved in China (2004) Ergo we are somewhat ahead but still in the pioneering phase ! a a a a a a

26 Aussichte: GT wird fortschreiten trotz den gängigen und zhukuenftiken Zwischenfälle
UNIFR Rusconi 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 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 Meinen 'Proust's questionnaire' bezüglich Gentherapie
UNIFR 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, intrabodies, aptamers, ribozymes, DNAzymes, SiRNA, designer Zn Fingers, triple helix, small drugs, ...whatever? ...whatever M Proust a a a a a a

28 ...Danke, und ... let's remain optimistic
UNIFR Rusconi 2005 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, or visit: a a a a a a

29 That's all, folks! UNIFR Rusconi 2005 a a a a a a

30 UNIFR Rusconi 2004 a a a a a a

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