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Sandro Rusconi (09.03.52) a aa a aa UNIFR Rusconi 2005 'Therapeutische' Gentherapie: Stand 2005 Sept 15, 2005 Uniklinik Balgrist 1972-75School teacher.

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Presentation on theme: "Sandro Rusconi (09.03.52) a aa a aa UNIFR Rusconi 2005 'Therapeutische' Gentherapie: Stand 2005 Sept 15, 2005 Uniklinik Balgrist 1972-75School teacher."— Presentation transcript:

1 Sandro Rusconi (09.03.52) a aa a aa UNIFR Rusconi 2005 'Therapeutische' Gentherapie: Stand 2005 Sept 15, 2005 Uniklinik Balgrist 1972-75School teacher (Locarno, Switzerland) 1975-79Graduation in Biology UNI Zuerich, Switzerland 1979-82PhD curriculum UNI Zuerich, molecular biology 1982-84Research assistant UNI Zuerich 1984-86Postdoc UCSF, K Yamamoto, (San Francisco) 1987-93Principal Investigator, UNI Zuerich, PD 1994-todayProfessor Biochemistry UNI Fribourg 1996-2002Director Swiss National Research Program 37 'Somatic Gene Therapy' 2002-03Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department 2002-05President Union of Swiss Societies for Experimental Biology (USGEB) 2002-06Euregenethy Network (EU-harmonsiation of biosafety and ethical aspects in gene therapy) 2005-xxDirector of Governmental Division for Culture and University Affairs of Canton Ticino Eigentlich gemeint war: Therapeutischer Gentransfer: Stand 2005

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

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

4 Recap: was ist ein Gen?: ein grosses Molekuel mit informativem Inhalt a aa a aa RNA(s)DNAProtein(s) GENEFUNCTION Transcription / translation codingspacer regulatory DNA RNA 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 UNIFR Rusconi 2005

5 1 Organismus -> mehr als 10 5 entwicklungsgenetisch kontrollierte Funktionen a aa a aa 2m 2 mm 0.2mm 0.02mm DNA RNAProtein 0.001mm Zur Erinnerung 1 Cm 3 Gewebe 1'000'000'000 Zellen! UNIFR Rusconi 2005

6 Gentherapie als logische Folge: die dritte Aera a aa a aa UNIFR Rusconi 2003 Eighties Genes as probes ok ** ok 12453 Nineties Genes as factories 8085909599 10 50 Y2K Genes as drugs 8085909500 1000 3000 Ergo gene transfer is a logical development of molecular biology

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

8 Wieso 'somatisch'? a aa a aa Germ Line Cells: the cells (spermatocytes and oocytes and their precursors) that upon fertilisation can give rise to a descendant organism Somatic Cells: all the other cells of the body 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 Ergo tranformierung von keimbahnzellen ist zur Zeit vermieden (technische und ethische Probleme) UNIFR Rusconi 2005

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

10 UNIFR Rusconi 2005 Pharmacologische Betrachtungen a aa a aa OH OH O OH OH O O OH OH O O 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 molecules Ångstrom/nm size rapidly reversible treatment Classical Drugs Mw 20 000- 100 000 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 Protein Drugs Mw N x 1000000 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 50-200 nm size slowly or not reversible Nucleic Acids Ergo: Therapy with nucleic acids Spezielle Formulierung Viel komplexer als konventionelle Medikament-Therapie Geringere Reversibilitaet

11 VIER grundlegende Fragen der SGT a aa a aa 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 Rusconi 2005

12 DREI Kategorien von anatomische Gen-Lieferung a aa a aa Ex-vivoIn-vivo topical delivery In-vivo systemic delivery V Examples: - bone marrow - liver cells - skin cells Examples: - brain - muscle - eye - joints - tumors Examples: - intravenous - intra-arterial - intra-peritoneal UNIFR Rusconi 2005 Ergo ex vivo or local delivery are currently preferred over systemic delivery

13 ZWEI Vektor-Typen: non-viral & viral a aa a aa a b Non-viral transfer (transfection of plasmids) Viral gene transfer (Infection by r-vectors) Nuclear envelope barrier! see, Nature Biotech December 2001 UNIFR Rusconi 2005 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 -...

14 Transfection versus Infection a aa a aa Transfection Infection exposed to 10 6 particles/cell 12 hours 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 Rusconi 2005

15 Kurze Liste von Vektoren a aa a aa r-Adenovirus r-Adeno-Associated V. r-Retrovirus (incl. HIV) Naked DNA Liposomes & Co. Oligonucleotides UNIFR Rusconi 2005

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

17 Gentherapie in der Klinik: Trials Worldwide (cumulative) a aa a aa cancer hered. Infect. vasc. 40 60 100 20 80 trials 500 1500 1000 patients 199219941996199819902000 20% overall still pending or not yet Initiated ! 66% phase I 19% phase I-II 13% phase II 0.8% phase II-III 1.7% phase III As of January 2005: 938 cumulative protocols (90-2005) 4700 treated /enrolled patients 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 I I-II II UNIFR Rusconi 2005 ! As of Jan 1, 2004: 1 approved product in China (Gendicine, by Sibiono Inc. 2004) 2600 Patients treated in 2004

18 Klinische Meilensteine der Gentherapie a aa a aa Anderson, 1990 Bordignon, 2000 (ESGT, Stockholm) 2002, science 296, 2410 ff) 1990, 1993, 2000, // ADA deficiency F Anderson, M Blaese 90/93/ C Bordignon 2000/2004 Isner, 1998 1997, 2000, Critical limb ischemia J Isner ( 4.11.2001), I Baumgartner, 1998 2000, Hemophilia M Kay, K High Fischer, 2000 2002 2000, 2002, X-SCID A Fischer, 2000/2002, Thrasher 2003 Kirn, 2000, 2001 2002 2003 Intravascular adenoviral agents in cancer patients: Lessons from clinical trials (review) dropped in 2004? licensed China 2005? 2001, 2003 ONYX oncolytic Viruses D Kirn (Cancer Gene Ther 9, p 979-86) Manuel Grez Hans Peter Hossle Reinhard Seger 2004/2005 very encouraging data from just initiated clinical trial, prospected >10 patients 2004, Chronic Granulomatous Disease M Grez Frankfurt; R Seger Zürich UNIFR Rusconi 2005 Approved commercialisation of Gendicine (Jan 2004) for cancer treatment in China. -> ! Hum Gene Ther 16, 1016 ff. Sibiono Shenzen 2004/2005 Gendicine (adeno-p53 vector) L Peng, Sibiono Inc, Shenzen, China 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)

19 Zwei persistierende Frustrationsfälle a aa a aa 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' UNIFR Rusconi 2005

20 Die meist befürchtete Nebeneffekte der Gentherapie a aa a aa 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 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 UNIFR Rusconi 2005 immune response or long term side effects from new or foreign gene product (-> autoimmunity)

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

22 SAEs2: Recent Autoimmunity Reports a aa a aa 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 UNIFR Rusconi 2005 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

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

24 16 25 Gentherapie Hoehe und Tiefe: a true roller-coaster ride! >90 a aa a aa high Low mood NIH Motulski report Lentivectors Adeno III J. Isner F Anderson R. Crystal Adeno I A. Fischer M. Kay AAV germline in mice? Ergo whenever a reasonable cruise speed was achieved, a major adverse event has brought us back «square one» or even below V.Dzau Paris I and II Leukaemias J. Gelsinger 909192939495969798990001020304 UNIFR Rusconi 2005 05 C Bordignon 5 lentivectors hopes Auto- immunity gendi cine Paris III ? ? ? 4 companies

25 Schlussfolgerungen: GT hat Konzepte bewiesen aber bleibt immer noch im Pionierzustand a aa a aa 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 ! UNIFR Rusconi 2005

26 Aussichte: GT wird fortschreiten trotz den gängigen und zhukuenftiken Zwischenfälle a aa a aa 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 UNIFR Rusconi 2005

27 Meinen 'Proust's questionnaire' bezüglich Gentherapie a aa a aa UNIFR Rusconi 2005 will GT ever make it into routine clinical practice ? yes The most worrying adverse-effect? immunity Which will bloom: viral or non viral transfer? combination thereof Is insertional mutagenesis an important hurdle? No 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 when will GT widely established ? not tomorrow

28 Thank you all for the patience and attention, or visit:, und... let's remain optimistic a aa a aa My UNIFR and TI collaborators Ch. Gerber, B. Fuchs Orthopedics Update UNIFR Rusconi 2005 Ergo let's look forward to a safe landing

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

30 a aa a aa UNIFR Rusconi 2004

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