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Sandro Rusconi (09.03.52)

UNIFR Rusconi 2005. Sandro Rusconi (09.03.52). 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) 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-harmonsation of biosafety and ethical aspects in gene therapy) March 1, 2005 ECPM course Gene Therapy growing teenage, what have we learned? 2005-xx Head 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 AAV germline Sept 2000 No previous medical procedure generated so many discussions so long before being ever clinically applicable 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 ... Oct 1998 VEGF ischemia second SAE Paris Feb 2003 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?) UNIFR Rusconi 2005 1 Gene -> 1 or more functions • Ergo • to say 'one gene->one function' is like pretending'one disease -> one drug' • Multifunctional character implies: • cross talk with different pathways • unclarified hyerarchical position • unclarified side-effects potential >300 ’000 functions (>150 ’000 functions) a a a a a a

  4. DNA RNA(s) Protein(s) GENE Transcription / translation FUNCTION RNA DNA UNIFR Rusconi 2005 Recap: what is a gene?:a regulated nanodevice for RNA production • Therefore, to fulfil its role, a transferred gene 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 Organism -> more than 105developmentally and genetically-controlled functions • Remember • 1 Cm3 of tissue • 1'000'000'000 cells! 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 2005 Reductionistic molecular biology paradigm(gene defects and gene transfer) • Gene transfer implies either: • transfer of new function, or • transfer of restoring function, or • transfer of interfering function a a a a a a a a a a a a

  7. genetics behaviour environment UNIFR Rusconi 2005 Examples of inheritable gene defects 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: • every person bears one or more latent genetic defects • many defects are not manifest but lead to predispositions • there are also protective predispositions 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 2005 Not only the genome determines the health status... • also acquired conditions may have a genetic component that modulates their healing • trauma • fractures • burns • infections a a a a a a

  9. 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 2005 • This major challenge means: • higher investments • more financial returns • long term treatment • customised treatment • social security dilemmawill molecular therapy boost the efficacy of treatment of age-related diseases? The major disease of the 21st century: Ageing aa getting oldcomp2.mov a a a a a a

  10. UNIFR Rusconi 2005 The THREE missions of medicine Prevention + 'Molecular Medicine' Application of the know-how in molecular genetics to medicine Diagnosis + + Therapy a a a a a a

  11. 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 The FOUR eras of molecular medicine genomeABC.mov a a a a a a

  12. UNIFR Rusconi 2005 Somatic Gene Therapy (SGT) 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

  13. UNIFR Rusconi 2005 The SGT principle is simple Yes,...but the devil is often in the 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 Geneva ... counting votes... ! ask Florida's officials ... gene therapy... look at progress in 13 years... a a a a a a

  14. UNIFR Rusconi 2005 Why 'somatic'? • 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. UNIFR Rusconi 2005 When/where/ may be SGT (currently) indicated? • 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: • there are many indications for SGT as stand-alone or as complementary therapy • Complement to conventional • increases specificity of conventional therapy (cancer) • increases efficacy of conventional therapy (hemophilia) • Perfid deviation dreams (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

  16. UNIFR Rusconi 2005 SGT's four fundamental questions & players 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

  17. UNIFR Rusconi 2005 Pharmacological considerations for DNA transfer Nucleic Acids Classical Drugs Protein Drugs • 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 • 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 O H O H O O O H O H O O O H • Therapy with nucleic acids • requires particulated formulation • is much more complex than previous drug deliveries • has a different degree of reversibility (intrinsic dosage / titration problem) O H O a a a a a a

  18. V UNIFR Rusconi 2005 THREE classes of anatomical gene delivery 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

  19. UNIFR Rusconi 2005 TWO classes of gene transfer vectors: non-viral & viral delivery Non-viral transfer (transfection of plasmids) a Viral gene transfer (Infection by r-vectors) b Nuclear envelope barrier! see, Nature Biotech December 2001 a a a a a a

  20. 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

  21. UNIFR Rusconi 2005 Comparing relevant issues in the two main 'vectorology' sectors (viral versus nonviral) • Viral vectors • Packaging capacity from 4 to 30 kb problem for some large genes (ex. dystrophin gene or CFTR gene) • important toxic load: ratio infectious/non-infectious particles from 1/10 to 1/100 • strong immunogenicity: capsid and envelope proteins, residual viral genes • contaminants: replication-competent viruses (ex. wild type revertant viruses) • Viral amount (titre) obtainable with recombinants (ex. 10exp5 = poor, 10exp10=excellent) • Complexity of manufacturing (existence or not of packaging cell systems) ('MAD' !) • Emotional problems linked to pathogenicity of donor vectors (ex. lentiviruses) • Nonviral vectors • Packaging capacity not an issue, even very large constructs can be used (example entire loci up to 150 kb) • minor toxic load: small percentage of non relevant adventitious materials • moderate immunogenicity: methylation status of DNA (example CpG motifs) • contaminants: adventitious pathogens from poor DNA purification (ex endotoxins) • Amount of DNA molecules is usually not a problem, the other components depends on chemical synthesis • No particular complexity, except for specially formulated liposomes • no particular emotional problems linked to the nature of the reagents • Ergo • problems that must be solved to be suitable for clinical treatment and for manufacturing are different between viral and non-viral vectors • when ignoring thir low efficiency, nonviral vectors appears largely superior a a a a a a

  22. UNIFR Rusconi 2005 Short list of popular vectors/methods 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

  23. Efficiency +++ Specificity Persistence Toxicity ++ UNIFR Rusconi 2005 Recombinant Adenoviruses • Manufacturing • Generation I/ II • Generation III • Hybrid adenos: • Adeno-RV • Adeno-AAV • Adeno-Transposase • Advantages / Limitations • 8 Kb capacity Generation I / II>30 Kb capacity Generation IIIAdeno can be grown at very high titers,However • Do not integrate in host genome • Can contain RCAs • Are toxic /immunogenic • Examples • OTC deficiency (clin, ---) • Cystic Fibrosis (clin, --- ) • Oncolytic viruses (clin, +++) a a a a a a

  24. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2005 Recombiant Adeno-associated-virus (AAV) Manufacturing 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 • Promotes autoimmunity? • Small capacity (<4.5 kb) which does not allow to accommodate large genes or gene clusters. • Examples • Hemophilia A (clin, animal, +++(autoimm?) • Gaucher (clin, animal, +++) • Brain Ischemia (animal, +++) • Cystic fibrosis (animal, +/-) • retinopathy (animal (+/-) a a a a a a

  25. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2005 Recombinant retroviruses (incl. HIV) Manufacturing 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

  26. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2005 Naked or complexed DNA 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

  27. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2005 Oligonucleotides 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

  28. UNIFR Rusconi 2004 Recap: current limitations of popular vectors 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 • Ergo • the future will probably see an increasing interest in viral-like, but artificial particles General - low transfer efficiency - no or little genomic integration General - antibody response - limited packaging - gene silencing - Manufacturing limitations Solutions: - improved liposomes with viral properties (“Virosomes”) Solutions: - synthetic viruses (“Virosomes”) a a a a a a

  29. Chronic Metabolic (ex. OTC, Gaucher, Haemophilia, hematopoietic) AAV, Lenti, Adeno III, r-retroviruses, repair oligo persistence of expression of the transferred gene, minimize readministration Local chronic or progressive (ex. CNS, joints, eyes) AAV, nonviral, Lenti No rapid expression necessary, persistence required, low toxicity Solid tumors +/- metastat.(cervical, breast, brain, skin) Adeno II, Plasmid, oncolytic recombinant viruses rapid & transient expression of cytotoxic or immunomodulators Trauma or infection (Ischemia, fracture, burn, wound, acute infection, anaphyllaxis) Adeno II, Plasmid, modulatory oligonucleotides Rapid and transient action required UNIFR Rusconi 2005 Which vector for which disease category Disease Type Most 'suitable' vector Justifications /Issues a a a a a a

  30. UNIFR Rusconi 2005 Technologies related to-, but not all genuinely definable as 'gene therapy' • Transiently bioactive oligonucleotides • antisense • decoy dsDNA, decoy RNA • ribozymes DNAzymes • Si RNA oligonucleotides • Genuine gene therapy oligos • chimeroplasts (*gene correction induction) • Oncolytic viruses • ONYX-15, ONYX-638 (r-adeno) • r-HSV • r-FSV from www.nature.com • Ergo • among all these, SiRNA seems to be the most promising inhibitor factor, and can be permanently expressed from DNA vectors • Implants of encapsulated cells • neurotrophic factor producer cell implants • hormone-producing cells a a a a a a

  31. ADA deficiency(Immunodeficiency) ADA normal gene(enzyme)retrovirus, ex-vivo BM 1990 F. Anderson, 2002 C. Bordignon Cystic Fibrosis(Lung, Pancreas) CFTR gene(chlorine transpor-ter), retrov., aav, adenoII, local no significant resultsin spite of several trials Haemophilia B(Blood) Factor IX gene (clotting factor), aav, adenoIII, intramuscular 1999-2000 M. Kay, K. High SCID(Immunodeficiency) IL2R gene (gamma-C receptor) retrov., ex vivo BM 2000 A. Fischer2002, UK trials Cardiac ischaemia(Heart) Limb ischaemia(Hands, Feet) VEGF gee (vascular growth factor), plasmid, intracardiac VEGF gene (vascular growth factor), plasmid, intramuscular 2000 J. Isner 1998 J. Isner UNIFR Rusconi 2005 'Classical' SGT models and strategies Disease transferred function Clinical Results additional 'popular' and emerging examples: Morbus Gaucher, Morbus Parkinson, Crigler Njiar, OTC deficiency, Duchenne's MD, Restenosis control a a a a a a

  32. 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 Gene Therapy in the clinics: 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 Jan 2005:938 cumulative protocols (90-2004) 4600 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 21% overall still pending or not yet Initiated ! www.wiley.com/genetherapy a a a a a a

  33. Isner, 1998 Anderson, 1990 Dzau, 1999 Fischer, 2000 2002 Kirn, 2000, 2001 2002 2003 Manuel Grez Hans Peter Hossle Reinhard Seger 2004 Intravascular adenoviral agents in cancer patients: Lessons from clinical trials (review) 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 UNIFR Rusconi 2005 Gene Therapy Clinical and Preclinical Milestones 1990, 1993, 2000, 2004 // 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 2000, Hemophilia M Kay, K High 21 lives saved 21 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 trial ~xxx lives saved or quality-improved?by Gendicine (still undocumented) 2000, 2002, X-SCID A Fischer, Science April 2000, UK trials 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, Gendicine (adeno-p53 vector) L Peng, Sibiono Inc, Shenzen, China a a a a a a

  34. UNIFR Rusconi 2005 Two persisting major SGT frustration cases • 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

  35. UNIFR Rusconi 2004 The most feared potential side-effects of gene transfer • 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». • SGT enjoyed a side-effect-free illusion during its first 10-year of non-working early period • Many side effects are still related to the rather primitive state of the vectorology/delivery a a a a a a

  36. UNIFR Rusconi 2005 SAEs1: best documented cases: acute and long term SAEs: from Gelsingers' death to Paris' Leukaemias caused by insertional mutagenesis 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 at: www.unifr.ch/nfp37/adverse03.html it is now rather established (2004) that the Paris' leukaemia events were caused by treatment-specific circumstances (type of transferred gene, dosing, type of vector, predisposition) The third SAE might delay the nextly planned restart of patients recruitment 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

  37. UNIFR Rusconi 2005 Future solutions to insertional mutagenesis: targeted gene transfer approaches • 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 integrases (ZnFinger proteins) • Transient, non integrating vectors • adenovirus • plasmid • RNA virus based • oligonucleotides (SiRNA, antisense, ribozymes) • artificial chromosomes Ergo vector systems that allow specific or at least better location-controlled gene delivery are experimentally well advanced (see accompanying text) • Gene correction vectors • chimeroplasts (RNA-DNA chimeric oligos) • single stranded DNA (homologous recom) a a a a a a

  38. UNIFR Rusconi 2005 SAEs2: mid-term effects: Recent Autoimmunity Reports Blood, 1 May 2004, Vol. 103, No. 9, pp. 3248-3249 Autoimmunity in EPO gene transfer (macaques) Els Verhoeyen and François-Loïc Cosset - 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. - delivered the homologous EPO cDNA driven by ubiquitous and/or regulatable promoters via AAV vectors injected in muscle or aerosolized in lung, resulting in supra-physiologic serum levels of EPO, from 10- to 100 000- fold over the baseline Ergo somatic gene transfer can generate mid-term self immunity under inappropriate circumstances 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 a a a a a a

  39. UNIFR Rusconi 2005 SAEs3: Non-science factors that have disturbed the public perception and progress of gene therapy • '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 • Money and media: an explosive cocktail, just like for sports or arts,... the field tends to degenerate as soon as excessive financial speculations are involved and when the mass media become overly interested in it. • The fundamental error: we pretended making a business issue out of a scientific issue a a a a a a

  40. ? ? ? UNIFR Rusconi 2005 A. Fischer M. Kay Ups and Downs of Gene Therapy: a true roller-coaster ride! high R. Crystal V.Dzau C Bordignon lentivectors in clinics? Adeno I J. Isner promising results 2003-2004 F Anderson AAV germline in mice? NIH Motulski report Adeno III mood • Ergo • whenever a reasonable cruise speed was achieved, a major adverse event has brought us back «square one» or even below Lentivectors Auto-immunity Low Paris I and II Leukaemias J. Gelsinger Paris III 90 91 92 93 94 95 96 97 98 99 00 01 02 03 04 05 a a a a a a

  41. UNIFR Rusconi 2004 Conclusions 1: in spite of the many hurdles, GT has already saved >20 condemned lives and keeps producing positive signals • X- SCID trials • France: 9/10 patients permanently cured of the lethal disease X-SCID • UK: 6/6 patients cured of X-SCID lethal condition • ADA deficiency • C Bordignon trials 4/4 patients permanently corrected + detoxified • Others • significant amelioration of CLI condition in Phase II trials • important therapeutic benefit with oncolytic viruses • promising amelioration in hemophilia patients • promising results from Chronic Granulomatosis treatment • First gene medicine product registered in China by Sibiono Inc. (see www.unifr.ch/sibiono.html) • Ergo • gene therapy's principle works • we better know limitations and potential of individualvectors a a a a a a

  42. UNIFR Rusconi 2005 Conclusions 2: GT has proven several concepts, has several tools, but is still in the pioneering phase • 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 • Viral vectors have the advantage of efficiency • nonviral vector the advantage of lower toxicity/danger. • Viral vectors have the disadvantage of limited packaging and some toxicity • nonviral vectors have the major disadvantage of low efficiency of transfer • Ergo • we are somewhat ahead but still in the pioneering phase ! • «failure of evidence» does not mean «evidence of failure» ! • Clinically • over 900 trials and >4000 patients in 14 years • only a handful of trials is now reaching phase III • Progress further slowed down by periodical pitfalls a a a a a a

  43. UNIFR Rusconi 2005 Perspectives: somatic gene therapy will progress in spite of all past, present and future incidents/accidents • Fundamental level & vectorology • Better understanding of gene interactions and networking • Gene inhibition through Si RNA • specifically integrating gene constructs • artificial chromosomes become more realistic • Preclinically • scaling up to larger animal models (dog and monkey) • new transgenic models may give improved similarities to human diseases • Ergo • many adverse events were due rather to human errors than to intrinsic dangers • other undesired effects are due to prototypic state of tools • hurdles can be overcome • the genuine potential of SGT is intact • Clinically • Use of recombinant lentiviruses • 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 a a a a a a

  44. UNIFR Rusconi 2005 ...Thanks, and let's remain optimistic ECPM education program Fritz Bühler, Annette Mollet Swiss National Research Foundation Thank you all for the attention, sandro.rusconi@unifr.ch or visit: www.unifr.ch/nfp37/ a a a a a a

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

  46. UNIFR Rusconi 2004 a a a a a a

  47. 100 nm L1 L1 L2 L2 capsid E E UUNIFR Rusconi 2002 Discussion: Recap: what is a virus ? -> A superbly efficient replicating nanomachine docking entry disassembly genome replication early genes exp replication late genes exp assembly standard viral genome Spread Etc... a a a a a a

  48. L1 L2 rp rp Normal target cells X E E E E E E E Recombinant genome Packaging cells Normal target cells UNIFR Rusconi 2002 Discussion: Engineering of replication-defective, recombinant viruses (Principle) E Wild type genome Virions Packaging Packaging Packaging R-Virions a a a a a a

  49. UNIFR Rusconi2003 Discussion: The Paris' trial (see also www.unifr.ch/nfp37/adverse.html) 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 HLA-matching bone marrow • Gene Therapeutical approach • explant BM (3-6 month old) • select CD34+/CD38- • transduce with retroviral vector encoding gamma(c) • re-infusion, follow-up a a a a a a

  50. UNIFR Rusconi2005 Discussion: The Paris' odyssey(see also www.unifr.ch/nfp37/adverse.html) Chronology • 1998 A Fischer's team starts 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 oncogene, • October 2003, public disclosure, chemotherapy, good response, report at ESGT congress. • October 2003 3 US and 3 EU trials on hold Adverse 2 • december 2002, T cell hyper-proliferation in a second, 36 months patienthyper-proliferatory cells also contain insertion of transgene close to LMO2 gene • January 2003, notification to authorities, public disclosure, treatment chemotherapy • January 2003, 27 US and 5 EU trials on hold Adverse 3 • January 2005, T cell hyper-proliferation in a third, ~36 months patient Fischer's trial is again on hold a a a a a a

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