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Sandro Rusconi

UNIFR Rusconi 2003. Sandro Rusconi. 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)

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Sandro Rusconi

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  1. UNIFR Rusconi 2003 Sandro Rusconi 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) 1987-91 Principal Investigator, UNI Zuerich 1994-todayProfessor Biochemistry UNI Fribourg 1995-today Director Swiss National Research Program 37 'Somatic Gene Therapy' 2002-03 Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department 2002-05 President Union of Swiss Societies for Experimental Biology (USGEB) Feb 19, 2003 ECPM Basel 2003: Gene therapy turning teenage, what have we learned? a a a a a a

  2. UNIFR Rusconi 2003 Genetics has been used since millennia,Molecular Biology, only since 30 years 100’000 b.C. Empirical genetics 10’000 b.C. Biotechnology 2000 a.d. Molecular biology 2001 a.d, Genomics 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 Gene -> 1 or more functions a a a a a a

  4. DNA RNA Protein GENE Transcription / translation FUNCTION RNA DNA UNIFR Rusconi 2003 Recap: what is a gene?:a regulated machine for RNA production • To fulfil its role, a transferred gene must include: • regulatory sequences for Tx initiation • proper signals for RNA maturation/transport • proper signals for mRNA translation 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 2003 1 Organism -> more than 105genetically-controlled Functions • 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 2003 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 2003 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 2003 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 2003 The major disease of the 21st century: Ageing • This major challenge means: • higher investments • more financial returns • long term treatment • customised treatment • social security dilemma a a a a a a

  10. UNIFR Rusconi 2003 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 Rusconi2003 Now, let's talk about 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 Rusconi2003 Gene therapy turns teenage in 2003, but:has it really grown up? The SGT principle is simple Yes,...but the devil is often in the details There are many things that are simple in principle, like... 1990 First clinical trial of a monogenic disease F. Anderson & Co: ADA deficiency getting a train ticket... ...does not work ! 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 ... 2002 Same protocol as Anderson's for ADA gene therapy (C. Bordignon) ...it works! counting votes... ! ask Florida's officials ... gene therapy... look at progress in 13 years... a a a a a a

  14. UNIFR Rusconi2003 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 Rusconi2003 When/where/ may be SGT 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 • increase specificity of conventional therapy (cancer) • increase efficacly of conventional therapy (hemophilia) • Life quality burden of patient • costs of enzyme therapy (ex. ADA) • burden of daily injections (ex. Insulin) a a a a a a

  16. Remember! Efficiency Specificity Persistence Toxicity UNIFR Rusconi2003 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 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 • 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

  18. UNIFR Rusconi 2003 Pharmacological considerations for DNA transfer 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 • Therapy with nucleic acids • requires particulated formulation • is much more complex than previous drug deliveries • has a different degree of reversibility (dosage problem) O a a a a a a

  19. V UNIFR Rusconi 2003 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

  20. UNIFR Rusconi 2003 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

  21. UNIFR Rusconi 2003 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 times more efficent than nonviral transfer (when calculated in terms of transfer/particle) a a a a a a

  22. UNIFR Rusconi2003 Most 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 production (existence or not of packaging cell systems) • 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 industrial production are different between viral and non-viral vectors • when ignoring thir low efficiency, nonviral vectors appears largely superior a a a a a a

  23. UNIFR Rusconi2003 Ideal properties of a systemically delivered non-viral formulation • Stability • particle should resist serum inactivation • particle should be inert to immune inactivation • Ergo • several independent problems must be solved for a nonviral formulation to be suitable for clinical treatment and for industrial production • most viral vectors include many, if not all those properties • Addressability • particle should possess a vascular addressing signature • particle should bear a tissue-docking specificity • DNA construct should include tissue-specific regulatory elements • Efficiency • cargo should be protected from cytoplasmic inactivation (ex. lysosomes) • cargo should contain nuclear-translocating signals • DNA cargo should include genome-integration functions • DNA element must be guaranteed to function after genomic integration (no silencing) • Other properties • Particle should not include immunogenic/toxic surfaces • cargo should not encode immunogenic/toxic products • Cargo should include anti-apoptotic functions a a a a a a

  24. UNIFR Rusconi 2003 Small parade of popular vectors/methods Naked DNA Liposomes & Co. Oligonucleotides Adenovirus Adeno-associated V. Retrovirus (incl. HIV) but remember... "Nobody's perfect "! a a a a a a

  25. Efficiency +++ Specificity Persistence Toxicity ++ UNIFR Rusconi 2003 Recombinant Adenoviruses • 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

  26. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2003 Recombinant 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 problem is: • 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

  27. Efficiency Specificity Persistence Toxicity UUNIFR Rusconi 2003 Recombinant Retroviruses (includes HIV-based) 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

  28. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2003 Naked / 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

  29. Efficiency Specificity Persistence Toxicity UNIFR Rusconi 2003 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

  30. UNIFR Rusconi 2003 Recap: current limitations of popular vectors Adenovirus - no persistence - limited packaging - toxicity, immunogenicity Biolistic bombardment or local direct injection - limited area Electroporation - limited organ access Retrovirus (incl. HIV) - limited packaging - random insertion - unstable genome Liposomes, gene correction & Co. - very inefficient transfer General - antibody response - limited packaging - gene silencing General - low transfer efficiency - no or little genomic integration Solutions: - synthetic viruses (“Virosomes”) Solutions: - improved liposomes with viral properties (“Virosomes”) • Ergo • the future will see increasing interest in viral-like, but artificial particles a a a a a a

  31. UNIFR Rusconi2003 Not all gene therapy approaches are 'random shooting' • Ergo • genotoxic • non-genotoxic • Random integrating vectors • r-lentiviruses • r-retroviruses • 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

  32. 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 Rusconi2003 Which vector for which disease category Disease Type Most suitable vector Justifications /Issues a a a a a a

  33. UNIFR Rusconi2003 Technologies related to-, but not genuinely definable as 'gene therapy' • Bioactive oligonucleotides • antisense • decoy dsDNA • decoy RNA • ribozymes DNAzymes • Si RNA • Oncolytic viruses • ONYX-15, ONYX-638 (r-adeno) • r-HSV • r-FSV • Implants of encapsulated cells • neurotrophic factor producer cell implants • hormone-producing cells a a a a a a

  34. 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. Fischer Cardiac ischaemia(Heart) Limb ischaemia(Hands, Feet) VEGF gene (vascular growth factor), plasmid, intramuscular VEGF gene (vascular growth factor), plasmid, intracardiac 1998 J. Isner 2000 J. Isner UNIFR Rusconi2003 '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

  35. 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 Gene Therapy in the clinic: Trials Wordldwide • Ergo • in spite of 13 year- research only less than 1% of the trials has reached phase III As of December 2002:632 registered protocols 3472 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

  36. 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

  37. 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 Gene Therapy Clinical and Preclinical Milestones 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

  38. UNIFR Rusconi2003 Two 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) • 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 • Although genes discovered in the 90ties: • no suitable vector • no satisfactory delivery method a a a a a a

  39. UNIFR Rusconi2003 The most feared potential side-effects of gene transfer • 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 • Most side effects are still related to the rather primitive state of the vectorology/delivery a a a a a a

  40. UNIFR Rusconi2003 Three (four) bitter lessons, but only one treatment-related death so far 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

  41. UNIFR Rusconi2003 Public perception problems • Negative perception of manipulative genetics • general aversion of genetic manipulation • fear of catastrophic scenarios • Confusion with other gene-based and non-gene-based technologies • stem cell technology • human cloning procedures • genetically modified food • Deception after excessive promises • hopes reinforced by media spectacularisation and over-simplification • deception after non-complied deadline a a a a a a

  42. UNIFR Rusconi2003 Other factors that have negatively influenced the public perception and progress of gene therapy • 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 • An explosive cocktail, just like for sports or arts,... • the field tends to degenerate as soon as huge amounts of money are involved and when the mass media become interested in it. a a a a a a

  43. high mood Low NFP37 UNIFR Rusconi2003 A. Fischer M. Kay Ups and Downs of Gene Therapy: a true roller coaster ride! 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

  44. UNIFR Rusconi2003 Genes, cells, tissue transplants...some people fear possible negative developments Amelioration instead of therapy? aa beauty woman.mov Too High-tech too expensive robot woman.mov2 Bioweapons? military biolabs1.mov a a a a a a

  45. V UNIFR Rusconi2003 Somatic Gene Therapy is facing fierce competition • 1. Cell Therapy (Stem cells (SC)) • identified in many tissues • cell transfer could be combined with gene transfer • there would be no anatomical barriers for gene transfer • Selection /amplification of desired transformants • Current limitations of SC • Lack of control on differentiation and trans-determination • Difficulties in complex organ-reconstruction • Future of SC: • Increasing number of SC types will be characterised • culturing conditions will be perfectioned • May replace in vivo gene transfer for treatment of chronic conditions? • 2. Breakthroughs from the small/medium molecules • STI571 (Glivec) • anti HER2 (Herceptin) • Si RNA? • ... • 3. Challengers from the biomechanics world • bone reconstruction • intelligent protheses (stents) • micropumps • artificial organs a a a a a a

  46. UNIFR Rusconi2003 Conclusions • Fundamentally • a gene encodes usually more than one function • 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 • Vectors and models • 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 • Clinically • 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

  47. UNIFR Rusconi2003 Perspectives • Fundamental level & vectorology • 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 becoime more realistic • Preclinically • 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 • Clinically • 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 a a a a a a

  48. UNIFR Rusconi2003 ...Thanks ! ECPM My collaborators at UNIFR Swiss National Research Foundation Thank you all for the attention, and... if you are too shy to ask send an e-mail to: sandro.rusconi@unifr.ch or visit: www.unifr.ch/nfp37 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 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

  50. UNIFR Rusconi2003 Discussion: The Paris' odyssey(see also www.unifr.ch/nfp37/adverse.html) 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. • October 2003 3 US and 3 EU trials on hold 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 • January 2003,27 US and 5 EU trials on hold a a a a a a

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