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Technical Remarks

UNIFR Rusconi 2002. Technical Remarks. This slide show was prepared with Power Point 98 for Macintosh therefore, 'thank' to the limited portability it may halt or improperly display on windows-based machines.

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Technical Remarks

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  1. UNIFR Rusconi2002 Technical Remarks This slide show was prepared with Power Point 98 for Macintoshtherefore, 'thank' to the limited portability it may halt or improperly display on windows-based machines. This is not due to our bad will but to the sloppiness of software suppliers who are apparently unable to produce a platform that is genuinely interchangeable in the third millenium ! This is an internet-streamlined version (without heavy graphics or movies). The movie clips listed below accompany the full slide show Please write to: sandro.rusconi@unifr.ch to get the full size document. sruSki2.mov genet walk on DNA.mov sport boxe 01.mov aa getting oldcomp2.mov molecular_therapycardio1.mov sport GIRO98.MOV sport football 01.mov sport maradona 01.mov sport motorbike 01.mov sport snowboard slalom.mov sport hunter jones.mov sport weigh women 2.mov sport sydney2000.mov sport weights advert.mov iceage.sub.mov The slide show willdisplay better if your machine has sufficient live memoryand a speedy processor S. Rusconi a a a a a a

  2. UNIFR Rusconi 2002 Sandro Rusconi 1972-75 Primary 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 (mol. bio.) 1994-todayProfessor Biochemistry UNI Fribourg 1996-today Director Swiss National Research Program 37 'Somatic Gene Therapy' 2001 Participant Swiss Natl. Res Program 50 'Endocrine disruptors' 2002 Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department 2002 President Union of Swiss Societies for Experimental Biology (USGEB) Bern, Nov 22, 2002 Swiss Olympic gene doping workshop Sports doping: Is there a realistic application for gene transfer? sruSki2.mov a a a a a a

  3. UNIFR Rusconi 2002 Schedule Basic understanding of 'genes': what is a gene, how many genes, molecular biology dogma genetic diseases, environmental factors, ageing Essential concepts on 'molecular medicine' & molecular doping:applications and problems, Techniques of gene transfer (Gene Therapy)problems and solutions, vectors, clinical achievements Gene-based doping applications, comparison with other doping, detection Conclusions plausibility table a a a a a a

  4. UNIFR Rusconi 2001 Genetics has been used since millennia,Molecular Biology, only since 30 years 100’000 b.C. Empirical genetics genet walk on DNA.mov 10’000 b.C. Biotechnology 2000 a.d. Molecular biology 2001 a.d, Genomics a a a a a a

  5. 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 2001 1 Gene -> 1 or more functions a a a a a a

  6. DNA RNA Protein GENE Transcription / translation FUNCTION RNA DNA UNIFR Rusconi 2002 What is in fact a gene?: a segment of DNA acting as a regulated machine for RNA production spacer regulatory coding spacer a a a a a a

  7. 2 mm 0.2mm 2m 0.02mm 0.001mm DNA RNA Protein UNIFR Rusconi 2002 1 Organism -> more than 105genetically-controlled Functions a a a a a a

  8. DNA Protein GENE FUNCTION(s) GENE OK FUNCTION OK GENE KO FUNCTION KO GENE transfer FUNCTION transfer UNIFR Rusconi 2002 Reductionistic molecular biology paradigm(gene defects and gene transfer) sport boxe 01.mov a a a a a a a a a a a a

  9. UNIFR Rusconi 2002 Gene amplification / manipulation techniques(genetic engineering, recombinant DNA) segments of genomic DNA can be specifically cut and isolated Science-grade material can be essentially prepared in your cellar ...not so clinical-grade material! isolated segment can be recombined with a plasmid vector nucleus & DNA.mov plasmid vector is transferred into bacteria where it can multiply isolated recombinant DNA can be further recombined to obtainthe final desired molecule Final molecule is transferred into cells or organisms a a a a a a a a a a a a

  10. 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 2002 The FOUR eras of molecular medicine genomeABC.mov a a a a a a

  11. 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 2002 The major disease of the 21st century: Ageing many treatments that slow down ageing or age-related degenerative diseases are also potential doping treatments aa getting oldcomp2.mov a a a a a a

  12. UNIFR Rusconi2002 Now, let's talk about Somatic Gene Therapy (somatic gene transfer) Chronic treatment Definition of GT: '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 molecular_therapycardio1.mov Loss-of-function Gain-of-function a a a a a a

  13. Remember! Efficiency Specificity Persistence Toxicity UNIFR Rusconi2002 Somatic gene therapy’s (gene transfer) four fundamental questions Efficiency of gene transfer Specificity of gene transfer Persistence of gene transfer Toxicity of gene transfer a a a a a a

  14. UNIFR Rusconi2001 Why 'somatic'? • Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise to a descendant organism i.e. somatic gene transfer is a treatment aiming at somatic cells and conse-quently does not lead to a hereditary transmission of the genetic alteration dolly1.mov2 • Somatic Cells: all the other cells of the body a a a a a a

  15. UNIFR Rusconi 2001 Pharmacological considerations Classical Drugs Protein Drugs Nucleic Acids O H • 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 • 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 • 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 O H O O O H O H O O O H O H O a a a a a a

  16. V UNIFR Rusconi 2001 THREE classes of physiological 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

  17. UNIFR Rusconi 2001 TWO classes of gene transfer vehicles: non-viral & viral Non-viral transfer (transfection) a Viral gene transfer (Infection) b Nuclear envelope barrier! see, Nature Biotech December 2001 a a a a a a

  18. UNIFR Rusconi 2001 Transfection with recombinant DNAVs Infection with recombinant viruses Transfection exposed to 106 particles/cell 12 hours Infection exposed to 3 particle/cell 30 min a a a a a a

  19. UNIFR Rusconi 2002 Quick 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

  20. 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 Efficiency +++ Specificity Persistence • Examples • OTC deficiency (clin, ---) • Cystic Fibrosis (clin, --- ) • Oncolytic viruses (clin, +++) Toxicity ++ UNIFR Rusconi 2002 Recombinant Adenoviruses a a a a a a

  21. 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. Efficiency Specificity Persistence • Examples • Hemophilia A (clin, animal, +++) • Gaucher (clin, animal, +++) • Brain Ischemia (animal, +++) • Cystic fibrosis (animal, +/-) Toxicity UNIFR Rusconi 2002 Recombinant AAV (adeno-associated-virus) a a a a a a

  22. 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 Efficiency Specificity Persistence • Examples • SCID (IL2R defect, Paris) (clin, +++) • Adenosine Deaminase deficiency (clin, +++!!!) • Parkinson (preclin, +++) • Anti cancer (clin +/-) Toxicity UUNIFR Rusconi 2002 Recombinant Retroviruses (includes HIV-based) a a a a a a

  23. 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 Efficiency Specificity Persistence • Examples • Critical limb Ischemia (clin, +++) • Cardiac Ischemia (clin, +/-) • Vaccination (clin, +/-) • Anti restenosis (preclin. +/-) Toxicity UNIFR Rusconi 2002 Naked / complexed DNA a a a a a a

  24. 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) Efficiency Specificity Persistence • Examples • Anti cancer (clin,preclin., +/-) • Restenosis (clin, +++) • Muscular Distrophy (animal, +++) Toxicity UNIFR Rusconi 2002 Oligo-nucleotides √ ! a a a a a a

  25. UNIFR Rusconi 2002 Recap: current limitations of popular gene transfer vectors Adenovirus - no persistence - limited packaging - toxicity - immunogenicity Biolistic bombardment or local direct injection - limited area Electroporation - limited organ access Retrovirus (incl. HIV) - limited package - random insertion - unstable genome Liposomes, gene correction & Co. - very inefficient transfer General - antibody response - limited packaging - gene silencing General - low transfer efficiency 1/10’000 of viruses’ in vivo Solutions: - synthetic viruses (“Virosomes”) Solutions: - improved liposomes with viral properties (“Virosomes”) a a a a a a

  26. UNIFR Rusconi2002 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 dolly1.mov2 • Random integration in genome-> insertional mutagenesis (-> cancer risk) a a a a a a

  27. trials patients 100 1500 cancer 80 1000 60 hered. 40 500 vasc. 20 Infect. 1990 1992 1994 1996 1998 2000 UNIFR Rusconi2002 Gene Therapy in the clinic: Trials Wordldwide As of Sept. 2002:599 registered protocols 4000 treated patients 86% phase I 13% phase II 1 % phase III 21% overall still pending or not yet Initiated ! www.wiley.com a a a a a a

  28. Isner, 1998 Anderson, 1990 Fischer, 2000 Dzau, 1999 Dickson, 2000 Kmiec, 1999 Aebischer, 2000 Kirn, 2001 Clinical trials with ONYX-015, what we learned? (Review) Bordignon, 2000 (ESGT, Stockholm)proves efficacy of the same protocol UNIFR Rusconi2002 Gene Therapy 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, ESGT congress, 7.10.2000 Stockholm 2000, correction Parkinson (animal model) P Aebischer, Science, Nov 2000 2001, ONYX oncolytic Viruses D Kirn (Gene Ther 8, p 89-98) a a a a a a

  29. UNIFR Rusconi2002 Gene Therapy Adverse events: NY 1995 // UPenn 1999 // Paris 2002 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 was interrupted and many others were put 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). dolly1.mov2 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. a a a a a a

  30. high mood Low NFP37 90 91 92 93 94 95 96 97 98 99 00 01 02 UNIFR Rusconi2002 Ups and Downs of Gene Therapy: a true roller coaster ride! A. Fischer M. Kay lentivectors in clinics? R. Crystal Adeno I V.Dzau C Bordignon J. Isner ADA AAV germline in mice? NIH Motulski report Adeno III Lentivectors in pre-clinic Paris Ergo: in spite of its respectable age, gene transfer is still in its infancy and still produces more controversies than clinical results J. Wilson J. Gelsinger a a a a a a

  31. + + + UNIFR Rusconi 2002 The THREE levels of doping Before thecompetition (anabolic enhancers) 'Molecular treatments Application of the know-how in molecular genetics to doping During the competition (perfomance enhancers) After the competition (repair enhancers) sport GIRO98.MOV sport football 01.mov a a a a a a

  32. UNIFR Rusconi2002 Which gene transfer approaches would be compatible with doping strategies • ex vivo, hematopoietic tissue:erythropoietin? • in vivo local (example muscle):metabolic enhancers, growth factors, muscular fiber changers • in vivo local (example joints):pain reducers, inflammation inhibitors, recovery and repair factors • in vivo systemic:anabolic factors, endocrine factors, pain killers a a a a a a

  33. UNIFR Rusconi2002 Which are the objective current limitations ingene-based doping strategies Viral gene transfer • immune problems • limited readministration • general toxicity, genotoxicity sport maradona 01.mov Nonviral gene transfer • generally inefficient • lack of persistence, requires readministration sport motorbike 01.mov Strategy-independent problems • laborious, not readily available • long term gene expression difficult to control • irreversible effects or permanent tagging sport snowboard slalom.mov a a a a a a

  34. UNIFR Rusconi2002 Which side effects could be feared ingene-based doping strategies Short -mid term • Autoimmunity • Hyperimmunity • Toxic shock sport hunter jones.mov Long term • Fibrosis • Cancer • Conventional effects of administered factors • Inaccessibility to future gene therapy interventions (immunity) Intrinsic to reckless application (probably the biggest danger) • malpractice (unsuitable vector/administration route) • non-clinical grade material(adventitious pathogens or allergens) • lack of follow-up a a a a a a

  35. UNIFR Rusconi2002 Which detection methods would be (or not) evisageable for gene-based doping strategies • Antibody detection (viral antigens or other epitopes) • recombinant-nucleic acids detection (PCR) • recombinant protein detection(MALDI-TOF / proteomics) sport weigh women 2.mov • Gene transfer may be anatomically difficult to detect (if locally administered) but leaves permanent genetic marking • the detection of nucleic acids cannot be performed in body fluids (except for systemically administered treatments) and might require specific tissue biopsy sport sydney2000.mov a a a a a a

  36. UNIFR Rusconi2002 Final side-by-side comparison:gene-based doping versus drug- or protein-based doping CategoryDrug/proteinGene-based Rapidity of effectsrapidslow Reversibilityrapidslow/none Ergo: The odds speak currently ratheragainst the adoption of gene-based doping, but this applies to common-sense clinical practice, and this aspect is not guaranteed in the doping field Dosagestraightforwarddifficult Complexity of treatm.simplecomplex Associated risksdependshigh Detectabilityarduous 'straightforward' a a a a a a

  37. UNIFR Rusconi2002 ...Thanks ! Swissolympics Our own project/goal may indeed appear very small and harmless... This does not necessarily apply to its consequences... My collaborators at UNIFR Swiss National Research Foundation iceage.sub.mov 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

  38. UNIFR Rusconi2002 discussion slides a a a a a a

  39. genetics behaviour environment UNIFR Rusconi 2002 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% cures from the genome?.mov Sum of incidences min - max (all defects) 32 - 83% a a a a a a

  40. UNIFR Rusconi 2002 The long way to drug/procedure registration is the principal cause of financial burden, but we cannot avoid it yearevent costs U$D 0Idea 0 2Cell culture assays 0.5 Mio 5Pre-clinical tests animal models 2 Mio 7Clinical phase I 5-20 patients verify side effects 6 Mio 10Clinical phase II 30-100 patients dosis escalation 12 Mio 15Clinical Phase III >300- 1000 patients multicentric double blind 80 Mio 16>>Registration /Availability This means: assuming 20% of new developments makes it to final registration, the average investment is 300-500 Mio U$D for each approved drug/procedure Molecular therapy is NO EXCEPTION ! a a a a a a

  41. genetics behaviour environment Muscle distrophy Familial Breast Cancer Lung Cancer Sporadic Breast Cancer Obesity Artherosclerosis Alzheimer Parkinson ’s Drug Abuse Homosexuality UNIFR Rusconi 2002 Not only the genome determines the health status... a a a a a a

  42. 100 nm L1 L1 L2 L2 capsid E E UUNIFR Rusconi 2002 Recap: what is a virus ? -> A superbly efficient replicating machine docking entry disassembly genome replication early genes exp replication late genes exp assembly standard viral genome Spread Etc... a a a a a a

  43. 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 Engineering of replication-defective, recombinant viruses (Principle) E Wild type genome Virions Packaging Packaging Packaging R-Virions a a a a a a

  44. ADA deficiency(Immunodeficiency) • ADA normal gene(enzyme) • 1990 F. Anderson, 2002 C. Bordignon • Cystic Fibrosis(Lung, Pancreas) • CFTR gene(chlorine transporter) • no significant resultsin spite of several trials • Haemophilia B(Blood) • Factor IX gene(blood clotting factor • 1999-2000 M. Kay and K. Horwitz • SCID(Immunodeficiency) • IL2R gene(gamma-C receptor) • 2000 A. Fischer • Cardiac ischaemia(Heart) • Limb ischaemia(Hands, Feet) • VEGF gene(vascular promoter) • VEGF gene(vascular promoter) • 2000 J. Isner • 1998 J. Isner UNIFR Rusconi2002 'Classical' GT models and strategies • Disease • transferred function • Clinical Results dolly1.mov2 a a a a a a

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