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Sandro Rusconi

UNIFR Rusconi 2003. Geneva 30.09.03 AISTS 'genes & sport' workshop. 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

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Sandro Rusconi

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  1. UNIFR Rusconi 2003 Geneva 30.09.03 AISTS 'genes & sport' workshop 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) 1987-94 Group leader, UNI Zuerich (mol. bio., PD) 1994-todayProfessor Biochemistry UNI Fribourg 1996-02 Director Swiss National Research Program 37 'Somatic Gene Therapy' 2001-todaySwiss Natl. Res. Program 50 'Endocrine disruptors' 2002-03 Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department 2002-05 President Union of Swiss Societies for Experimental Biology (USGEB) Gene transfer: limits and potential as doping vehicle movie clip deleted a a a a a a

  2. UNIFR Rusconi 2003 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

  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 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

  5. 2 mm 0.2mm 2m 0.02mm 0.001mm DNA RNA Protein UNIFR Rusconi 2003 1 Organism -> more than 105genetically-controlled Functions 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) movie clip deleted sport boxe 01.mov a a a a a a a a a a a a

  7. 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

  8. UNIFR Rusconi 2003 Gene amplification / manipulation techniques(genetic engineering, recombinant DNA) are simple 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

  9. UNIFR Rusconi 2003 The THREE missions of medicine, impact of molecular techniques Prevention + 'Molecular Medicine' Application of the know-how in molecular genetics to medicine Diagnosis + + Therapy 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 2003 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 2003 The major disease and medical challenge of the 21st century: Ageing movie clip deleted NB: 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 Rusconi2003 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 movie clip deleted Loss-of-function Gain-of-function a a a a a a

  13. UNIFR Rusconi2003 Why 'somatic'? • Germ Line Cells: the cells (and their precursors) that upon fertilisation can give rise to a descendant organism Ergo: somatic gene transfer is a post-natal treat-ment aiming at somatic cells and consequently does not lead to a hereditary transmission of the genetic alteration --> is NOT a GENETIC SELECTION! • Somatic Cells: all the other cells of the body a a a a a a

  14. Remember! Efficiency Specificity Persistence Toxicity UNIFR Rusconi2003 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

  15. 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

  16. V UNIFR Rusconi 2003 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 2003 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 2003 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 2003 List 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. UNIFR Rusconi 2003 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

  21. trials patients 100 1500 cancer 80 1000 60 hered. 40 500 vasc. 20 Infect. 1990 1992 1994 1996 1998 2000 UNIFR Rusconi2003 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

  22. 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 Rusconi2003 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

  23. 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) • side effects of newly acquired gene product • Contamination of germ line cells movie clip deleted • Random integration in genome-> insertional mutagenesis (-> cancer risk) • Ergo • many effects are due to 'primitiveness' of the today's protocols • for the moment side effects would (should) ethically limit GT to serious diseases without valid alternatives movie clip deleted a a a a a a

  24. UNIFR Rusconi2003 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. !! Most Recent Paris' Trial News www.unifr.ch/nfp37/adverse.html 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

  25. high mood Low UNIFR Rusconi2003 A. Fischer M. Kay Ups and Downs and current status 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

  26. + + + UNIFR Rusconi 2003 The THREE levels of doping Before thecompetition (anabolic enhancers) 'Molecular treatments Application of the know-how in molecular genetics to doping During the competition (performance enhancers) After the competition (repair enhancers) movie clip deleted movie clip deleted a a a a a a

  27. UNIFR Rusconi2003 Which gene transfer approaches would be compatible with doping strategies • ex vivo, hematopoietic tissue:pro hematopoietic(Epo receptor, oxygen transport...) • in vivo local (example muscle):metabolic enhancers, growth factors, muscular fiber changers, cardio-modulators(glucose/oxygen, MGF, anti-myostatin,...) • in vivo local (example joints):pain reducers, inflammation inhibitors, recovery and repair factors (anti-TNF, BMPs, ...) • in vivo systemic:anabolic enhancers, endocrine factors, pain killers, vascular controllers, (hormone metabolising enzymes, proenkephalins, ...) a a a a a a

  28. UNIFR Rusconi2003 Which would be the objectivecurrent limitationsin gene-based doping strategies Viral gene transfer • immune problems • limited readministration possibilities • general toxicity, genotoxicity movie clip deleted Nonviral gene transfer • generally inefficient • lack of persistence, requires readministration Strategy-independent problems • laborious, not readily available • long term gene expression difficult to control • irreversible effects or permanent tagging a a a a a a

  29. UNIFR Rusconi2003 Which side effects could be feared ingene-based doping strategies Short -mid term • Autoimmunity • Hyperimmunity • Toxic shock movie clip deleted sport hunter jones.mov Long term • Fibrosis • Cancer • conventional side- 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(pathogens or allergens) • lack of follow-up a a a a a a

  30. UNIFR Rusconi2003 Putative detection methods for gene-transfer-based doping strategies and their linked problems • Antibody detection (viral antigens) • r-nucleic acids detection (PCR) • recombinant protein / post-translational modification detection (MALDI-TOF ) movie clip deleted sport weigh women 2.mov • Anatomically difficult to detect(if locally administered) -> but leaves permanent genetic marking • Detection of nucleic acids cannot be performed in body fluids (except in early phase after systemic administration) -> might require specific tissue biopsy sport sydney2000.mov a a a a a a

  31. UNIFR Rusconi2003 Final side-by-side comparison:gene-based doping versus drug- or protein-based doping CategoryDrug/proteinGene-based Rapidity of effectsrapidslow Ergo: The odds would speak currently ratheragainst the adoption of gene-based doping, Reversibilityrapidslow Dosagestraightforwarddifficult but: this applies to common-sense clinical practice, and this aspect is not guaranteed in the doping field Complexity of treatm.simplecomplex Associated risksdependshigh and: ... there are several sporting disciplines where doping is not rigourously (or not at all) verified. Concealabilitypossibledifficult /impossible a a a a a a

  32. UNIFR Rusconi2003 Somatic gene transfer:conclusions • somatic gene transfer has been originally developed for the treatment of diseases (genetical or acquired) • must be distinguished from genetic selection • has the potential to be applied for pre- during- and post-performance enhancement • currently still experimental and not technically mature for applications in non-lethal conditions • has already raised the interest of doping field • major risk linked with premature application • single gene transfer for enhancement will create more problems than it could solve a a a a a a

  33. UNIFR Rusconi2003 ...Thanks, and let's hope that fair sports will continue to rise genuine emotions:yesterday, today and tomorrow! AISTS, MSA program My collaborators at UNIFR movie clips deleted Swiss National Research Foundation 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

  34. UNIFR Rusconi2002 a a a a a a

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