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Technical remarks UNIFR Rusconi 2002 this document has been prepared with Powerpoint version 98 for the macintosh as a consequence, some graphics may display inappropriately when run on a windows-based system to display the video clips you need to have them copied in the same disk support as the main file and an installed application compatible with quicktime player (min. version 3). If you intend to use this presentation or part thereof for public disclosure, please contact: [email protected] iceage.sub.mov aa zoom-inplanetsDNA.mov aa secret bioweaponlab.mov aa robotwoman.mov aa beautywoman.mov aa balance-world.mov aa damocles.mov bb DNArepairModel.mov bb DNAerror+replcells.mov affy_tech.mov aa drugsmanufact.mov sequencing.swf Timeline.swf bb icelandic pedigree.mov aa getting oldcomp2.mov aa genetomato.mov bb pedigree iceland.mov genet walk on DNA.mov intro.swf sruSki2.mov UNIFR Sandro Rusconi Rusconi 2002 1972-75 1975-79 1979-82 1982-84 1984-86 1987-91 1994-today 1996-today 2001 2002 2002 Primary school teacher (Locarno, Switzerland) Graduation in Biology UNI Zuerich, Switzerland PhD curriculum UNI Zuerich, molecular biology Research assistant UNI Zuerich Postdoc UCSF, K Yamamoto, (San Francisco) Principal Investigator, UNI Zuerich (mol. bio.) Professor Biochemistry UNI Fribourg Director Swiss National Research Program 37 'Somatic Gene Therapy' Participant Swiss Natl. Res Program 50 'Endocrine disruptors' Sabbatical, Tufts Med. School Boston and Univ. Milano, Pharmacology Department President Union of Swiss Societies for Bormio, Experimental Biology (USGEB) March 2002 Milano, Nov 29, 2002 Scienze Farmacologiche Progetto Genoma Umano Analisi del genoma umano e applicazioni per la medicina: impatto a corto, medio e lungo termine Schedule Basic understanding of 'genes and genomes': what is a gene, how many genes, molecular biology dogma genetic diseases, environmental factors, Essential concepts on 'molecular medicine' applications and problems, ageing as the major disease Genomics and other 'omics' essential technologies Applications and Impacts diagnosis, prevention, therapy Conclusions concerns, plausibile developments UNIFR Rusconi 2002 Did I talk about ... 'Future' ... ? UNIFR Rusconi 2002 There are many different ways to guess the future, not all of them being equally reliable... 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 UNIFR Rusconi 2001 UNIFR 1 Gene -> 1 or more functions Rusconi 2002 DNA RNA(s) Protein(s) Transcription / translation Gene expression GENE 2-5 FUNCTIONS 100 ’000 genes (50 ’000 genes?) >300 ’000 functions (>150 ’000 functions) UNIFR 1 Organism -> more than 105 genetically-controlled Functions Rusconi 2002 2 mm 2m 0.2mm 0.02mm 0.001mm DNA RNA Protein But...how many genes exactly? The debate is still hot UNIFR Rusconi 2002 Human genome consortium 30'000 identified Celera 30'000 identified HGC x Celera -> 45'000 (only 15'000 overlap) How many gene products are we still missing? Over 150'00 unique EST cDNAs have been identified My own personal experience: 50% of genes of my research interest yet unannotated What is in fact a gene?: a segment of DNA acting as a regulated machine for RNA production DNA RNA(s) Protein(s) GENE Transcription / translation FUNCTION(s) protein(s) RNA(s) DNA spacer regulatory coding spacer UNIFR Rusconi 2002 Reductionistic molecular biology paradigm (gene defects and gene transfer) DNA Protein GENE FUNCTION(s) GENE OK FUNCTION(s) OK GENE KO FUNCTION(s) KO GENE transfer FUNCTION(s) transfer UNIFR Rusconi 2002 Gene amplification / manipulation techniques (genetic engineering, recombinant DNA) segments of genomic DNA can be specifically cut and isolated Basic manipulations can be essentially isolated segment can be recombined with a plasmid vector performed in your house cellar additional sophistications: plasmid vector is transferred into bacteria where it can multiply - Polymerase chain reaction (PCR) - customDNA synthetic oligonucleotides isolated recombinant can be further recombined to obtain - labeling and hybridisation techniques the final desired molecule - custom sequencing Final molecule is transferred intogene cells or organisms - pre-formulated transfer cocktails - recombinant viral vectors UNIFR Rusconi 2002 UNIFR Rusconi 2002 The THREE missions of medicine Prevention + 'Molecular Medicine' Diagnosis Application of the know-how in molecular genetics to medicine + + Therapy UNIFR The FOUR eras of molecular medicine Rusconi 2002 Eighties Genes as probes Nineties Genes as factories Y2K Genes as drugs 1 2 3 4 5 ok ** ok ** ** 50 10 3000 80 85 90 95 99 1000technologies Y2K+n Post-genomic improvements of former 80 85 90 95 00 UNIFR Rusconi 2002 100% 10 1 E2/E Ergo: Modern medicine faces a major challenge: M 4 understanding and treatment E3/E4 of age-related conditions 20 40 60 80 E4/E4 80 70 60 1900 2000 20 40 60 50 1900 100 Alzheimer’s free % Life expectancy (CH) cancer incidence The major disease of the 21st century: Ageing 1920 1940 1960 1980 199 1900 80 2000 UNIFR Not only the genome determines the health status... Rusconi 2002 genetics Muscle distrophy Familial Breast Cancer Sporadic Breast Cancer Lung Cancer Obesity Artherosclerosis Alzheimer Parkinson ’s Drug Abuse Homosexuality behaviour environment Genomics: the importance of population homogeneity for functiuonal-locus detection UNIFR Rusconi 2002 Fact Several genome mining companies are analysing genetically coherent ethnic groups: example icelandic population Example Millenium (owned by Roche) has apparently already identified over twenty genetic loci linked to delayed ageing Problem The major question: 'who owns which fraction of the patent rights'? UNIFR The gen(OME) (the ensemble of genes) and other characters of the 'omics' saga TCACCATGCGT Rusconi 2002 CCTAGG Structural genomics ACGGATCA AAAGTA TTTAGAACAGGT Functional g. 1: (transcriptomics) Functional g. 2: (Proteomics) you said...comics? Functomics Morphomics further ??? glycomics? spliceomics metabolomics lipidomics Genomics milestones The adventure started quite long time ago, but took speed only recently UNIFR Rusconi 2002 Part 1: structural genomics... UNIFR Rusconi 2002 Structural Genomics Determination of genome sequence Determination of genome variability Determination of genomic evolution virus, bacteria, yeast, plants, mammals intra-species polymorphism, inter-species polymorphism Structural genomics aims and techniques Automated, high throughput fluorescent sequencing (size-dependent time) micro-array-based resequencing (size independent time) single nucleotide polymorphism detection techniques ultra-microscope-based sequencing (SNP) direct polymerase output sequencing Relevant criteria: precision (new sequencing) rapidity & convenience (re-sequencing) annotation of sequence electronic storage/retrieval UNIFR Rusconi 2002 Structural genomics: current impact on research UNIFR Rusconi 2002 Good: Paradigmatic shift: from gene hunters to function hunters Cross comparison: human, mouse, drosophila, C elegans, yeast, Arabidopsis Rapid contextualisation of newly identified sequences Availability of cloned genomic segments Easy design of analytical or preparative PCR amplicons In silico search for paralogues and homologues and gene families Rapid assessment of inter-species conserved elements Excellent source of working hypotheses about functionally relevant sequences Bad: Limited access to Celera and other privately-owned project data Limited a priori-gene recognition mistakes or mis-annotation can slow down some specific projects too many teams turn to genomics without proper knowledge Structural genomics current impact on clinics UNIFR Rusconi 2002 Good: More thorough assessment of cancer-linked predispositions Discovery of genes linked to neuro-degenerative conditions microsatellite polymorphism markers chromosomal painting Identification of age-linked gene loci amelioration and speeding up of molecular diagnostics Bad: possible funds withdrawal from other productive research too much faith combined with too much determinism could lead to major initial failures UNIFR Molecular diagnostics: three levels Rusconi 2002 Prenatal hereditary disorders severe predispositions Real time (monitoring) pathogens profile Immune status cancer status Pre-symptomatic Predispositions to: cancer autoimmune d. degenerative d. behavioural d. pharmacogenetics UNIFR Molecular diagnosis I: prenatal Rusconi 2002 Hematological defects - Thalassemias - Hemophilias Conventional Metabolic disorders most of these assays are - Phenylketonuria - Ornithine TC already available, but are currently adopted only when there is a familial Immunodeficiencies - ADA history - SCID Neurological defects - fragile X - Ataxia Genomically-driven Muscolo-skeletalcould defects improve and extend diagnosis - muscular dystrophy by rapid re-sequencing technologies - osteogenesis imperfecta that detect subtle mutations Cardiovascular defects ... even if they are spontaneous -> mass screening UNIFR Impact on molecular diagnosis II: pre-symptomatic Rusconi 2002 Predispositions to: Convenional - colon many of these diseases can be - breast efficiently treated if precociously Autoimmune diseases - asthma diagnosed (currentlky limited to - rheumatoid A. familial cases) Degenerative diseases Cancer ? - Alzheimer - Parkinson Genomically-driven: higher predictive value and massBehavioural diseases - Drug addiction screening through molecular Pharmaco-sensitivity - hyper reactivity structural and functional genomic - toxicity analyses - hypo reactivity Molecular diagnosis III: 'real time' molecular diagnosis UNIFR Rusconi 2002 better monitoring of: Conventional pathogens' profile many of these conditions can be - differentiation efficiently treated only if correctly - antibiotica resistance - virulence monitored and distinguished - biofilm (example antibiotic resistance) Immune status - antigens load Genomically-driven - antibody diversity more rapid and precise verification cancer follow-up of the steady-state status of the - clonal status disease through molecular profiling - tumor markers (example leukemias) - evolution - MRD -> better prognosis / treatment Structural genomics mid/long-term impact UNIFR Rusconi 2002 Mid term: Differential diagnosis of neoplastic conditions Complete panel of pathogen's genomes Bedside, precise analysis of infectious diseases (Pocket analysers) Angelo Nicolin (this meeting) the genome and new pharmacological therapies Long term: New anti-microbial drugs through bacterial genomics Identification of novel drug targets Understanding of single nucleotide polymorphism Comprehension of polygenic diseases Part 2: Functional genomics... UNIFR Rusconi 2002 Functional Genomics Collection of expressed cDNA Measurement of mRNA expression differentials Determination of differential Protein profiles Knock-out knock-in conditional KO of genes EST collections Transcriptomics Proteomics functomics Conventional, 'brute force' genomics (analysis of steady-state RNA) stimulus UNIFR Rusconi 2002 No stimulus Uridine several hours, or days RNA1 RNA2 Very complex mixture of: transcriptional + post-tr. effects primary + secondary events functional genomics (display or array) Label cDNA1 cDNA2 UNIFR Molecular diagnosis : microarrays Rusconi 2001 mg tissue Extract RNA Label fluorescently Hybridise to microarray Computer-assisted inspection UNIFR Principle of microarrays Rusconi 2002 differential analyses: SideAlternative view Top view SAGE Silicon display Differential mRNA Nylon Subtractive cDNA cloning Glass Micro-spotting baits collection criteria Relevant probes / surface (dots/mm2) Hybridize apparatuses, time) Costs (materials, labelled mixture Sensitivity (low/high expressors) NO HYB Selectivity (gene families, alternative splicing etc) A>B Representativity Rinse/detect A<B A=B Principle of proteomics Tissues/cells from different status Cell/ tissue lysis 2D gel electrophoresis separation Already known proteins: Spot identification / intensity Novel proteins/modificatins: Spot elution / microsequencing UNIFR Rusconi 2002 Functional genomics milestones UNIFR Rusconi 2002 1975 Britten and Davidson hybridisation kinetics mRNA-genomic DNA 1980 transgenic mice 1985 Subtractive cDNA libraries (tissue specific genes) 1998 knockout transgenics 1990 mRNA differential display 1995 oligonucleotide microarrays 1999 MALDI TOF, proteomics 2000 wide commercialisation of: arrays, dedicated bait filters, spotters, readers, custom analyses transgenic custom companies 2002 proteomic arrays with antibodies Functional genomics: current impact on research UNIFR Rusconi 2002 Good: Financial availability of microarray services & tools Possibility to tackle functional gene patterns alterations Novel hypotheses on primary and subsequent responders (infections, stresses, stimuli, drugs, development) Bad: too deterministic interpretation blocks understanding too many research teams join in the 'fashion field' insufficient attention and understanding on the mechanistic aspects (real time kinetics) Functional genomics current impact on clinics UNIFR Rusconi 2002 clinical experimental leukemia profiling early clinical assessment of pharmacogenomics principles strong interest of pharmaceutical companies increasing interest of practicting clinicians UNIFR Impact on prevention: vaccination Rusconi 2002 Recent & prospected Conventional Vaccines a posteriori identification of antigens Hepatitis B preparation of vaccine by - 1995 inactivated pathogen or single recombinant gene fermentation AIDS - 2008 ? Cancers - 2005-2010 ? Malaria - 2010 ? Alzheimer - 2010 ? Massimo Di Nicola (this meeting) immunotherapy of cancer Genomically driven a priori or custom identification of vaccines vaccine cocktails with recombinant DNA Impact on drug administration: Principles in pharmacogenetics variant Side-E Effects desired dosis Good Acceptable Unacceptable UNIFR Rusconi 2002 Individualised treatment through genomic profiling - Dosing - Side effects - Hyper sensitivity -Diego HypoFornasari sensitivity (this meeting) -Indicated / contraindicated pharmacogenetics - Drugs combination Preventive approaches - functional food, vaccination - behavioural medical coaching UNIFR Impact on Therapy I: Biopharmaceuticals Rusconi 2002 Isolate gene of interest (GOI) Conventional disease -> gene -> product single product treatments Redesign GOI Transform redesigned GOI Into micro-organisms Production through large-scale Fermentation processes Genomically driven product 1990 -> gene ->disease 1980 2000 multiple product treatments improvements in gene therapy Impact on Therapy II: improvements on GENE THERAPY? IMPROVED REGULATION OF TRANSGENES Discovery of new promoters / enhancers / LCR / etc Construction of therapeutical mini-chromosomes SPECIFICALLY INTEGRATING TRANSGENES Identify target sites for integrases Identify target sites for triple-stranded inhibitors PROVIDING NEW MOLECULAR TARGETS Clarify disease-causing pathways Use of gene combination cocktails UNIFR Rusconi 2001 Functional genomics mid/long term influence UNIFR Rusconi 2002 Mid term gene patterns triggered by basic signal transduction gene patterns involved in major pathologies gene patterns in pathogenic micro-organisms knock-outs of all known genetic function fundamental genetic response to nutrients/drugs Long term Gaetano Finocchiaro (this meeting) making sense of polymorphic response to nutrients and nervous system therapy drugs clearer picture on complex pathologies (cancer, alzheimer, parkinson, rheumatoid arthritis, atherosclerosis) understanding of genetic background/modifiers follow-up / prediction of genetic drift of microbial pathogens (eg influenza) identification of new emerging infectious pathogens Genomics: public understanding and public concern Major current public opinion issues different concerns in different cultural backgrounds general fear of 'gene manipulation' technology concerns about unsolicited genetic practices (screening) confusion of genetics/ reproductive technology privacy / data protection (insurances, employers) More hypothetical but still plausible issues eugenic practices novel epidemics through artificial gene shuttling ethnically-targeted poisons novel forms of behavioural screening/control UNIFR Rusconi 2002 Genes, cells, transplants, ... some may ask: «where are we going»? UNIFR Rusconi2 2002 Amelioration instead of therapy? Too sophisticated too expensive? Bioweapons? Genomics conclusion UNIFR Rusconi 2002 Facts The genomics era is a logical consequence of gene technology progress There is a good basic understanding of involved molecules There is a wealth of sequence data However: There is a deluge of functional data (transcriptomics) We still dont know how many genes/genome The post- genomics era has already started We still dont know how many functions/gene The first impact has been at the research level Clinical impact is currently minor There are social / ethical / legal concerns Nevertheless: Genomics will keep advancing exponentially there will be applications in diagnosis within few years There may be improvements of therapeutical applications within about a decade ...Thanks ! UNIFR Rusconi 2002 University of Milano CEND Our personal project/goal may indeed appear very small and harmless... My collaborators at UNIFR This does not necessarily apply to its consequences... Swiss National Research Foundation Thank you all for the attention, and... if you are too shy to ask send an e-mail to: [email protected] or visit: www.unifr.ch/nfp37