Razvoj genomike in molekularne biologije oz. biotehnologije znanstveni, etični in pravni vidiki. Public attitude in biomedicine Molecular medicine

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1 Razvoj genomike in molekularne biologije oz. biotehnologije znanstveni, etični in pravni vidiki Public attitude in biomedicine Molecular medicine Radovan Komel Medical Center for Molecular Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000 Ljubljana, Slovenia Phone: ; Fax: ; radovan.komel@mf.uni-lj.si

2 Also: Explanation of biological consequences of complementary base-pairing. Pomembno: dokaz, da informacija izhaja iz materialne osnove (zgradbe DNA), se ohranja, preoblikuje in izraža ter prenaša iz generacije v generacijo (celic, osebkov).

3 Odkritje restrikcijskih endonukleaz: rezanje DNA na specifičnih mestih fragmentiranje DNA. Odkritje možnosti združevanja fragmentov DNA različnega porekla rekombinacija.

4 Recombinant DNA technology: cloning into a plasmid Genska tehnologija uporablja in združuje molekulsko kloniranje (vnos genske informacije preko vektorja (npr. plazmida) v enostavno gostiteljsko celico (npr. bakterija), tam njeno pomnožitev na več kopij) in celično kloniranje (delitev take, gensko spremenjene celice na veliko število potomcev - hčerinskih celic). Biotehnologija izrablja možnost izražanja pomnožene informacije v obliki velikih količin produktov (rekombinantni proteini).

5 Asilomar State Beach Conference Center, 1975: Conference on biohazards presented by recombinant DNA technology - containment should be made an essential consideration in the experimental design - containment should match the estimated risk as closely as possible - different levels of risk associated with the experiment, which would require different levels of containment - use of biological barriers to limit the spread of recombinant DNA - bacterial hosts unable to survive in natural environments - nontransmissible vectors able to grow in only specified hosts - physical containment: e.g. use of hoods, limited access, negative pressure laboratories - strict adherence to good microbiological practices - prohibition of cloning of recombinant DNAs from highly pathogenic or cloning of DNA containing toxin genes - no large scale experiments using recombinant DNAs that are able to make products potentially harmful to life - education and training of all personnel

6 Basic recomendations, guidelines and directives for the handling of recombinant DNA molecules NIH Guidelines for research involving DNA molecules (NIH Guidelines). EC directive 91/356/EEC on GMP (good manufacturing practice). EU Council Directive 90/219/EEC, of 23 April 1990 on the deliberate release into the environment of genetically modified organisms. EU Council Directive 90/220/EEC, of 23 April 1990 on the contained use of genetically modified organisms. OECD: Recombinant DNA Safety Considerations including a number of recommendations. National Legislations Ethical Considerations

7 Rekombinantna zdravila - primeri uspeha genske tehnologije: javnost jo v tem pogledu široko sprejema. Growth hormone: - Cadaver harvested: 70,000 80,000 anterior pituitary glands, G.B. - Recombinant: first, 1981; commercially available,1985. Insulin: - Animal-sourced: from 1922, possible allergic reactions. - Recombinant: first, 1977; commercially available,1982. Factor VIII: - Blood harvested: HIV contamination scandal, Recombinant: first, 1984; commercially available,1990. Recombinant Therapeutic Proteins: Delivering a $100 Billion Mature Market by Global biopharmaceutical market expected to surpass US$ 167 Billion by 2015.

8 Produkti genske tehnologije so prisotni tudi v drugih sferah vsakdanjika. Nekaj primerov: Lipases: - Fungal, bacterial: yogurt and cheese fermentation - Recombinant, modern applications: - baking, laundry detergents - biocatalysis - conversion of vegetable oil into fuel Most of actual industrial enzymes are recombinant. - Food and beverage industry: The world wide enzyme market is a $1.3 billion industry. - Pulp and paper industry. - Detergents: laundry processes; high-temperature industrial processes; bioremediation. - Textile manufacturing processes. The global market for industrial enzymes is estimated at $3.3 billion in 2010.

9 Transgeneza gensko spremenjeni organizmi (stabilna vgraditev genske informacije v genom): velika pričakovanja, vendar tudi odpor, zavračanje... Recombinant DNA and higher eukaryotes Genetic modification Transgenic plants and animals Medicine: -production of monoclonal antibodies -animal models for human diseases -tissue/organ transplants. Industry: - production of bioactive compounds in plants ( pharmaceutical crops ) and animals. Agriculture: - increasing growth rate - improving resistance to patogens and parasites - improving tolerance to extreme metorological conditions (heat, cold, dryness, salt) - genetically modified food

10 Šaljiva obravnava pričakovanj genske tehnologije.

11 Strašenje z gensko tehnologijo in njenimi posledicami.

12 Katerega od paradižnikov bomo raje izbrali za obrok: transgenskega ali biološkega? Kaj pa toksične naravne obrambne snovi v biološko pridelani rastlini? Se bomo v razpravi posvetili bolj nevarnosti, da nas napadejo z Marsa, kot znanstveno preverjenim argumentom o možnih (ne)posledicah genske tehnologije?

13 Ko polž leze čez salatni list, v vesolje stotisočih vrst proteinov salate spusti tudi sled nekaj svojih proteinov. Je to bolj tvegano za zdravje, kot vnos 1-2 polževih proteinov z gensko tehnologijo v salato?

14 Gensko spremenjena živila morajo biti označena, da se potrošnik lahko odloča (možne alergije na dodaten protein). Verjetno bi morala biti označena tudi za drugačne vrste tveganja?!

15 Človek je v zgodovini s križanjem in fizičnimi stresi premeščal cele nize genov, za razliko od (sicer bolj specifične in natančne) genske tehnologije, ki zaenkrat premešča zelo omejeno število genov).

16 Genetically modified crops, genetically modified food Public debate Mass media Different legislation Admission The Cartagena Protocol on Biosafety (CPB), an environmental treaty legally binding for its Parties, regulates transboundary movements of living modified organisms (LMOs). In view of the dynamics of the debate on GM foods, legislation is likely to continue to evolve.

17 EU Legislation on Genetically Modified Organisms Directive 2001/18 on the deliberate release into the environment of genetically modified organisms. Directive on the Deliberate Release into the Environment of Genetically Modified Organisms (90/220/EEC). Directive on the contained use of genetically modified micro-organisms (90/219). Regulation 258/97 on Novel Foods and Novel Food Ingredients. Regulation 1830/2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms. Guidelines on the new rules on genetically modified food and feed. Regulation 1829/2003 on genetically modified food and feed. Regulation 1946/2003 on transboundary movements of genetically modified organisms.

18 The Human Genome Project, 1990 Start projekta Človeški genom: kartiranje genoma in določitev zaporedja njegovih 3 milijarde nukleotidov.

19 1990 Desetletno tekmovanje med akademskim konzorcijem Človeški genom (James Watson, v nadaljevanju Francis Collins) in zasebno družbo Celera Genomics (Craig Venter). Je možno/dobro genom oz. genomske informacije patentirati? 2001

20 Istočasni, vendar ločeni objavi osnutka zaporedja človeškega genoma, 2001.

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22 Proičakovanja medicine po razkritju zgradbe človeškega genoma: Molecular Medicine: molecular basis of disease molecular daignostics gene therapy other therapeutical issues

23 Molecular medicine and ethical issues Reproductive Technology Embryo research Trait Genomes for Sale Social and ethical Genetic determinism - Genetic testing - Genetic screening - The privacy of genetic information

24 The use of somatic cell interventions to treat disease is generally regarded as ethically acceptable, because such interventions are consistent with the purpose of medicine, and because risks are localized to a single patient. Germline interventions involve more significant ethical concerns, because risks will extend across generations, magnifying the impact of unforeseen consequences. If a test is positive, what options, medical or otherwise, will be available to ameliorate the condition? Will the patient s relatives be informed that they too may be affected by the ailment? As more genetic tests become available, which ones should be universally administered to newborns? What role should parental consent play in determining when children are screened? What if the disease is not easily treatable, or can only be treated at great expense that parents may not want to incur? What if an ailment is late onset and untreatable, as is the case with Huntington s disease? What if a test can only determine a probability, not a certainty, that a child will develop a disease? What if a parent refuses to consent to a test that is clearly in their child s best interest? What if a parent decides to pursue a genetic enhancement that involves significant risks for a child, or that may limit a child s life prospects? Etična in pravna vprašanja medicine v zvezi z uporabo informacij in spoznanj človeškega genoma: genska diagnostika, gensko zdravljenje...

25 Po razkritju zgradbe človeškega genoma je nastopilo po-genomsko obdobje funkcijske genomike, ki celico obravnava kot sistem sočasnosti molekulske navzočnosti in molekulskih dogodkov oz. interakcij Red = modified Green = reference Functional Genomics transcriptome Also: Proteomics Metabolomics

26 Vsaka točka na bio-čipu (mikromreži) ima večje število pritrjenih molekul sonde (krajša enoverižna DNA, komplementarna nukleotidnemu zaporedju nekega gena oz. njegove mrna). Zaradi možnosti primerjave imajo vse točke na čipu enako število molekul specifičnih sond.

27 Delamo diferenčno: mrna iz celic v 1. fiziološkem stanju (npr. preiskovana bolezen) pretvorimo v ustrezne enoveružne cdna z uporabo fluorescentno označenih dntp ene barve, mrna iz celic v 2. fiziološkem stanju (npr. zdravo tkivo) pa z uporabo druge barve.

28 Povezava na spletno stran s prikazom principa ekspresijskega bio-čipa (mikromreže):

29 Diagnostična uporaba pristopov transkriptomike / proteomike: Z gručenjem informacij iz analize transkriptoma oz. proteoma z biočipi (horizontalne vrstice) in postavitvijo hierarhično postavljenih vprašanj (vertikalni stolpci) dobimo bioinformatsko sliko nekega fizoiološkega stanja. Odgovori na vprašanja so: da (zeleno), ne (rdeče), ne vem (sivo). Slika na desni prikazuje 3 histološko enake primere neke malignosti, vendar analiza transkriptoma/proteoma razkriva različne molekulske poti nastanka/razvoja bolezni morda indikacija za izbiro različnih pristopov zdravljenja... Plate-forme Puces-à-ADN Gif/Orsay

30 Transkriptomika/prote omika/metabolomika lahko razkrijejo molekule proteinov, ki so ključni igralci v neki metabolni oz. komunikacijski poti preučevanje njihove vloge in razvoj tarčnih zdravil. cdc42

31 In nastopilo je obdobje kloniranja... Dolly, the sheep: 1996

32 Šale in strašenje z možnostmi kloniranja...

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34 Razlika med naravno reprodukcijo in kloniranjem s prenosom celičnega jedra

35 These three little kittens were born to the world's first cloned cat. Two of the kittens take after their father, while the third, in front of her mother, has a gray-white coat unlike his father. Genes may contribute 50% and environment (during pregnancy) 50% to the phenotype. Theory Fact

36 ?

37 Mankind Uniques and diversity Curiosity and creativity

38 Reprodukcijsko kloniranje človeka ni dovoljeno (iz bioloških, varnostnih in etičnih razlogov), glede terapevtskega kloniranja pa so si stališča zelo nasprotujoča.

39 Human Genome, Medicine and Legislation U.S.: H.R. 493 Genetic Information Nondiscrimination Act. EC Convention for the protection of Human Rights and dignity of the human being with regard to the application of biology and medicine: Convention on Human Rights and Biomedicine. United Nations Declaration on Human Cloning. The Islamic Fiqh Academy issued a Fatwā stating that human cloning is prohibited by the faith.

40 May 2010 Craig Venter uvede tehnologijo za ustvarjanje umetnega življenja : nastopi obdobje sintezne biologije.

41 nucelotide sequence databases DNA synthesis platforms PUBLIC DATABASES INTERNET INFORMATION DNA SYNTHESIS ARTIFICIAL GENOME

42 What defines life, both "real" and artificial? What would be the consequences of the use of new powerful postgenomic technologies on the human society and environment.

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44 No. of persons Basic researcher Garage bio-hacker Under-resourrced researcher Disappointed researcher Bio-weapons developer Constructive Intent Disruptive Enable people to be constructive! Most people do not want to destroy the world! It is boring and you get to do it only once!

45 We have just one world and one life. Take care!

46 Cancer research and medicine Strategy for cancer biomarker discovery

47 Cancer Biomarker Names HER2 PSA Alfa-fetoprotein Alternative Names ErbB2, NEU, CD340 Human chorionic gonadotropin-β β-hcg Calcitonin Prostate-specific antigen, Kallikrein 3, KLK3 AFP, α-fetoprotein Thyrocalcitonin Cancer biomarkers in clinical use Cancer type Breast cancer Prostate cancer Germ-cell cancer Hepatoma cancer Testicular cancer Medullary thyroid cancer CA125 mucin 16, MUC16 Ovarian cancer CA 15-3 CA 19-9 Carcinoembryonic antigen Carcinoma Antigen 15-3 cancer antigen 19-9, sialylated Lewis (a) antigen CEA Breast cancer Clinical use based on ASCO and/or NACB recommendations Select patients for trastuzumab therapy Screening (with DRE) Diagnosis (with DRE) Diagnosis Differential diagnosis of NSGCT Staging Detecting recurrence Monitoring therapy Diagnosis Staging Detecting recurrence Monitoring therapy Diagnosis Monitoring therapy Prognosis Detecting recurrence Monitoring therapy Monitoring therapy Pancreatic cancer Monitoring therapy Colon cancer ER Estrogen receptor Breast cancer PgR PR, NR3C3 Breast cancer Lactate dehydrogenase LDH, LD Germ cell cancer Monitoring therapy Prognosis Detecting recurrence Screening for hepatic metastases Select patients for endocrine therapy Select patients for endocrine therapy Diagnosis Prognosis Detecting recurrence Monitoring therapy Thyroglobulin Tg Thyroid cancer Monitoring Abbreviations Recommended Quality Products HER2 / ErbB2 protein, antibody, ELISA kit & cdna clone KLK3 protein, antibody & cdna clone AFP protein, antibody & cdna clone Human HCG Gene cdna Clone DRE, digital rectal examination; ER, estrogen receptor; NACB, National Academy of Clinical Biochemistry; NSGCT, nonseminomatous germ cell tumor; PgR, progesterone receptor.

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49 Array comparative genome hybridization Genotyping microarrays Gene expression arrays Protein arrays Affimetrix Agilent Mycooarray CustomArray Arrayit...

50 CANCER IS A GENETICALLY COMPLEX DISEASE NO TWO CANCERS ARE IDENTICAL A key aspect in almost all cancer genomic analyses performed to date is that tumors are highly complex. No two tumors are identical and it is impossible to predict which genetic alterations are present in an individual cancer without direct sequence analysis of the tumor. Genome-wide analyses of the first 100 tumors from cancers of the breast, colon, pancreas, brain, and ovaries show that many of the identified mutations were novel and could not have been predicted without direct sequencing of the tumor tissue.

51 Comparison of Three Web-Based Microarray Tests Company Name Navigenics 23andMe decode Genetics Test Name Health Compass decodeme Genotyping platform Affymetrix Illumina Illumina # of SNPs interrogated One million 0.58 million plus some proprietary SNPs One million Date of service launched April 8, 2008 Nov. 19, 2007 Nov. 16, 2007 Sample type Saliva Saliva Buccal swab # of predicted diseases & physical traits 18(Alzheimer's disease, Breast cancer, Celiac disease, Colon cancer, Crohn's disease, Type 2 diabetes, Glaucoma, Graves' disease, Heart attack, Lupus, Macular degeneration, Multiple sclerosis, Obesity, Osteoarthritis, Prostate cancer, Psoriasis, Restless legs syndrome, Rheumatoid arthritis) 16 (Breast Cancer, Crohn's Disease, Heart Attack, Multiple Sclerosis, Obesity, Prostate Cancer, Restless Legs Syndrome, Rheumatoid Arthritis, Type 1 Diabetes, Type 2 Diabetes, Venous Thromboembolism, Alcohol Flush Reaction, Bitter Taste Perception, Earwax Type, Lactose Intolerance, and Muscle Performance) 20 (Age-related Macular Degeneration, Asthma, Alzheimer's Disease, Atrial Fibrillation, Breast Cancer, Celiac Disease, Colorectal Cancer, Exfoliation Glaucoma XFG, Crohn's Disease, Multiple Sclerosis, Myocardial Infarction, Obesity, Prostate Cancer, Psoriasis, Restless Legs, Rheumatoid Arthritis, Type 1 Diabetes, Type 2 Diabetes, eye color and hair color) Follow-up strategy Offer free genetic consultation Provide referrals to genetic counselors Provide referrals to genetic counselors Aim of the test Emphasis on disease prognosis Emphasis on providing personalized information Emphasis on providing personalized information Cost $2500 $999 $985

52 SLOVENIA Inhabitants: 2,058 milllion Capital: Ljubljana Area - Total 20,273 km2 (7,827 sq mi) - Water (%) 0.7[ GDP - Total $ billion - Per capita $28,195

53 Ministry for Education Ministry for Science Slovenian Research Agency Ministry for Health 600 employees 270 students/year 120 employees 80 students/year 4,700 employees 2,147 beds 961 employees 293 beds Local hospitals

54 13 institutes 26 pedagogic chairs 10 research centres University of Ljubljana 25,000 students

55 Gynecological Clinic Pediatric Clinic Institute of Oncology Faculty of Medicine, Ljubljana: Medical Centre for Molecular Biology (MCMB) CFGBC: Centre for Functional Genomics and Biochips

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