Osnove in razvoj tehnologije rekombinantne DNAZgodovinski pregled razvoja bioznanosti (biomedicina):

varnostni, etični in pravni vidiki

Radovan Komel

Medicinski center za molekularno biologijo, Medicinska fakulteta, Univerza v LjubljaniVrazov trg 2, 1000 Ljubljana

tel.: (01) 543 7662; fax: (01) 543 7641; e-naslov: radovan.komel@mf.uni-lj.si

Se opravičujem: samo del slik je opremljen s slovenskim, večji del pa z angleškim besedilom.

Ta PPT predstavitev spremlja besedilo članka R.Komel: “Ethical and safety issues with recent developments in genomics and biotechnology” (JOURNAL of BIOETHIC, 2012 – v tisku).

Pozor: Razložita tudi biološke posledice komplementarnega parjenja baz.

Restrikcijski encim

Restrikcijski encim ustvari fragmente DNA z “lepljivimi”komplementarnimi konci.

= značilno zaporedje 4-7 nukleotidov

DNA 1 DNA 2

Tehnologija rekombinantne DNA: povezovanje DNA iz različnih lokacij / izvorov.

Tehnologija rekombinantne DNA: molekulsko kloniranje s plazmidom = pomnoževanje fragmenta DNA (lahko tudi gena).

Izražanje nekega (pomnoženega) gena v gostiteljskih (npr. bakterijskih, kvasnih...) celicah uporablja biotehnologija za proizvodnjo velikih količin “rekombinantnih” proteinov.

Asilomar State Beach Conference Center, 1075:Konferenca o biološkem tveganju zaradi tehnologije r-DNA

- zadrževalni ukrepi morajo biti nujen del načrtovanja poskusa

- zadrževalni ukrepi morajo biti skladni z oceno tveganja

- različne ravni tveganja v poskusu potrebujejo različne ravni zadrževanja

- nujna je uporaba bioloških ovir, da preprečino širjenje rekombinantne DNA

- bakterijski gostitelji, ki ne preživijo v naravnem okolju

- neprenosljivi vektorji, ki lahko rastejo samo v izbranem gostitelju

- fizične ovire: npr. laminariji, omejen dostop, podpritisk v laboratoriju

- strogo upoštevanje dobre laboratorijske prakse

- prepoved kloniranje r-DNA iz zelo patogenih organizmov, kot tudi kloniranja DNA s toksinskimi geni

- nič poskusov v večjem merilu, v katerih je r-DNA, iz katere lahko nastanejo za življenje nevarni produkti

- nujno izobraževanje in usposabljanje osebja

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, 1980.

- Recombinant: first, 1984; commercially available,1990.

Recombinant Therapeutic Proteins: Delivering a $53 Billion Mature Market by 2010.

Global biopharmaceutical market expected to surpass US$ 167 Billion by 2015.

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 enzymemarket 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.

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 deliberaterelease 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

Integriranje tuje DNA v genom gostitelja = transgeneza.

“Transgen” je tuji vnešeni gen, ki se stabilno vgradi v genom gostitelja.

Gensko spremenjeni organizem je “transgenski organizem”.

Različne metode vnosa tuje DNA.

oz. vektor

Recombinant DNA and higher eukaryotesGenetic 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

Šaljivo obravnavanje transgeneze v javnosti: gensko spremenjeni organizmi.

Strašenje javnosti s posledicami transgeneze: gensko spremenjena hrana, transgenski človek.

Ali nas bodo res napadli...?

“Vesolje” polževih proteinov na “vesolju” proteinov slabo oprane salate ....

Za razliko od označevanja gensko spremenjene hrane, s kemikalijami obdelanih živil ne označujemo ...

Ustvarjanje novih pasem skozi zgodovino ... premeščanje celih nizov genov ?

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.

EU Legislation on Genetically Modified Organisms

•Directive 2001/18 on the deliberate release into the environment of geneticallymodified organisms.

•Directive on the Deliberate Release into the Environment of Genetically ModifiedOrganisms (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 geneticallymodified 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 modifiedorganisms.

Projekt Človeški genom, 1990

1990

2001

HGP: zgodovina določanja zgradbe (sekvenciranja) človeškega genoma.

Osnutek zaporedja človeškega genoma je bil objavljen leta 2001.

text.

Molecular Medicine:

• molecular basis of disease

•molecular daignostics

•gene therapy

•other therapeutical issues

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

•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?

Nastopilo je post-genomsko obdobje.

Cell as a system of simultaneous (qualitative and quantitative) presence of biological molecules and their communication (interplaying molecular events), under a studied physiological condition.

The world of “omics”:

• transcriptomics

• proteomics

• metabolomics

• interactomics / systems biology

FUNCTIONAL GENOMICS

“Normalno fiziološko stanje” “Bolezen”

N T

Funkcijska genomika: s prekrivanjem slik bomo lahko ugotovili razlike (lučke = “ izraženi geni/proteini”), ki so morda vzrok prehoda iz normalnega v patološko stanje.

Red = reference

Green = modified

transcriptome

Also:

• Proteomics

• Metabolomics

Plate-forme Puces-à-ADN Gif/Orsay

Uporaba podatkov iz transkriptomskih / proteomskih raziskav v molekulski diagnostiki.

cdc42

Iz transkriptomskih / proteomskih raziskav najdena proteina (zeleno) sta lahko tarči za načrtovanje biološkega zdravila.

Dolly, the sheep: 1996

These three little kittens were born to the world's first cloned cat. Two of the kittens take after their mother, while the third, left, has a gray coat like his father.

Theory

Fact

?

Mankind

• Uniques and diversity

• Curiosity and creativity

“Terapevtsko kloniranje.

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.

May 2010

PUBLIC DATABASES

INTERNET

INFORMATION DNA SYNTHESIS

ARTIFICIAL GENOME

nucelotide sequence databases

DNA synthesis platforms

What defines life, both "real" and artificial?

What would be the consequences of the use of new powerful post-genomic technologies on the human society and environment.

Constructive Intent Disruptive

No. ofpersons

Basic researcher

Garage bio-hacker

Under-resourrced researcher

Disappointed researcher

Bio-weapons developer

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!

Bodimo razumni in konstruktivni! Imamo samo en planet!