1.Biopharmaceutical

1. Biopharmaceuticals

Introduction

• Biopharmaceuticals are medical drugs produced using biotechnology.

• They are proteins (including antibodies), nucleic acids (DNA,RNA or antisense oligonucleotides) used for therapeutic or in vivo diagnostic purposes, and are produced by means other than direct extraction from a native (non-engineered) biological source.

• The first such substance approved for therapeutic use was biosynthetic 'human' insulin made via recombinant DNA technology.

• Sometimes referred to as rHI, under the trade name Humulin, was developed by Genentech, but licensed to Eli Lily and Company, who manufactured and marketed the product starting in 1982.

Biotherapeutic Agents

• Virtually all biotherapeutic agents in clinical use are biotech pharmaceuticals

• A biotech pharmaceutical is simply any medically useful drug whose manufacture involves microorganisms or substances that living organisms produce (e.g., enzymes).

• Most biotech pharmaceuticals are recombinant—that is, produced by genetic engineering

Biopharmaceutical Categories

• Biopharmaceuticals can be grouped into six categories.– Cytokines– Enzymes– Hormones– Clotting Factors– Vaccines– Monoclonal Antibodies

Cytokines

• Cytokines - Cytokines are hormone-like molecules that can control reactions between cells. They activate immune-system cells such as lymphocytes and macrophages.– Interferon - potent glycoprotein cytokine that act

against viruses and uncontrolled cell proliferation– Interleukins function as messengers for various

steps in the immune process. Ex. interleukin-2 (IL-2) stimulates T lymphocytes, a recombinant variant of IL-2, aldesleukin (Proleukin), for treating renal cell carcinoma. IL-3 stimulates bone marrow stem cells. IL-1 secreted by macrophages induces fever.

Cytokines

• Cytokines– Granulocyte-colony stimulating factor (G-CSF)

stimulates the bone marrow to produce neutrophils (antibacterial leukocytes), used for cancer treatments that are immunodepressants

– Granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulates the bone marrow to produce neutrophils and macrophages. For chemo and radio therapy that suppresses bone marrow function

Enzymes

• Enzymes – are complex proteins that cause a specific chemical change in other substances without being changed themselves.– Alteplase – dissolves blood clots– Dornase alfa – a recombinant DNAse I that digests

DNA in the mucous secretions in lungs– Imiglucerase. A recombinant glucocereborsidase for

Gaucher’s disease (bone destruction and enlargement of the liver and spleen)

Other Biopharmaceutical categories

• Hormones – chemicals that transfer information and instructions between cells in animals and plants. Ex. insulin, HGH.

• Clotting Factors – any factor in the blood that is essential for the blood to coagulate.

• Vaccines – microorganisms that can be used to stimulate resistance in a human to specific diseases. Ex. – Hepatitis B virus, Ebola virus

• Monoclonal antibodies – immortal cells created by fusion of a cancer cell with an antibiotic producing spleen cell.

Cloning Insulin

Human Growth Hormone

• Facts– Peak production during adolescence– 60 year old secretes 25% of a 20 year old– Anterior lobe of the pituitary gland– Primarily released in pulses during beginning phases of sleep– Converted in the liver into insulin-Like Growth Factor type I– Incidence: 1 out of 4,000 to 10,000 births


• Also known as somatotropin

• Most abundant hormone secreted by the pituitary gland

The Pituitary gland is a small, pea-sized gland that is located in the middle of your skull, just below the hypothalamus.

Pituitary Gland

• The pituitary gland has two lobes, anterior and posterior

• The anterior lobe also produces– Prolactin – breast milk– FSH, LS – sexual reproduction– Thyrotropin – thyroid function– Adrenocorticotropin - adrenal function – Growth Hormone - growth


• Usually the first hormone to be lost or reduced

• Can be congenital• Can be acquired

– Inflammation – Surgery – Radiation – Autoimmune disease – Tumors


• 191 amino acid (approx. 22 kdal)• Appears to be 5 variants of the gene on chromosome 17• Takes 80 cadavers for one year’s worth of therapy• Creutzfeldt -Jacob syndrome (odds – 1:500)

Protein Pharmaceuticals• Natural sources are often rare and expensive

– Difficult to keep up with demand– Hard to isolate product– Lead to immune reactions (diff. species)– Viral & pathogen contamination

• Most protein pharmaceuticals today are produced recombinantly– Cheaper, safer, abundant supply

Recombinant Methods

• Developed in 1970’s &1980’s

• Paul Berg (1973) restriction enzymes

• Herbert Boyer (1978) cloning human insulin into E. coli – Genentech

• Two general approaches– Expression in isolated cells– Expression in transgenic plants/animals

Six step process

• Isolation of gene of interest

• Introduction of gene to expression vector

• Transformation into host cells

• Growth of cells through fermentation

• Isolation & purification of protein

• Formulation of protein product

Cloning Process• Gene of interest is cut

out with restriction enzymes (RE)

• Host plasmid (circular chromosome) is cut with same REs

• Gene is inserted into plasmid and ligated with ligase

• New (engineered) plasmid inserted into bacterium (transform)

Cloning (Details)

Cloning (Details)

protein

Recombinant Protein Expression Systems

• Escherichia coli• Other bacteria• Pichia pastoris• Other yeast• Baculovirus• Animal cell culture• Plants• Sheep/cows/humans

Polyhedra

Expression System Selection

• Choice depends on size and character of protein– Large proteins (>100 kD)? Choose eukaryote

– Small proteins (<30 kD)? Choose prokaryote

– Glycosylation essential? Choose baculovirus or mammalian cell culture

– High yields, low cost? Choose E. coli

– Post-translational modifications essential? Choose yeast, baculovirus or other eukaryote

Which Vector?• Must be compatible with host cell system (prokaryotic vectors for

prokaryotic cells, eukaryotic vectors for eukaryotic cells)• Needs a good combination of

– strong promoters– ribosome binding sites– termination sequences– affinity tag or solubilization sequences– multi-enzyme restriction site

Plasmids and Vectors

• Circular pieces of DNA ranging in size from 1000 to 10,000 bases

• Able to independently replicate and typically code for 1-10 genes

• Often derived from bacterial “mini” chromosomes (used in bacterial sex)

• May exist as single copies or dozens of copies (often used to transfer antibiotic resistance)

Key Parts to a Vector

• Origin of replication (ORI) – DNA sequence for DNA polymerase to replicate the plasmid

• Selectable marker (Amp or Tet) – a gene, when expressed on plasmid will allow host cells to survive

• Inducible promoter – Short DNA sequence which enhances expression of adjacent gene

• Multi-cloning site (MCS) – Short DNA sequence that contains many restriction enzyme sites

A Generic Vector

Which Vector?• Promoters

– arabinose systems (pBAD), phage T7 (pET), Trc/Tac promoters, phage lambda PL or PR

• Tags– His6 for metal affinity chromatography (Ni)

– FLAG epitope tage DYKDDDDK– CBP- calmodulin binding peptide (26 residues)

– E-coil/K-coil tags (poly E35 or poly K35)

– c-myc epitope tag EQKLISEEDL– Glutathione-S-transferase (GST) tags– Celluluose binding domain (CBD) tags

Gene Introduction (Bacteria)

Bacterial Transformation

Bacterial Transformation

• Moves the plasmid into bacterial host• Essential to making the gene “actively” express the

protein inside the cell• 2 routes of transformation

– CaCl2 + cold shock

– Electroporation• Typical transformation rate is 1 in 10,000 cells (not very

efficient) for CaCl2, but 1 in 100 for electroporation

Electroporator

25 microfarads = 2500 V@ 200 ohms for 5 ms

Electroporation

• Seems to cause disruption in cell membrane

• Reconstitution of membrane leads to large pores which allow DNA molecules to enter

• Works for bacteria, yeast and animal cells

Bacterial Systems

• Grow quickly (8 hrs to produce protein)

• High yields (50-500 mg/L)

• Low cost of media (simple media constituents)

• Low fermentor costs

• Difficulty expressing large proteins (>50 kD)

• No glycosylation or signal peptide removal

• Eukaryotic proteins are sometimes toxic

• Can’t handle S-S rich proteins

Advantages Disadvantages

Cloning & Transforming in Yeast Cells

Pichia pastoris

Pichia Pastoris• Yeast are single celled eukaryotes• Behave like bacteria, but have key advantages

of eukaryotes• P. pastoris is a methylotrophic yeast that can

use methanol as its sole carbon source (using alcohol oxidase)

• Has a very strong promoter for the alcohol oxidase (AOX) gene (~30% of protein produced when induced)

Pichia Cloning

Pichia Pastoris Cloning

• Uses a special plasmid that works both in E. coli and Yeast

• Once gene of interest is inserted into this plasmid, it must be linearized (cut open so it isn’t circular)

• Double cross-over recombination event occurs to cause the gene of interest to insert directly into P. pastoris chromosome where the old AOX gene used to be

• Now gene of interest is under control of the powerful AOX promoter

Pichia Systems

• Grow quickly (8 hrs to produce protein)

• Very high yields (50-5000 mg/L)

• Low cost of media (simple media constituents)

• Low fermentor costs

• Can express large proteins (>50 kD)

• Glycosylation & signal peptide removal

• Has chaperonins to help fold “tough” prtns

• Can handle S-S rich proteins

Advantages More advantages

Baculovirus Expression• Baculoviruses are rod-shaped dsDNA viruses found mainly in insects. The most

common baculovirus used for expression studies is Autographa californica multiple nuclear polyhedrosis virus, which relies on the lepidopteran species Spodoptera frugiperda and Trichoplusia ni as host insects.

• AcMNPV particles surround themselves with a protective matrix consisting of the protein polyhedrin, which permits survival in the environment and efficient spread to new hosts.

• Under the control of the extremely strong promoter pPolh, polyhedrin is expressed at extremely high levels (up to 50% of all cellular protein) at the end of the baculovirus life cycle.

• The baculovirus expression system makes use of the fact that in cell culture a polyhedrin coat is not essential for virus propagation and thus heterologous proteins can be expressed under the control of the pPolh promoter.

• Autographica californica multiple nuclear polyhedrosis virus (Baculoviurs)• Virus commonly infects insects cells of the alfalfa looper (small beetle) or

armyworms (and their larvae)• Uses super-strong promoter from the polyhedron coat protein to enhance

expression of proteins while virus resides inside the insect cell

Baculovirus Expression

Baculovirus Expression

~12 days

Baculovirus Successes

• Alpha and beta interferon• Adenosine deaminase• Erythropoietin• Interleukin 2• Poliovirus proteins• Tissue plamsinogen activator (TPA)

Baculovirus Systems

• Grow very slowly (10-12 days for set-up)

• Cell culture is only sustainable for 4-5 days

• Set-up is time consuming, not as simple as yeast


• Correct glycosylation & signal peptide removal


• Very high yields, cheap

Disadvantages Advantages

Mammalian Expression Systems

Mammalian Cell-line Expression

• Sometimes required for difficult-to-express proteins or for “complete authenticity” (matching glycosylation and sequence)

• Cells are typically derived from the Chinese Hamster Ovary (CHO) cell line

• Vectors usually use SV-40 virus, vaccinia virus promoters and DHFR (dihydrofolate reductase) as the selectable marker gene

Mammalian Expression

• Gene initially cloned and plasmid propagated in bacterial cells

• Mammalian cells transformed by electroporation (with linear plasmid) and gene integrates (1 or more times) into random locations within different CHO chromosomes

• Multiple rounds of growth and selection using methotrexate to select for those cells with highest expression & integration of DHFR and the gene of interest

Methotrexate (MTX) Selection

Gene of interest DHFR

TransfectDfhr - cells

Grow inNucleosideFree medium

Culture aColony of cells

Grow in0.05 uM Mtx


Methotrexate (MTX) Selection

Grow in5.0 uM Mtx

Grow in0.25 uM Mtx



Foreign geneexpressed inhigh level inCHO cells

Mammalian Systems

• Selection takes time (weeks for set-up)

• Cell culture is only sustainable for limited period of time

• Set-up is very time consuming, costly, modest yields


• Correct glycosylation & signal peptide removal, generates authentic proteins


Disadvantages Advantages

Mammalian Cell Successes

• Factor IX

• Factor VIII

• Gamma interferon

• Interleukin 2

• Human growth hormone

• Tissue plamsinogen activator (TPA)

Conclusion

• Isolation of gene of interest

• Introduction of gene to expression vector

• Transformation into host cells

• Growth of cells through fermentation

• Isolation & purification of protein

• Formulation of protein product

What About the TermGenetic Engineering?

Genetic engineering involves:

Isolating genes Modifying genes so they function better Preparing genes to be inserted into a new species Developing transgenes

Genetic engineering is the basic tool set of biotechnology

What is a transgenic?

Transgene – the genetically engineered gene added to a species

Ex. – modified epsp synthase gene (encodes a protein that functions even when plant is treated with Roundup)

Transgenic – an organism containing a transgene introduced by technological (not breeding) methods

Ex. – Roundup Ready Crops

Concept Based on the Term Transgene

We can develop organisms that express a “novel” trait not normally found in the species

Why are transgenics important?

Extended shelf-life tomato (Flavr-Savr)

Herbicide resistant soybean (Roundup Ready)

Agriculture Transgenics On the Market

Source: USDA

Insect resistant cotton – Bt toxin kills the cotton boll worm• transgene = Bt protein

Insect resistant corn – Bt toxin kills the European corn borer• transgene = Bt protein

Normal Transgenic

Virus resistance - papaya resistant to papaya ringspot virus• transgene = virus coat protein

Source: Monsanto

Herbicide resistant crops Now: soybean, corn, canola Coming: sugarbeet, lettuce, strawberry alfalfa, potato, wheat (transgene = modified EPSP synthase or phosphinothricin-N-acetyltransferase

Biotech chymosin; the enzyme used to curdle milk products• transgene = genetically engineered enzyme

bST; bovin somatotropin; used to increasemilk production• transgene = genetically engineered enzyme

Source: Rent Mother Nature

Source: Chr. Hansen

Some Ag Biotech Products Are Discontinued

Poor Quality• FlavrSavr tomatoes (Calgene)

Negative Consumer Response• Tomato paste (Zeneca)

Negative Corporate Response• NewLeaf (Monsanto)

Universal Negative Publicity• StarLink corn (Aventis)

Next Generation of Ag Biotech Products

Source: Minnesota Microscopy Society

Golden Rice – increased Vitamin A content (but not without controversy)transgene = three pathway enzymes

Sunflower – white mold resistancetransgene = oxalate oxidase from wheat

Turfgrass – herbicide resistance; slower growing (= reduced mowing)

Bio Steel – spider silk expressed in goats; used to make soft-body bullet proof vests (Nexia)

Biotechnology is Not Just on the Farm

Disease Treatment

Diagnostics

Environmental Cleanup

Human Applications

Human Applications

• Pharmaceutical products New solutions to old problems • Disease diagnosis Determine what disease you have or may get

• Gene therapy Correcting disease by introducing a corrective gene

Biotechnology and Health

Product Use

Insulin Diabetes

Interferon Cancer

Interleukin Cancer

Human growth hormone Dwarfism

Neuroactive proteins Pain

The genes for these proteins are:

• Cloned• Inserted into bacteria• Product isolated using biofermentation

Environmental Applications

Bioremediation - cleanup contaminated sites; uses microbes designed to degrade the pollution

Indicator bacteria – contamination can be detected in the environment

Tooth decay – engineered Streptococcus mutans, the bacteria that destroys enamel

Future Health-related Biotech Products

Vaccines – herpes, hepatitis C, AIDS, malaria

Edible VaccinesTransgenic Plants Serving Human Health Needs

• Works like any vaccine • A transgenic plant with a pathogen protein gene is developed• Potato, banana, and tomato are targets• Humans eat the plant • The body produces antibodies against pathogen protein• Humans are “immunized” against the pathogen• Examples:

DiarrheaHepatitis BMeasles

A Popular Term We Need To Know

GMOs - Genetically modified organisms

• GMO - an organism that expresses traits that result from the introduction of foreign DNA

• Originally a term equivalent to transgenic organism

Important Plant Improvement Methods

• Breeding Crossing two individuals from the same species; produces a new, improved variety; not a biotechnology procedure

• Transformation Adding a gene from another species; the essential biotechnology procedure to produce transgenics

Source: USDA

Source: USDA

Wheat Rye

Triticale

X

Interspecific Cross

New species, but NOT biotechnology products

Mutagenesis

A useful procedure to produce a new trait

But the normal gene is modified

A transgene is not involved

The product of mutagenesis is not a GMO

ATTCGA

ATTGGA

SusceptibleNormalGene

ResistantMutantGene

MutagenesisTreatment

Mutagenesis Changes the DNA Sequence

The Roundup Ready Story

• Glyphosate is a broad-spectrum herbicide• Active ingredient in Roundup herbicide • Kills all plants it come in contact with• Inhibits a key enzyme (EPSP synthase) in an amino acid pathway

• Plants die because they lack the key amino acids

• A resistant EPSP synthase gene allows crops to survive spraying

+ Glyphosate

X

Roundup Sensitive Plants

X

X

Shikimic acid + Phosphoenol pyruvate

3-Enolpyruvyl shikimic acid-5-phosphate(EPSP)

Plant EPSP synthase

Aromaticamino acids

Without amino acids, plant dies

X

BacterialEPSP synthase

Shikimic acid + Phosphoenol pyruvate

3-enolpyruvyl shikimic acid-5-phosphate(EPSP)

Aromaticamino acids

Roundup Resistant Plants

+ Glyphosate

With amino acids, plant lives

RoundUp has no effect;enzyme is resistant to herbicide

The Golden Rice Story

• Vitamin A deficiency is a major health problem

• Causes blindness• Influences severity of diarrhea, measles

• >100 million children suffer from the problem

• For many countries, the infrastructure doesn’t existto deliver vitamin pills

• Improved vitamin A content in widely consumed cropsan attractive alternative

-Carotene Pathway Problem in Plants

IPP (Isopentynyl diphosphate)

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Problem:Rice lacks

these enzymes

NormalVitamin A

“Deficient”Rice

The Golden Rice Solution

IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

ξ-carotene desaturase

Daffodil gene

Single bacterial gene;performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin APathway

is completeand functional

GoldenRice

Introducing the Geneor

Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

Transformation Cassettes

Contains

1. Gene of interest

• The coding region and its controlling elements

2. Selectable marker

• Distinguishes transformed/untransformed plants

3. Insertion sequences• Aids Agrobacterium insertion

Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissue• Develop shoots• Root the shoots

Field test the plants

• Leaf, germinating seed, immature embryos

• Tissue must be capable of developing into normal plants

• Agrobacterium or gene gun

• Multiple sites, multiple years

• Transformation cassettes are developed in the lab

• They are then introduced into a plant

• Two major delivery methods

Delivering the Geneto the Plant

• Agrobacterium

• Gene GunTissue culturerequired to generatetransgenic plants

The Lab Steps

The Next Test Is The Field

Non- transgenics

Transgenics

Herbicide Resistance

Final Test of the TransgenicConsumer Acceptance

RoundUp Ready Corn

Before After

• In nature, plant cells often live in close association with certain bacteria, which may provide a convenient vehicle for introducing cloned DNA into plants.

• Agrobacterium tumefaciens, for example, attaches to the cells of dicotledonous plants and causes the formation of plant tumours known as galls.

• This bacterium introduces a circular DNA molecule called the Ti (tumour -inducing) plasmid into the plant cell in a manner similar to bacterial conjugation.

• The plasmid DNA then recombines with the plant DNA. Since the Ti plasmid has been isolated, new genes can be inserted into it using recombinant DNA techniques and the Ti genes causing tumours can be disrupted.

• The resulting recombinant plasmid can then transfer desired genes into plant cells.An especially useful characteristic of plants for transgenic studies is the ability of cultured plant cells to give rise to mature plants.

• Meristemativ (growing) cells from dissected plant tissue or cells within excised parts of a plant will grow in culture to form callus tissue, an undifferntiated lump of cells.

• Under the influence of plant growth hormones, different plant parts (roots, stems, and leaves) develop from the callus and eventually grow into whole fertile plants.

• When an agrobacterium containing a recombinant Ti plasmid infects a cultured plant cell, the newly incorporated foreign gene is carried into the plant genome.

• A. tumefaciens readily infects dicots (petunia, tobacco, carrot) but not monocots; reliable techniques for introducing genes into monocots are still being developed.

• Direct introduction of DNA by electroporation has been successful in rice plants, (which are monocots), and the future looks bright for the manipulation of other commercially important monocotyledonous crop plants.

• Also available for gene transfer experiments are cells of a tiny, rapidly growing member of the mustard family called Arabidopsis thaliana.

• This plant appears to be well suited to genetic analysis of a variety of developmental and physiological processes.

• It takes up little space, is easy to grow, and has a small genome, and genes defined by mutations can be cloned by positional cloning strategies.

• A transgenic animal is one that carries a foreign gene that has been deliberately inserted into its genome. The foreign gene is constructed using recombinant DNA methodology. In addition to a structural gene, the DNA usually includes other sequences to enable it

• to be incorporated into the DNA of the host and • to be expressed correctly by the cells of the host.• Transgenic sheep and goats have been produced that express

foreign proteins in their milk. • Transgenic chickens are now able to synthesize human

proteins in the "white" of the eggs. • These animals should eventually prove to be valuable sources

of proteins for human therapy.

http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/RecombinantDNA.html

• To date, there are three basic methods of producing transgenic animals:

• DNA microinjection • Retrovirus-mediated gene transfer

• Embryonic stem cell-mediated gene transfer

• 1. DNA Microinjection• The mouse was the first animal to undergo successful gene

transfer using DNA microinjection. This method involves:• transfer of a desired gene construct (of a single gene or a

combination of genes that are recombined and then cloned) from another member of the same species or from a different species into the pronucleus of a reproductive cell

• the manipulated cell, which first must be cultured in vitro (in a lab, not in a live animal) to develop to a specific embryonic phase, is then transferred to the recipient female

• 2. Retrovirus-Mediated Gene Transfer• The second method produces chimeras, altered animals with mixed

DNA.• A retrovirus is a virus that carries its genetic material in the form of

RNA rather than DNA. This method involves• retroviruses used as vectors to transfer genetic material into the

host cell, resulting in a chimera, an organism consisting of tissues or parts of diverse genetic constitution

• chimeras are inbred for as many as 20 generations until homozygous (carrying the desired transgene in every cell) transgenic offspring are born

• The method was successfully used in 1974 when a simian virus was inserted into mice embryos, resulting in mice carrying this DNA.

• 3. Embryonic Stem Cell-Mediated Gene Transfer• The presence of transgenes can be tested at the embryonic

state in this third method.• This method involves:• isolation of totipotent stem cells (stem cells that can develop

into any type of specialized cell) from embryos • the desired gene is inserted into these cells • cells containing the desired DNA are incorporated into the

host’s embryo, resulting in a chimeric animal • Unlike the other two methods, which require live transgenic

offspring to test for the presence of the desired transgene, this method allows testing for transgenes at the cell stage.

Medical Applications• Transplant organs may soon come from transgenic animals.• a) xenotransplantation

Patients die every year for lack of a replacement heart, liver, or kidney. For example, about 5,000 organs are needed each year in the United Kingdom alone.

• Transgenic pigs may provide the transplant organs needed to alleviate the shortfall.

• Currently, xenotransplantation is hampered by a pig protein that can cause donor rejection but research is underway to remove the pig protein and replace it with a human protein.

• Milk-producing transgenic animals are especially useful for medicines.

• b) nutritional supplements and pharmaceuticals Products such as insulin, growth hormone, and blood anti-clotting factors may soon be or have already been obtained from the milk of transgenic cows, sheep, or goats.

• Research is also underway to manufacture milk through transgenesis for treatment of debilitating diseases such as phenylketonuria (PKU), hereditary emphysema, and cystic fibrosis.

• In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk at 2.4 grams per litre. This transgenic milk is a more nutritionally balanced product than natural bovine milk and could be given to babies or the elderly with special nutritional or digestive needs. Rosie’s milk contains the human gene alpha-lactalbumin.

• A transgenic cow exists that produces a substance to help human red cells grow.

• c) human gene therapy Human gene therapy involves adding a normal copy of a gene (transgene) to the genome of a person carrying defective copies of the gene. The potential for treatments for the 5,000 named genetic diseases is huge and transgenic animals could play a role. For example, the A. I. Virtanen Institute in Finland produced a calf with a gene that makes the substance that promotes the growth of red cells in humans.

What are the ethical concerns surrounding transgenesis?

• These ethical issues, better served in their own article, include questions such as:

• Ethical concerns must be addressed as the technology grows, including the issue of lab animal welfare.

• Should there be universal protocols for transgenesis?• Should such protocols demand that only the most promising research be

permitted?• Is human welfare the only consideration? What about the welfare of other

life forms?• Should scientists focus on in vitro (cultured in a lab) transgenic methods

rather than, or before, using live animals to alleviate animal suffering?• Will transgenic animals radically change the direction of evolution, which

may result in drastic consequences for nature and humans alike?• Should patents be allowed on transgenic animals, which may hamper the

free exchange of scientific research?

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