biopharming training

8/2/2019 Biopharming Training

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Concept of Biopharming

Knowledge on structure and function of cellularmacromolecules

Isolation and characterization of genes, cloning a

gene and studying its structure and expression throughrecombinant DNA (rDNA) technology

rDNA technology in biological research

rDNA technology in pharmaceutical industry


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The concept of biopharming is not new.

Common medicines, such as codeine, morphine, bulk laxatives, and the anticancerdrugs such as taxol and vincristine have long been purified from plants.

But biopharmings great promise lies in using genetic modification i.e., techniques tomake wild (nontransformed) plants to do drastic new things.

Biopharming offers tremendous advantages over traditional methods for producingpharmaceuticals.

Great potential for reducing the costs of production.

The energy for product synthesis comes from the sun, and the primary raw materialsare water and carbon dioxide.

To expand production, it is much easier to plant a few additional hectares than tobuild a new bricks-and-mortar manufacturing facility.

Vaccines produced in this way will be designed to be heat-stable so that norefrigeration chain from manufacturer to patient will be required. This would have agreat application in developing countries, especially in the tropics and throughoutAsia and Africa.


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Use of Microbes Earlier days microbes were well exploited among which bacteria are

highly exploited.

Eg. human growth hormone and insulin.

Prior to the advent of genetic engineering, human growth hormonewas produced from pituitary glands removed from cadavers. Itresulted in recipients contracting CreutzfeldJakob syndrome.

The recombinant approach resulted in unlimited supplies of safematerial. This safety aspect has been extended to various clottingfactors that were originally isolated from blood but now carry the riskof HIV infection.

As the methods for cloning genes became more and more

sophisticated, an increasing number of lymphokines and cytokineswere identified and significant amounts of them produced for the firsttime.


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Use of microbes for production of recombinant therapeutic proteins hasseveral problems

The foreign gene may contain sequences that act astermination signals.

The codon usage of the gene may not be ideal for translation

in bacterial system (Codon bias).

Lack of post translational modification and correct folding ofthe human recombinant proteins in microbial system.

Degradation of recombinant proteins since it is somewayrecognized as foreign protein in bacteria.


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Selective examples of recombinant proteins and their applications


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Transgenic animals and plants as Bioreactors

Recombinant-protein synthesis in animal cells has a number ofadvantages over microbial expression systems, the most importantof which is the authentic post-translational modifications that areperformed in animal cells.

However, large scale culture of animal cells is expensive.

The production of growth hormone in the serum of transgenic miceprovided the first evidence that recombinant proteins could beproduced, continuously, in the body fluids of animals.

Secretion of recombinant proteins in mouse milk was reported. This

was achieved by joining the transgene to a mammary-specificpromoter, such as that from the casein gene.

The first proteins produced in this way were sheep -lactoglobulinand human tissue-plasminogen activator (tPA).


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Selective examples of therapeutic compounds produced usinganimals as bioreactors


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Plants as bioreactors

Plants are a useful alternative to animals for recombinant-

protein production because they are inexpensive

Therefore, there is much interest in using plants as productionsystems for the synthesis of recombinant proteins and otherspeciality chemicals.

There is some concern that therapeutic molecules producedin animal expression systems could be contaminated withsmall quantities of endogenous viruses or prions, a risk factorthat is absent from plants.

Furthermore, plants carry out very similar post-translationalmodification reactions to animal cells, with only minordifferences in glycosylation patterns. Thus plants are quitesuitable for the production of recombinant human proteins fortherapeutic use.


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Selective examples of recombinant human therapeutic proteinsexpressed in plants


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The first such report was the expression of human growth hormone,as a fusion with the AgrobacteriumNopaline synthase enzyme, intransgenic tobacco and sunflower.

Tobacco has been the most frequently used host for recombinant-protein expression although edible crops, such as rice, are nowbecoming popular, since recombinant proteins produced in suchcrops could in principle be administered orally without purification.

The expression of human antibodies in plants has particular

relevance in this context, because the consumption of plant materialcontaining recombinant antibodies could provide passive immunity(i.e. immunity brought about without stimulating the host immunesystem).

Antibody production in plants was first demonstrated by Hiatt and

During team, who expressed full-size immunoglobulins in tobaccoleaves. Since then, many different types of antibody have beenexpressed in plants, predominantly tobacco, including full-sizeimmunoglobulins, Fab fragments and single-chain Fv fragments(scFvs).


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A fully humanized antibody against herpes simplex virus-2 (HSV-2) has been expressed in soybean.

Even secretory IgA (sIgA) antibodies, which have fourseparate polypeptide components, have beensuccessfully expressed in transgenic tobacco plants.

Plants producing recombinant sIgA against the oralpathogen Streptococcus mutanshave been generated,and these plant-derived antibodies (plantibodies) haverecently been commercially produced as the drug

CaroRxTM, marketed by Planet Biotechnology Inc.


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Isolation of gene sequence

Codon optimization andConstruction of Recombinant vector

Transformation

Plant/Animal/Microbes

High throughput

Expression ofCloned genes

Separation and purificationof medicinal protein


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Microbial production of Pharmaceutical compounds

Human disorders due to the absence or malfunction of a protein

normally synthesized in the body.

Treatment by supplying the patient with the correct version of theprotein, but for this to be possible the relevant protein must beavailable in relatively large amounts.

Obtaining sufficient quantities will be a major problem unlessdonated blood can be used as the source in some cases.

Animal proteins are used whenever these are effective, but thereare not many disorders that can be treated with animal proteins andthere is always the possibility of side effects such as an allergenicresponse.


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Microbial production of Insulin

Insulin, synthesized by the -cells of the islets of Langerhans in the

pancreas, controls the level of glucose in the blood.

Deficiency leads to Diabetes mellitus.

Treatment with insulin injections and supplementing the limited amount ofhormone synthesized by the patients pancreas.

Traditionally obtained from the pancreas of pigs and cows slaughtered formeat production.

Although animal insulin is generally satisfactory, problems may arise in itsuse to treat human diabetes.

slight differences between the animal and human proteins may lead to sideeffects in some patients.

the purification procedures are difficult and potentially dangerouscontaminants can not always be completely removed

l Z A l cZ B gene


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LacpromoterlacZ A gene

LacpromoterlacZ B gene

Vector carrying the

Artificial A and B genes

met

galactosidasesegment

A chain

galactosidasesegment

met

B chain

AB

TransformedE. colisynthesize

A and B fusion proteins

Purification of A and B chains

Attach by disulphide bridges

Synthesis of insulin protein

Cyanogen bromide

Cleaved fusion proteins Cleaved fusion proteins

BA


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Synthesis of human growth hormone in E. coli

Somatostatin and somatotropin

Somatostatin - somatotropin release-inhibiting factor (SRIF) expressed in the central and peripheral nervous systems, the gut, and other

organs also inhibit the release of thyroid-stimulating hormone; prolactin; insulin; and

glucagon besides acting as a neurotransmitter and neuromodulator.

Agromegaly (uncontrolled bone growth) and dwarfism.

Somatostatin was the first human protein to be synthesized in E. coli.

Somatostatin - very short protein, only 14 amino acids in length, it wasideally suited for artificial gene synthesis.

Strategy - insertion of the artificial gene into a lacZ reading frame of thepBR 322 vector, synthesis of a fusion protein and cleavage with cyanogensbromide.


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Lacpromoter

lacZ Artificial somatostatin gene

galactosidase

segment

met

Somatostatin fusion

protein

Transformation intoE. coli

Cyanogen bromide

Cleaved somatostatin

Production of recombinant somatostatin


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Somatotropin

Somatotropin -191 amino acids in length, equivalent to almost 600 bp,

Combination of artificial gene synthesis and cDNA cloning was used toobtain a Somatotropin-producing E. colistrain.

mRNA was obtained from the pituitarygland and a cDNA library wasprepared.

The Somatotropin cDNA turned out to have a unique site for the restrictionendonucleases HaeIII, which cuts the gene into two segments.

The longer segment, consisting of codons 24 to 191, was retained for use inconstruction of the recombinant plasmid.

The smaller segment was replaced by an artificial DNA molecule thatreproduced the start of the Somatotropin gene and provided the correctsignals for translation in E. coli.

The modified gene was then ligated into an expression vector carrying thelacpromoter.


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Production of recombinant somatotropin cDNA fragmentCodons 0 24 191

HaeIII

0 24 24 191

Discard Retain

Synthetic leadersequence

Expression of somatotropin

LacpromoterlacZ somatotropin gene

E. colitransformationSomatotropin is

synthesized


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Recombinant Factor VIII

Central role in blood clotting.

Recombinant pharmaceutical protein produced in eukaryotic cells

Haemophilia

Treatment - injection of purified Factor VIII protein, obtained from humanblood provided by donors.

Purification is a complex procedure and the treatment is very expensive

Difficult to remove the virus particles that are present in the blood. Hepatitis and AIDS can and have been passed on to haemophiliacs via

Factor VIII injections.

Recombinant factor VIII, free from contamination problems, would besignificant achievement for biotechnology.


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The factor VIII gene is very large,

Over 186 kb in length, and is split into 26 exons and 25 introns.

The mRNA codes for a large polypeptide (2351 amino acids) whichundergoes a complex series of post-translational processing events,eventually resulting in a dimeric protein consisting of a largesubunit, derived from the upstream region of the initial polypeptideand a small subunit from the downstream segment.

The two subunit contain a total of 17 disulphide bonds and a numberof glycosylated sites.

It is not possible to synthesize an active version in E. coli.

Most attempts made on mammalian cells.


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First, entire cDNA was cloned in hamster cells, but yields of protein weredisappointingly low.

Because of the failure in post-translational events, (do not convert theentire initial product into an active form limiting the overall yield).

As an alternative, two separate fragments from the cDNA were used,one fragment coding for the large subunit polypeptide, the second forthe small subunit.

Each cDNA fragment was ligated into an expression vector,downstream of the Ag promoter(a hybrid between the chicken -actinand rabbit -globin sequences) and upstream of a polyadenylationsignal from SV40 virus.

The plasmid was introduced into a hamster cell line and recombinant

protein obtained.

The yields were over ten times greater than those from cells containingthe complete cDNA and the resulting factor VIII protein wasindistinguishable in terms of function from the native form


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Tissue Plasminogen Activator (tPA)

naturally occurring protease enzyme, helps to dissolveblood clots inside a blood vessel

Boon for patients suffer from thrombosis

1st pharmaceutical product to be produced bymammalian cell culture

Majority of the natural deaths due to blockade ofcerebral/coronary artery (thrombus)


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Production of recombinant tPASynthesize cDNA molecule for tPA

Attached to synthetic plasmid

Introduced into mammalian cells

Cultured and tPA producing cells were selected by using methotrexate to the medium

tPA producing cells were transferred to fermenter

tPA secreted into culture medium is isolated for therapeutic purpose


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Synthesis of other recombinant human proteins

Interferon and interleukins - cancer therapy. Serum albumin, are more easily obtained, but are

needed in such large quantities that production inmicroorganisms is still a more attractive option.

Erythropoietin hormone synthesized by the kidneystimulate the stem cells of bone marrow to producemature erythrocytes


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Interferon

Antiviral substance

First line of defense against viral attacks

Glycoprotein in nature

Containing a group of > 20 substances with

mol. Wt of 20000 - 30000 daltons

Interferon

Interferon

Interferon


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rDNA derived therapeutic agents (approved by FDA) with tradenames and their applications in humans

rDNA product Trade name Application/uses

Insulin Humulin Diabetes

Growth hormone Protropin/Humatrope Pituitary dwarfism

Hepatitis Bvaccine Recombinax HB/Engerix B Hepatitis B

Tissue

Plasminogen

Activator

(tPA)

Activase Myocardial infarction

Factor VIII Kogenate/Recombinate Hemophilia

DNase Pulmozyme Cystic fibrosis

Erythropoietin Epogen Severe anaemia with kidney

damage


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Some otherrDNA derived therapeutic agents approvedby USFDA

rDNA product Application/uses

Coagulation factor VIII Hemophilia A

Coagulation factor IX Christmas disease (Hemophilia B)

Interferon Leukemia

Interferon Multiple sclerosis

Interferon Chronic granulomatous disease

Interleukin 2 Renal cell carcinoma (Kidney cancer)

Interleukin 10 Thrombocytopenia (few platelets inblood)


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Restriction endonucleases

> 500 different restriction endonucleases

Synthesized by a wide range of microorganisms and for eachorganism, a detailed fermentation protocol, has to bedeveloped and optimized

To avoid having to maintain a large number of differentmicroorganisms, stock a very wide range of mediumcomponents, design several different fermenters and spendan inordinate amount of time developing optimal growthconditions for a large number of different organisms,researchers often clone restriction enzyme gene into E.coli.

Because it is easy to standardize the conditions and E. colicells grow rapidly to high cell densities and can be engineeredto significantly over express the target restriction enzymes.


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However, host organism is dramatically affected

by the production or presence of a heterologousprotein.

Over expression of heterologous protein maydrain the host organism of important metabolic

resources and this may affect its growth orsometimes it may be lethal to the host.

Eg. There is a possibility to digest the host DNA

by the heterologous restriction enzyme unless aprotection mechanism is present.


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Microbial system has also been used to synthesizeseveral industrially important

low molecular weight molecules L-ascorbic acid (Vitamin C)

indigo

amino acids

antibiotics and

high molecular weight molecules biopolymers (Xanthan gum)

melanin

rubber

polyhydroxyalkanoates etc.


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Transgenic animals

Gene transfer to animal cells has been practiced for thepast 40 years.

Techniques are available for the introduction of DNA intomany different cell types

Animal cells - advantageous for the production ofrecombinant animal proteins (authentic post-translational modifications) that are not carried out by

bacterial cells and fungi.Cell cultures - commercial scale to synthesize products


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Intense research - efficient vector systems andtransformation methods for animal cells.

Baculovirus expression system - in insects.

More recently, introduction of DNA into animal cells in

vivoto treat disease (in vivogene therapy).

Viral gene-delivery vectors are favoured for therapeuticapplications because of their efficiency, but safetyconcerns have prompted research into alternative DNA-mediated transfer procedures.

G


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Gene-transfer strategies

(1) Direct DNA transfer - the physical introduction of foreign

DNA directly into the cell. microinjection - in cultured cells bombardment with tiny DNA-coated metal particles - for

cells in vivo.

(2) Transfection - chemical and physical, which can be

used to persuade cells to take up DNA from theirsurroundings.

(3) Transduction: Packaging the DNA inside an animalvirus

Transformation can be transient or stable, depending onhow long the foreign DNA persists in the cell.


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Proteins with therapeutic and industrial value have been produce(not commercialized) in the milk of transgenic animals

Protein Animal Use

Antithrombin III Goat Reduce the amount of blood needed in somesurgeries

Factor VIII and IX Goat, Pig,Sheep

Treatment of Hemophila

CFTR (Cystic

FibrosisTransmembrane

conductance

regulator)

Sheep Treatment of Cystic Fibrosis

Lactoferrin Cow Natural antibiotic and used in coronary surgery

Alpha 1 anti trypsin Sheep Treatment of Cystic Fibrosis and emphysema

Lysostaphin Cow Antibacterial compound that prevents mastitis incows

Spider silk protein Goat Production of ultra strong and light weight

industrial materials


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Transgenic animals:

Animals which have been genetically engineered tocontain one or more genes from an exogenoussource.

Transgenes are integrated into the genome.

Transgenes can be transmitted throughgermlineto progeny.

First transgenic animal produced = Founder Animal


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Mouse animal of choice for transgenic expt.

Easily handled and researcher friendly

Transgenic mice contributed Understanding of

Mol. Biol

Genetics Immunology

Cancer studies

Animal models for human genetic diseases


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Methods for introducing foreign gene/Transgenic mice production

Retroviral vector

Microinjection

ES cell method


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Retroviral vector

Transfer small pieces of DNA (8kb)

Not suitable for large DNA

Risk of losing regulatory sequences

Risk of retroviral contamination

A commercial product is to be synthesizedby the transgenic organism or thetransgenic organism to be used as food, itis absolutely necessary that there be noretroviral contamination.

Retroviral vector method


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Cleavage stage embryos (eight-cell stage)

infected with defective

retrovirus carrying a transgeneImplanted females (foster mothers)

Transgenic pups

Matings are carried out to determine which pups have the

transgene in their germ line cells.

Transgenic lines can be established from these foundertransgenic animals


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Microinjection method


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Microinjection method

(Super ovulated female) Young virgin female mice (4-5 weeks)FSH (pregnant mares serum)

2 days latter human chorionic gonadotropin

Produce 30 35 eggs

Mated with males and sacrificed on the following day

Fert. Eggs removed from the fallopian tubesmicroinjection needle

DNA inserted into male pronucleus of fert. Eggs

Eggs with transgene kept overnight in incubator to develop 2 cell stage

Implanted in foster mother (Pseudomouse pregnant female mouse mated with avasectomised male)

3 weeks after implantation

Transgenic pups

PCR/Southern blot hybridisation


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Limitations of microinjection method

Low efficiency (3 5 % success rate)

Foreign DNA randomly integrates into the host genome

Many pieces of DNA get incorporated at single site

Transgene may not be expressed / over expressed /under expressed and affect normal physiology

Time consuming

Costly

Labour intensive

E b i St C ll M th d


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Embryonic Stem Cell Method Introduction of foreign DNA into ES cell by

electroporation/micro injection

Cells from the blastocyst stage of a developing mouseembryo can proliferate in cell culture and have thecapability of differentiating into all other cell types-including germ line cells-after they are reintroduced into

another blastocyst embryo (Pluripotency).

Pluripotency in the broad sense refers to "having morethan one potential outcome".

Pluripotent stem cells can give rise to any fetal or adultcell type.

Embryonic stem cells (ES cells) are harvested from theinner cell mass (ICM) of mouse blastocysts.


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1. Make your DNA Using recombinant DNA methods, DNA containing the structural gene,

vector DNA, Promoter and enhancer sequences to enable the gene tobe expressed by host cells

2. Transform ES cells in culture Expose the cultured cells to the DNA so that some will incorporate it.

3. Inject these cells into the inner cell mass (ICM) of mouse blastocysts

4. Embryo transfer(Implantation) Prepare a pseudopregnant mouse (to make uterus receptive). Transfer the embryos into her uterus. develop into healthy pups (no more than one-third will).

5. Test her offspring

Remove a small piece of tissue from the tail and examine its DNA for thedesired gene.

6. Establish a transgenic strain Mate two heterozygous mice and screen their offspring for the 1:4 that will

be homozygous for the transgene.

Mating these will found the transgenic strain.


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Transgenesis in large animals

More difficult than with miceLess number of eggsTechnical difficulties in handlingLong gestational period

Early expts. not satisfactory

Transgenic sheep overproducing growth hormone susceptible toinfection, infertile and tend to die at early stage

Due to ineffective control of gene regulation

Resistance to diseases, enhancing the milk production, production ofcommercial and pharmaceutical compounds

T i ttl


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Transgenic cattle Mammary gland of the dairy cattle- ideal bioreactor

Transgenic cow over expressed K - casein transgene milk withhigher content of casein

Lactase transgene lactose free milk lactose intolerant people

Resistance to viral, bacterial and parasitic diseases

Attempts inherited immunological protection through transgenesis

Introduction of genes that code for heavy and light chains ofmonoclonal antibodies has met with some success

In vivoimmunization ideal for disease protection, which involvesthe insertion of a transgene for an antibody that specifically binds toan antigen


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Mature oozytes are collected from cows and fertilized withsemen collected from a bull in vitro.

Fertilized oozytes are centrifuged to settle the yolk at onepole of the oozytes

Foreign gene is microinjected into male pronucleus in theoozytes

Oozytes grown in vitrotill blastocyst stage

Implanted in uterus of foster mother

T i h


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Transgenic sheep

Mostly involve development of mammary glands asbioreactors for the production of proteins forpharmaceutical use. Eg. 1-antitrypsin, Factor IX

1-antitrypsin is fused with -lactoglobin promoter

microinjected into male pronucleus of fertilized egg.

1-35 g of 1-antitrypsin per litre of milk

Gene for factor IX

Isolated and purified from milk to treat haemophilia

Transgenic sheep


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Transgenic sheep

Keratin wool protein, highly cross linked disulfide bridges

Cysteine is required in large quantities - quality wool

Cysteine supply inadequate

Microbes harbouring in the rumen utilize it and release in the form ofsulfide

Transgenic sheep containing bacterial genes for synthesis ofcysteine

Two enzymes , synthesized by the transgenes trap the H2S andliberate in the intestine to produce the cysteine

Good supply of cysteine to the sheep improves the quality andquantity of wool


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Transgenic goats

Transgenic goats with tPA gene producestissue Plasminogen activator in milk

Transgenic pigs


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Transgenic pigs

Gene for Factor VIII introduced into zygote of pig by microinjection

Zygote implanted in uterus of a sow

Factor VIII in milk

For haemophilia treatment


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Human haemoglobins (globulin gene)

Human haemoglobins can be separated from pig haemoglobins byanalytical techniques

Haemoglobin O2 carrying protein of RBC can be used as a substitute inblood transfusion expts.

Haemoglobin can be stored for longer time than whole blood

No problem of contamination compared to whole blood

Free haemoglobins can not transport O2 as likehaemoglobins of RBC

Naked haemoglobin is easily degraded and breakdown products causedamage to kidney

Contamination by pig viruses and other compounds cause allergic reactions

Xenotransplantation


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Xenotransplantation

Transplantation of animal organs in human system

Human organs such as liver, pancreas, kidney and lungs greatdemand for transplantation surgery

Shortage can be overcome by developing them in mammal

Pig favourite animal for harvesting human organs

But human body produced antibodies against pig organs reject thetransplantsHyperacute rejection

Imutran Company (USA) produced transgenic pig (Astrid) bymicroinjecting genes for human immune system into malepronucleus of the zygote

Transgenic rabbits


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Transgenic rabbits

Interleukin-2 gene along with -casein promoter

Interleukin-2 is produced in milk

To treat cancers

Transgenic rabbits with human growth hormone genegrow faster and produce more meat with in short

duration

Transgenic chickens


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Transgenic chickens

Complicated

Fertilization several sperms enter the ovum instead of one(contrast to mammals)

Identification of male pronuclei that will fuse with female pronuclei isquite difficult

ES cells have not been identified in chickens The blastoderm cells can be removed from a donor chicken and are

transfected with transgenes Modified blastoderm cells reintroduced into subgerminal space of

irradiated blastoderm of freshly laid eggs Transgenic lines are produced Transgenesis for low fat and cholesterol, high protein containing

eggs Resistance to viral and bacterial diseases , Production of

pharmaceutical proteins Transgenic chickens resistant to Avian Leukosis virus (ALV)

Transgenic animals as bioreactors for the productionf th ti t i


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of therapeutic proteins

Transgenic

animalProtein product Biological importance

Cow Lactoferrin To overcome iron deficiency anaemias and alsoposssses antibacterial activity

Cow Interferon Resistance against viral infections

Sheep 1 antitrypsin Treatment of emphysema in lungs(Promotes gas exchange in lungs)

Goat CFTR To treat CF (promotes transport of ions)

Goat tPA To treat myocardial infarctions (to dissolve blood clots)

Goat Antithrombin III Regulates blood clotting

Rabbits glucosidase To treat Pompes disease (A genetic disorder

characterized by block in glycogen degradation)Mouse Urokinase to dissolve blood clots

Mouse Immunoglobulins Nhances immunity


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Conventional vaccines Purified antigen vaccines Recombinant vaccines

Vaccines

Live vaccines Inactivated pathogen Recombinant proteins/Sub unit vaccines

DNA Vaccines

Whole protein molecule Polypeptide

Con entional accines


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Conventional vaccines

consist of whole pathogenic organisms (bacterial / viral vaccines), which

may be either killed or live (attenuated) (live vaccines) (most viral vaccines)

Highly effective and easy to produce at low cost.

Limitations:

In many cases live vaccine have to be used, killed pathogen vaccines are

ineffective

Live vaccines are generally based on cultured animal cells and expensive tissueculture set up is essential

Live vaccines are heat labile

Risk of disease development due to occasional presence of active virus particle orreversion to virulence

Purified antigen vaccines


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Based on purified antigens isolated from concerned pathogens

Since they do not contain the organism, risk of pathogenicity isavoided

Limitations:

Cost is higher due to steps involved in purification and vaccinepreparation

Many of isolated antigens are poorly immunogenic

Successful examples of such vaccines are mostly from bacteria

Eg. Vaccines based on polysaccharide antigens from bacterial cell wallcapsules of Neisseria meningitisand Streptococcus pneumoniae

Many bacteria produce exotoxins, which are highly immunogenic. Butthese toxins produce toxic effects and it may decrease with storage dueto heat and chemicals. Fortunately most exotoxins that have lost theirtoxicity retain their immunogenecity; they are called toxoids and areeffectively used as effective vaccines.

Eg. Toxoids of pathogens causing tetanus, diphtheria etc.


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Recombinant vaccines

Contains either a protein or a gene encoding aprotein of a pathogen origin that is immunogenic

and critical to pathogen function; the vaccine isproduced using recombinant DNA technology.

Vaccines based on recombinant proteins are

called as sub unit vaccines.


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Steps involved:

Identification of protein that is immunogenic andcritical for the pathogen

Gene encoding for the protein is identifiedisolated

Gene is inserted into a expression vector andintroduced in to suitable host where it produceslarge quantity

Protein isolated and purified from the host cells. It is used for the preparation of vaccine.

Comparison of different biological systems for

th ti t i d ti


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therapeutic protein production

Production of therapeutic proteins in plants


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Production of therapeutic proteins in plants

Until recently pharmaceuticals used for treatment of diseases

largely based on the production of relatively small organisms. Molecules synthesis by microbes or by organic chemistry

antibiotics, analgescis, hormones and others

Proteins are large molecules composed of long chains of subunits

called amino acids. Structure and functionality of given protein is determined by its

sequence of amino acids, which, in turn, determines its three

dimensional confrontation / structure.

Internal bonds (S and H bonds) among the amino acids give theproteins its final shape and form.


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Complex proteins undergo further processing such as

phosphorylation and glycosolation, which modify proteins functions.

Information stored in DNA directs the protein synthesizing

machinery of the cell to produce specific protein required for its

structure, function and metabolism.

Since proteins play critical roles in cell biology, they have many

therapeutic uses in preventing and curing diseases and disorders.

Short peptide chains can be synthesized chemically whereas large

peptides in living cells.

DNA that encodes the instructions for producing desired proteins is

inserted into cells and as the cells grow they synthesize the proteinsand subsequently they are harvested and purified.


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Recently transgenic plant expression systems were well

developed.

Use of plants as a means of lower cost of production and

easier expansion of larger volume of production than cell

culture systems.

However 50% of total cost of production goes forextraction and purfication.

Plant system able to produce complex proteins with

some extent of post translational modifications.

Therapeutic proteins


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p p

Antibodies

Passive immunization an immune response that results from injecting

another organisms antibodies into organism that is being challenged

by the pathogen

Passive immunization using MAB are largest category of biotech derived

drugs.

In passive immunization, rather than injecting an antigen and inducingthe body to produce antibodies against it, an antibody targetted

towards a specific antigen is administered as a therapeutic.

Eg. Multiple doses of Herceptin against breast cancer

Antibody therapes are available for lymphoma, rheumatoid arthritis,

respiratory synchytial virus

Clinical trials are underway.

Antibodies produced from transgenic plants are


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p g pused for the treatment of

Dental caries Rheumatoid arthritis Cholera E. colidiarrohea

Malaria Certain cancers HIV Norwalk virus Rhino virus

Influenza Hepatitis B Herpes simplex virus

Vaccines


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Protein antigens from various pathogens have beenexpressed in plants and used to produce immune

responses resulting in protection against diseases. Plant derived vaccines against Vibrio cholerae,

enterotoxigenic E. coli, Hepatitis B virus, Norwalk virus,rabies virus, human cytomegalo virus, respiratory

synchytial virus Insulin expression in plants produceda vaccine useful for

protection against insulin dependent auto immuneMellitus diabetes

Antigen specific to an individual patient tumor areexpressed in tobacco, harvested, purified andadministered to the patient.This entire process will takeplace as little as 4 weeks compared to 9 months for the

same process in mammalian cell culture.


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Plant derived antigens purified and usedas injectable vaccines.

Oral delivery of these vaccines with infoods also successful.

Edible vaccines


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Edible vaccines

Low cost delivery mechanism for immunisation

No need for injection, sterile needles, and refrigeration Edible vaccines successfully immunized test animals against

enterotoxigenic E. coli, V. cholerae, hepattitis B, norwalk virus rabies

virus etc.

Concentration of vaccine protein in edible vaccine is relatively low.

Research is underway to increase them in targeted sites. Eg.

Potato, Tomato, banana and carrot

Potato cooking inactivate the vaccine.

Tomato and banana short storage lifeCarrot few storage problems, can be eaten raw. (hepatitis B vaccine)

Ch ll


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Challenges:

Standardization of expression

Dosage level and immune responses

Distributed through health service channels

Decrease the cost of immunisation indeveloping countries

Eg. Golden rice program

P ibl l l f i d t i l d


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Possible molecular farming products include:

1) Primary Products 2) Derived Products

Monoclonal Antibodies,Immunogloblulin (Ig) fragments-

Fabs, scFv (Passive immunity)

Bio-plastics - PHAs(polyhydroxyalkanoates, chemically

related to polyesters).

Antigens (vaccines) (Active

immunity)

Structural: proteins, peptides,

hormones, (interleukins,

interferons and colony stimulating

factors)

Enzymes: food, feed, industrial,

therapeutic, diagnostic, cosmetic

Nutraceuticals:

Macro: Carbohydrates, Fats

Micro: Vitamins, co-factors, minerals,

Phytochemicals: carotenoids (beta-

carotene, lycopene, lutein), flavonoids

(quercetin, kaempferol, allicin),

isoflavones (phytoestrogens -

genistein and daidzein),

isothiocyanates (glucosinolates,

indoles, and sulforaphane), phenolics(reservatrol, catechin), tannins

Anti-disease therapeutics: Factor

VII,

Non-nutrient phytochemicals: fragrances,

flavors

Enzyme inhibitors Fibres: polymers, lignins

Th G ld Ri St


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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 doesnt exist

to deliver vitamin pills

Improved vitamin A content in widely consumed crops

an attractive alternative

Carotene Pathway Problem in Plants


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-Carotene Pathway Problem in PlantsIPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

-carotene desaturase

Problem:

Rice lacks

these enzymes

Normal

Vitamin A

Deficient

Rice

The Golden Rice Solution


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IPP

Geranylgeranyl diphosphate

Phytoene

Lycopene

-carotene(vitamin A precursor)

Phytoene synthase

Phytoene desaturase

Lycopene-beta-cyclase

-carotene desaturase

Daffodil gene

Single bacterial gene;

performs both functions

Daffodil gene

-Carotene Pathway Genes Added

Vitamin A

Pathway

is complete

and functional

Golden

Rice

I t d i th G


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Introducing the Gene

or

Developing Transgenics

Steps

1. Create transformation cassette

2. Introduce and select for transformants

Transformation Cassettes


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Transformation Cassettes

Contains

1. Gene of interest

The coding region and its controlling elements

2. Selectable marker

Distinguishes transformed/untransformed plants

3. Insertion sequencesAidsAgrobacterium insertion

Transformation Steps


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Transformation Steps

Prepare tissue for transformation

Introduce DNA

Culture plant tissueDevelop 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

D li i th G


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Transformation cassettes are developed in the lab

They are then introduced into a plant

Two major delivery methods

Delivering the Gene

to the Plant

Agrobacterium

Gene GunTissue culturerequired to generate

transgenic plants


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The Lab Steps


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The Next Test Is The Field

Non-transgenics

Transgenics

Herbicide Resistance

biopharming training

Documents