Concept of Biopharming

Knowledge on structure and function of cellular macromolecules Isolation and characterization of genes, cloning a gene and studying its structure and expression through recombinant DNA (rDNA) technology rDNA technology in biological research

rDNA technology in pharmaceutical industry

The concept of biopharming is not new.

Common medicines, such as codeine, morphine, bulk laxatives, and the anticancer drugs such as taxol and vincristine have long been purified from plants. But biopharmings great promise lies in using genetic modification i.e., techniques to make wild (nontransformed) plants to do drastic new things. Biopharming offers tremendous advantages over traditional methods for producing pharmaceuticals. Great potential for reducing the costs of production. The energy for product synthesis comes from the sun, and the primary raw materials are water and carbon dioxide. To expand production, it is much easier to plant a few additional hectares than to build a new bricks-and-mortar manufacturing facility.

Vaccines produced in this way will be designed to be heat-stable so that no refrigeration chain from manufacturer to patient will be required. This would have a great application in developing countries, especially in the tropics and throughout Asia and Africa.

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 hormone was produced from pituitary glands removed from cadavers. It resulted in recipients contracting CreutzfeldJakob syndrome.
The recombinant approach resulted in unlimited supplies of safe material. This safety aspect has been extended to various clotting factors that were originally isolated from blood but now carry the risk of HIV infection. As the methods for cloning genes became more and more sophisticated, an increasing number of lymphokines and cytokines were identified and significant amounts of them produced for the first time.

Use of microbes for production of recombinant therapeutic proteins has several problems
The foreign gene may contain sequences that act as termination 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 of the human recombinant proteins in microbial system. Degradation of recombinant proteins since it is someway recognized as foreign protein in bacteria.

Selective examples of recombinant proteins and their applications

Transgenic animals and plants as Bioreactors

Recombinant-protein synthesis in animal cells has a number of advantages over microbial expression systems, the most important of which is the authentic post-translational modifications that are performed in animal cells. However, large scale culture of animal cells is expensive. The production of growth hormone in the serum of transgenic mice provided the first evidence that recombinant proteins could be produced, 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-specific promoter, such as that from the casein gene. The first proteins produced in this way were sheep -lactoglobulin and human tissue-plasminogen activator (tPA).

Selective examples of therapeutic compounds produced using animals as bioreactors

Plants as bioreactors
Plants are a useful alternative to animals for recombinantprotein production because they are inexpensive

Therefore, there is much interest in using plants as production systems for the synthesis of recombinant proteins and other speciality chemicals.
There is some concern that therapeutic molecules produced in animal expression systems could be contaminated with small quantities of endogenous viruses or prions, a risk factor that is absent from plants. Furthermore, plants carry out very similar post-translational modification reactions to animal cells, with only minor differences in glycosylation patterns. Thus plants are quite suitable for the production of recombinant human proteins for therapeutic use.

Selective examples of recombinant human therapeutic proteins expressed in plants

 The first such report was the expression of human growth hormone, as a fusion with the Agrobacterium Nopaline synthase enzyme, in transgenic tobacco and sunflower. Tobacco has been the most frequently used host for recombinantprotein expression although edible crops, such as rice, are now becoming popular, since recombinant proteins produced in such crops 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 material containing recombinant antibodies could provide passive immunity (i.e. immunity brought about without stimulating the host immune system). Antibody production in plants was first demonstrated by Hiatt and During team, who expressed full-size immunoglobulins in tobacco leaves. Since then, many different types of antibody have been expressed in plants, predominantly tobacco, including full-size immunoglobulins, Fab fragments and single-chain Fv fragments (scFvs).

 A fully humanized antibody against herpes simplex virus2 (HSV-2) has been expressed in soybean.

Even secretory IgA (sIgA) antibodies, which have four separate polypeptide components, have been successfully expressed in transgenic tobacco plants. Plants producing recombinant sIgA against the oral pathogen Streptococcus mutans have been generated, and these plant-derived antibodies (plantibodies) have recently been commercially produced as the drug CaroRxTM, marketed by Planet Biotechnology Inc.

Isolation of gene sequence

Codon optimization and Construction of Recombinant vector

Transformation Plant/Animal/Microbes High throughput Expression of Cloned genes

Separation and purification of medicinal protein

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 the protein, but for this to be possible the relevant protein must be available in relatively large amounts.
Obtaining sufficient quantities will be a major problem unless donated blood can be used as the source in some cases. Animal proteins are used whenever these are effective, but there are not many disorders that can be treated with animal proteins and there is always the possibility of side effects such as an allergenic response.

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 of hormone synthesized by the patients pancreas. Traditionally obtained from the pancreas of pigs and cows slaughtered for meat production. Although animal insulin is generally satisfactory, problems may arise in its use to treat human diabetes.
slight differences between the animal and human proteins may lead to side effects in some patients. the purification procedures are difficult and potentially dangerous contaminants can not always be completely removed

Lac promoter

lacZ

A gene

Lac promoter

lacZ

B gene

Vector carrying the Artificial A and B genes

Synthesis of insulin protein

galactosidase segment met Cyanogen bromide Cleaved fusion proteins Cleaved fusion proteins Purification of A and B chains Attach by disulphide bridges
B A

A A chain

Transformed E. coli synthesize A and B fusion proteins galactosidase segment met

B chain

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 was ideally suited for artificial gene synthesis. Strategy - insertion of the artificial gene into a lacZ reading frame of the pBR 322 vector, synthesis of a fusion protein and cleavage with cyanogens bromide.

Production of recombinant somatostatin

Lac promoter lacZ Artificial somatostatin gene

Transformation into E. coli

galactosidase segment met

Somatostatin fusion protein

Cyanogen bromide

Cleaved somatostatin

Somatotropin
Somatotropin -191 amino acids in length, equivalent to almost 600 bp, Combination of artificial gene synthesis and cDNA cloning was used to obtain a Somatotropin-producing E. coli strain. mRNA was obtained from the pituitarygland and a cDNA library was prepared. The Somatotropin cDNA turned out to have a unique site for the restriction endonucleases HaeIII, which cuts the gene into two segments. The longer segment, consisting of codons 24 to 191, was retained for use in construction of the recombinant plasmid. The smaller segment was replaced by an artificial DNA molecule that reproduced the start of the Somatotropin gene and provided the correct signals for translation in E. coli. The modified gene was then ligated into an expression vector carrying the lac promoter.

Production of recombinant somatotropin cDNA fragment

Codons 0 24 HaeIII 0 Discard 24 24 191 191

Retain

Expression of somatotropin
Synthetic leader sequence lacZ somatotropin gene E. coli transformation Somatotropin is synthesized

Lac promoter

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 human blood 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 be significant achievement for biotechnology.

 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) which undergoes a complex series of post-translational processing events, eventually resulting in a dimeric protein consisting of a large subunit, derived from the upstream region of the initial polypeptide and a small subunit from the downstream segment.

The two subunit contain a total of 17 disulphide bonds and a number of glycosylated sites. It is not possible to synthesize an active version in E. coli.
Most attempts made on mammalian cells.

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

Because of the failure in post-translational events, (do not convert the entire 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 for the small subunit.
Each cDNA fragment was ligated into an expression vector, downstream of the Ag promoter (a hybrid between the chicken -actin and rabbit -globin sequences) and upstream of a polyadenylation signal 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 containing the complete cDNA and the resulting factor VIII protein was indistinguishable in terms of function from the native form

Tissue Plasminogen Activator (tPA)

naturally occurring protease enzyme, helps to dissolve blood clots inside a blood vessel Boon for patients suffer from thrombosis

1st pharmaceutical product to be produced by mammalian cell culture

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

Production of recombinant tPA

Synthesize 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

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 in microorganisms is still a more attractive option. Erythropoietin hormone synthesized by the kidney stimulate the stem cells of bone marrow to produce mature erythrocytes

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

rDNA derived therapeutic agents (approved by FDA) with trade names and their applications in humans
rDNA product Insulin Trade name Humulin Application/uses Diabetes

Growth hormone
Hepatitis B vaccine Tissue Plasminogen Activator (tPA)

Protropin/Humatrope
Recombinax HB/ Engerix B Activase

Pituitary dwarfism
Hepatitis B Myocardial infarction

Factor VIII
DNase Erythropoietin

Kogenate/Recombinate Hemophilia
Pulmozyme Epogen Cystic fibrosis Severe anaemia with kidney damage

Some other rDNA derived therapeutic agents approved by USFDA

rDNA product Application/uses

Coagulation factor VIII Coagulation factor IX

Interferon Interferon Interferon Interleukin 2 Interleukin 10

Hemophilia A Christmas disease (Hemophilia B)

Leukemia Multiple sclerosis Chronic granulomatous disease Renal cell carcinoma (Kidney cancer) Thrombocytopenia (few platelets in blood)

Restriction endonucleases
> 500 different restriction endonucleases Synthesized by a wide range of microorganisms and for each organism, a detailed fermentation protocol, has to be developed and optimized To avoid having to maintain a large number of different microorganisms, stock a very wide range of medium components, design several different fermenters and spend an inordinate amount of time developing optimal growth conditions 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. coli cells grow rapidly to high cell densities and can be engineered to significantly over express the target restriction enzymes.

 However, host organism is dramatically affected by the production or presence of a heterologous protein. Over expression of heterologous protein may drain the host organism of important metabolic resources and this may affect its growth or sometimes it may be lethal to the host. Eg. There is a possibility to digest the host DNA by the heterologous restriction enzyme unless a protection mechanism is present.

Microbial system has also been used to synthesize several industrially important low molecular weight molecules

L-ascorbic acid (Vitamin C) indigo amino acids antibiotics and

biopolymers (Xanthan gum) melanin rubber polyhydroxyalkanoates etc.

high molecular weight molecules

Transgenic animals
Gene transfer to animal cells has been practiced for the past 40 years. Techniques are available for the introduction of DNA into many different cell types Animal cells - advantageous for the production of recombinant animal proteins (authentic posttranslational modifications) that are not carried out by bacterial cells and fungi. Cell cultures - commercial scale to synthesize products

 Intense research - efficient vector systems and transformation methods for animal cells.

Baculovirus expression system - in insects.

More recently, introduction of DNA into animal cells in vivo to treat disease (in vivo gene therapy). Viral gene-delivery vectors are favoured for therapeutic applications because of their efficiency, but safety concerns have prompted research into alternative DNAmediated transfer procedures.

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 their surroundings. (3) Transduction: Packaging the DNA inside an animal virus

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

Proteins with therapeutic and industrial value have been produce (not commercialized) in the milk of transgenic animals
Protein Antithrombin III Factor VIII and IX CFTR (Cystic Fibrosis Transmembrane conductance regulator) Lactoferrin Alpha 1 anti trypsin Lysostaphin Spider silk protein Animal Goat Goat, Pig, Sheep Sheep Use Reduce the amount of blood needed in some surgeries Treatment of Hemophila Treatment of Cystic Fibrosis

Cow Sheep Cow Goat

Natural antibiotic and used in coronary surgery Treatment of Cystic Fibrosis and emphysema Antibacterial compound that prevents mastitis in cows Production of ultra strong and light weight industrial materials

 Transgenic animals:
Animals which have been genetically engineered to contain one or more genes from an exogenous source. Transgenes are integrated into the genome. Transgenes can be transmitted through germline to progeny. First transgenic animal produced = Founder Animal

 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

Methods for introducing foreign gene/ Transgenic mice production

Retroviral vector Microinjection ES cell method

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 synthesized by the transgenic organism or the transgenic organism to be used as food, it is absolutely necessary that there be no retroviral contamination.

Retroviral vector method

Cleavage stage embryos (eight-cell stage) infected with defective retrovirus carrying a transgene Implanted 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 founder transgenic animals

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 tubes microinjection 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 a vasectomised male) 3 weeks after implantation Transgenic pups PCR/Southern blot hybridisation

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

Embryonic Stem Cell Method

Introduction of foreign DNA into ES cell by electroporation/micro injection

Cells from the blastocyst stage of a developing mouse embryo can proliferate in cell culture and have the capability of differentiating into all other cell typesincluding germ line cells- after they are reintroduced into another blastocyst embryo (Pluripotency).
Pluripotency in the broad sense refers to "having more than one potential outcome".

Pluripotent stem cells can give rise to any fetal or adult cell type.
Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of mouse blastocysts.

1. Make your DNA Using recombinant DNA methods, DNA containing the structural gene, vector DNA, Promoter and enhancer sequences to enable the gene to be 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 the desired 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.

Transgenesis in large animals More difficult than with mice

Less number of eggs Technical difficulties in handling Long gestational period Early expts. not satisfactory

Transgenic sheep overproducing growth hormone susceptible to infection, infertile and tend to die at early stage
Due to ineffective control of gene regulation Resistance to diseases, enhancing the milk production, production of commercial and pharmaceutical compounds

Transgenic cattle
Mammary gland of the dairy cattle- ideal bioreactor Transgenic cow over expressed K - casein transgene milk with higher 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 of monoclonal antibodies has met with some success In vivo immunization ideal for disease protection, which involves the insertion of a transgene for an antibody that specifically binds to an antigen

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

Fertilized oozytes are centrifuged to settle the yolk at one pole of the oozytes
Foreign gene is microinjected into male pronucleus in the oozytes Oozytes grown in vitro till blastocyst stage Implanted in uterus of foster mother

Transgenic sheep
Mostly involve development of mammary glands as bioreactors for the production of proteins for pharmaceutical 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
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 of sulfide Transgenic sheep containing bacterial genes for synthesis of cysteine

Two enzymes , synthesized by the transgenes trap the H2S and liberate in the intestine to produce the cysteine
Good supply of cysteine to the sheep improves the quality and quantity of wool

Transgenic goats
Transgenic goats with tPA gene produces tissue Plasminogen activator in milk

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

Human haemoglobins (globulin gene)

Human haemoglobins can be separated from pig haemoglobins by analytical techniques Haemoglobin O2 carrying protein of RBC can be used as a substitute in blood 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 like haemoglobins of RBC Naked haemoglobin is easily degraded and breakdown products cause damage to kidney Contamination by pig viruses and other compounds cause allergic reactions

Xenotransplantation
Transplantation of animal organs in human system Human organs such as liver, pancreas, kidney and lungs great demand 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 the transplants Hyperacute rejection Imutran Company (USA) produced transgenic pig (Astrid) by microinjecting genes for human immune system into male pronucleus of the zygote

Transgenic rabbits
Interleukin-2 gene along with -casein promoter

Interleukin-2 is produced in milk

To treat cancers Transgenic rabbits with human growth hormone gene grow faster and produce more meat with in short duration

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 is quite 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 production of therapeutic proteins

Transgenic animal Cow Cow Sheep Goat Goat Goat Protein product Lactoferrin Interferon 1 antitrypsin CFTR tPA Antithrombin III Biological importance To overcome iron deficiency anaemias and also posssses antibacterial activity Resistance against viral infections Treatment of emphysema in lungs (Promotes gas exchange in lungs) To treat CF (promotes transport of ions) To treat myocardial infarctions (to dissolve blood clots) Regulates blood clotting

Rabbits
Mouse Mouse

glucosidase
Urokinase Immunoglobulins

To treat Pompes disease (A genetic disorder characterized by block in glycogen degradation)

to dissolve blood clots Nhances immunity

Vaccines

Conventional vaccines

Purified antigen vaccines

Recombinant vaccines

Live vaccines

Inactivated pathogen

Recombinant proteins/ Sub unit vaccines

DNA Vaccines

Whole protein molecule

Polypeptide

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 tissue culture set up is essential Live vaccines are heat labile Risk of disease development due to occasional presence of active virus particle or reversion to virulence

Purified antigen vaccines

Based on purified antigens isolated from concerned pathogens Since they do not contain the organism, risk of pathogenicity is avoided

Limitations:
Cost is higher due to steps involved in purification and vaccine preparation 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 wall capsules of Neisseria meningitis and Streptococcus pneumoniae Many bacteria produce exotoxins, which are highly immunogenic. But these toxins produce toxic effects and it may decrease with storage due to heat and chemicals. Fortunately most exotoxins that have lost their toxicity retain their immunogenecity; they are called toxoids and are effectively used as effective vaccines. Eg. Toxoids of pathogens causing tetanus, diphtheria etc.

Recombinant vaccines

Contains either a protein or a gene encoding a protein of a pathogen origin that is immunogenic and critical to pathogen function; the vaccine is produced using recombinant DNA technology.
Vaccines based on recombinant proteins are called as sub unit vaccines.

Steps involved: Identification of protein that is immunogenic and critical for the pathogen Gene encoding for the protein is identified isolated Gene is inserted into a expression vector and introduced in to suitable host where it produces large quantity Protein isolated and purified from the host cells. It is used for the preparation of vaccine.

Comparison of different biological systems for therapeutic protein production

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 the proteins its final shape and form.

 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 proteins and subsequently they are harvested and purified.

 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 for

extraction and purfication.

Plant system able to produce complex proteins with some extent of post translational modifications.

Therapeutic proteins
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 inducing the 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 used for the treatment of
Dental caries Rheumatoid arthritis Cholera E. coli diarrohea Malaria Certain cancers HIV Norwalk virus Rhino virus Influenza Hepatitis B Herpes simplex virus

Vaccines
Protein antigens from various pathogens have been expressed 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 immune Mellitus diabetes Antigen specific to an individual patient tumor are expressed in tobacco, harvested, purified and administered to the patient.This entire process will take place as little as 4 weeks compared to 9 months for the same process in mammalian cell culture.

 Plant derived antigens purified and used as injectable vaccines. Oral delivery of these vaccines with in foods also successful.

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 life Carrot few storage problems, can be eaten raw. (hepatitis B vaccine)

Challenges: Standardization of expression Dosage level and immune responses Distributed through health service channels Decrease the cost of immunisation in developing countries Eg. Golden rice program

Possible molecular farming products include:

1) Primary Products
Monoclonal Antibodies, Immunogloblulin (Ig) fragmentsFabs, scFv (Passive immunity)

2) Derived Products
Bio-plastics - PHAs (polyhydroxyalkanoates, chemically related to polyesters).

Nutraceuticals: Antigens (vaccines) (Active Macro: Carbohydrates, Fats immunity) Micro: Vitamins, co-factors, minerals, Phytochemicals: carotenoids (betaStructural: proteins, peptides, carotene, lycopene, lutein), flavonoids hormones, (interleukins, (quercetin, kaempferol, allicin), interferons and colony stimulating isoflavones (phytoestrogens factors) genistein and daidzein), isothiocyanates (glucosinolates, Enzymes: food, feed, industrial, indoles, and sulforaphane), phenolics therapeutic, diagnostic, cosmetic (reservatrol, catechin), tannins Anti-disease therapeutics: Factor VII, Enzyme inhibitors Non-nutrient phytochemicals: fragrances, flavors Fibres: polymers, lignins

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

IPP Geranylgeranyl diphosphate
Phytoene synthase

Phytoene

Problem: Rice lacks these enzymes

Phytoene desaturase

-carotene desaturase

Lycopene
Lycopene-beta-cyclase
Normal Vitamin A Deficient Rice

-carotene (vitamin A precursor)

The Golden Rice Solution

-Carotene Pathway Genes Added IPP Geranylgeranyl diphosphate
Daffodil gene Phytoene synthase

Phytoene

Vitamin A Pathway is complete and functional

Phytoene desaturase Single bacterial gene; performs both functions

-carotene desaturase

Lycopene
Daffodil gene Golden Rice

Lycopene-beta-cyclase

-carotene (vitamin A precursor)

Introducing the Gene or

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
Tissue must be capable of developing into normal plants Leaf, germinating seed, immature embryos

Introduce DNA
Agrobacterium or gene gun

Culture plant tissue

Develop shoots Root the shoots

Field test the plants

Multiple sites, multiple years

Delivering the Gene to the Plant

Transformation cassettes are developed in the lab
They are then introduced into a plant

Two major delivery methods

Agrobacterium Gene Gun
Tissue culture required to generate transgenic plants

The Lab Steps

The Next Test Is The Field

Herbicide Resistance

Non-transgenics

Transgenics