Plant molecular farming for recombinant therapeutic proteins

CONTENT
Introduction DNA Transformation Expression technology Host system Recombinant proteins produced Plantibodies Edible vaccine Acceptance of GM based drugs and firms involved Biosafety concerns Conclusion Future prospects
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What is Molecular farming?

Molecular farming, biofarming, greening of vaccine technology and plant molecular farming are expressions for the large scale production of recombinant proteins in living cells or organisms. Plant molecular farming is a novel approach to the production of pharmaceuticals, where valuable recombinant proteins can be produced in transgenic plants on an industrial scale. This can be considered as 3rd revolution.

Methodology

The DNA that encodes the instructions for producing the desired protein (transgene) is inserted into plant cells and as the cells grow they synthesize the protein which is subsequently harvested and purified.

The first steps.

The first pharmaceutically relevant protein made in plant was human growth hormone in 1986. Since then many other human proteins have been produced increasingly in an diverse range of crops. First antibody was expressed in tobacco in 1989 In 1992 plants were used first time to produce an experimental vaccine: hepatitis B virus surface antigen (HBV). Range of recombinant proteins has extended to include industrial enzymes, technological proteins used in research, milk proteins, biopolymers and many more
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DNA transformation

Stable transformation into the nuclear genome is done primarily using agrobacterium mediated transformation or particle bombardment method. Transient transformation by transduction using recombinant viral vector. DNA transformation to chloroplast.

Chloroplast based transformation

Several examples of chloroplast based molecular farming have been reported in tobacco. These include Production of human growth hormone at 8%TSP, human serum albumin at 11%TSP and cholera and tetanus toxin fragments at 25%TSP. DISADVANTAGES: Inability to carry out post translational modification and horizontal gene transfer to bacteria is also reported. ALTERNATIVE APPROACH: Express protein from nuclear genome but introduce a chloroplast targeting sequence. e.g. Expression of camelid heavy chain antibody in potatoes
Jobling, S. A. et al., (2003) 8

Figure 1. Pag A gene was inserted in chloroplast specific vector (pLD-Cty) after addition of upstream regulatory element. Biolistic process was used to transform to tobacco leaf chloroplast .
Construction of chloroplast transformation vector. A) Representation of the chloroplast vector after cloning of modified pagA in Eco RV and Not I enzyme sites.

B) Restriction analysis of the chloroplast transformation vector, PLD-PAG. M, 1kb Gene ladder; 1, pLD-Ctv digested with Eco RI; 2, pLD-PAG digested with Eco RI; 3, pLD-PAG digested with Eco RV; 4, pLD-PAG digested with Not I.

India

Mohammad A. et al.,(2005) 8

Expression technology

HIGH LEVEL TRANSGENE EXPRESSION.

Expression-construct design can help to achieve high yields by maximizing rate of transcription and translation. Dicots --- strong and constitutive cauliflower mosaic virus(CaMV35s) promoter. Cereals --- maize ubiquitin-1(ubi1) promoter, intron mediated enhancement. Regulated promoters can be used instead of constitutive promoters. Inducible promoters can also be used for time dependent expression.

1.

2. 3. 4. 5.

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High level translation.

Translation rate can be optimized by ensuring removal of sequences from construct that cause instability to mRNA sequences. Codon usage can be modify in some cases to maximize rate of protein synthesis and to eliminate introns.

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2.

Transcriptional and translational level can be maximized by taking precaution against transgene silencing.

Protein targeting
Targeting of recombinant protein to oil bodies. e.g. oleosin fusion protein developed by semBiosys in which target recombinant protein is expressed in oilseed crop as fusion with oleosin. Targeting of recombinant protein to plasma membrane. e.g. recombinant protein produced by fusing with t cell receptor membrane spanning domain. Targeting recombinant protein to exudates.

UK

11 Eva stoger et al., (2003) 11

Directing translational pathway

Sub cellular targeting can be used to increase yield. Secretary pathway is more suitable compartment for folding and assembly than cytosolic. Recombinant protein pass through ER. In absence of further signal, product is secreted in apoplast. Ab are less stable in apoplast. Ab protein can be retrieved in ER lumen using H/KDEL C-terminal tetra peptide tag. Ab produced by this are not modified in golgi body.
UK
12 Eva stoger et al., (2003) 11

Examples of recombinant proteins targeted to subcellular compartments in transgenic plants

Proteins
amylase Avidin Secretary antibodies glucouronidase Anti- oxazolone

Host plants
Tobacco Corn Tobacco Brassica Tobacco

Tissue expression
Leaves Seeds Leaves Seeds Leaves

Sub cellular targets

Apoplast Apoplast Apoplast Oil bodies ER

Xylanase
Anti-phytochrome Anti -1,4Endoglucanase Hirudin Bulgaria

Brassica
Tobacco Potato

Seeds
Leaves Roots

Oil bodies
Cytosol Cytosol

Brassica

Seeds

Oil bodies kunka kamenarova et al., (2005)

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Humanization of proteins

Plant derived recombinant protein tend to have carbohydrate groups (12)xylose and (13) fucose. It Lacks terminal galactose and sialic acid residues which are found in many mammalian glycoprotein. Change in glycan structure may turn recombinant protein immunogenic when administered to human.

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Strategies to humanize protein

The use of purified human (14) galactosyl transferase enzyme for the in vitro modification of plant derived recombinant protein. Expression of human (1,4) galactosyl transferase in transgenic plants to produce recombinant Ab with galactose extended glycans. Inhibition of fucosyl transferase and xylyl transferase using Ab, ribozyme, iRNA helps in removing plant specific carbohydrates.

Gene targeting by homologous recombination has been used to produce recombinant proteins lacking plant specific glycans in moss phycomitrella patens.
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Host system
Choice of host system affects overall cost, product quality , production timescale, scale up capacity and biosafety. Various host system are used like bacteria, yeast, transgenic animals, plant cell cultures, transgenic plants. Plants are ideal host systems which are cost effective, rapidly scaled up, fewer ethical issues and better public acceptance than transgenic animals.
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Comparison of host system

Expression system
Bacteria

Advantages
Established regulatory track; wellunderstood genetics; cheap and easy to grow Recognized as "safe;" long history of use; fast; inexpensive; posttranslational modifications

Disadvantages
Proteins not usually secreted; contain endotoxins; no posttranslational modifications Overglycosylation can ruin bioactivity; safety; potency; clearance; contains immunogens/antigens

Cost per gram

Yeast

$50-100

Insect cells

Posttranslational modifications; properly folded proteins; fairly high expression levels Usually fold proteins properly; correct posttranslation modifications; good regulatory track record; only choice for largest proteins Complex protein processing; very high expression levels; easy scale up; lowcost production

Minimal regulatory track; slow growth; expensive media; baculovirus infection (extra step); mammalian virus can infect cells Expensive media; slow growth; may contain allergens/contaminants; complicated purification Little regulatory experience; potential for viral contamination; long time scales; $500-5,000

Mammalian cells

Transgenic animals

$20-50

transgenic plants

Shorter development cycles; easy seed storage/scaling; good expression levels; no plant viruses known to infect humans

Potential for new contaminants (soil fungi, bacteria, pesticides); posttranslational modifications; contains possible allergens

$10-20

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Aziz Elbehri (2006)

PLANT EXPRESSION HOST

The range of plant species amenable to transformation is growing at a unique rate.

Many factors need to be taken into consideration. They are

Yield of functional protein in given species.

Transformable capacity.
Biosafety concerns. Storage and distribution of protein. Cost of grain storage and distribution.
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Cost of extraction and purification.

Leafy crops tobacco

Advantages well established technology for gene transfer. High biomass yield. Prolific seed production. Existence of large scale processing infrastructure. Little risk to contaminate food chain.

Disadvantages Biosafety concerns. Interfere downstream processing due to presence of phenolic compounds. Recombinant protein is often unstable.

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Bulgaria

kunka kamenarova et al., (2005)

Leafy crops continued

Alfa alfa and soybean give advantage of large dry biomass.

These crops also use atmospheric nitrogen through nitrogen fixation thus reducing cost of fertilizers. Lettuce is also used for edible vaccines production.

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Cereals and legumes

Rice, wheat , pea, maize and soybean are used. Maize gives high biomass yield , ease of transformation, in vitro manipulation facilities and convenience of scale up. Rice among cereals gives highest yield. Disadvantage is of gene transfer via pollen transfer.

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Fruits and vegetables

The main benefit of fruit and vegetable is that they can be consumed raw or particularly processed which makes them particularly suitable for subunit edible vaccines. Potatoes have been widely used for production of plant derived vaccines and have been administered to humans in most of clinical trials.

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Tomatoes were used to produce the first plantderived rabies vaccine and are more palatable than than potatoes and offer high yield. Bananas have been grown in developing countries where vaccines are most needed. It can be consumed raw or as puree by both adults and children.

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Alternative plant based production system

Plant cell suspension culture (derivation of hairy roots, shoot teratomas, immobilized cells, suspension cell culture) Requires simple, synthetic media, defined and sterile production conditions, inexpensive, carry out proper glycosylation and folding of proteins. Wide group of recombinant antibodies produced in BY2 tobacco and rice cell suspension culture including full size Ig, Fab Fragment, ScFv and fusion proteins.
Fischer et al.,.(1999)

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Industrial proteins and enzymes. Therapeutic and pharmaceutical proteins. Plantibodies. Plantigens.

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INDUSTRIAL PROTEINS AND ENZYMES

This group include hydrolyses encompassing both glycosidases and proteases.

Avidin and -glucouronidase (GUS) were the first successful commercial recombinant protein produced in corn and are marketed by sigma as research reagents. All of these products are usually characterized by the fact that they are needed in large amount and dont require high levels of purification.
A.S.Rishi (2001)

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industrial enzymes and proteins produced in different plant host system

Industrial enzyme
- amylase Phytase Manganese peroxide (1,4)xylanase (1,3)glucanase

Potential Use
Industry Industry Industry industry Industry

Host
Tobacco Alfaalfa, tobacco Alfalafa, tobacco Tobacco, canola Tobacco, barley

Avidin
Glucouronidase Cellulase

Research reagent
Research reagent Industry

Maize
Maize Alfalafla, potato, tobacco
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A.S.Rishi (2001)

THERAPEUTIC AND PHARMACEUTICAL

PROTEINS
o Includes all proteins used directly as pharmaceuticals along with those proteins used in the making of pharmaceuticals. The list of such proteins is long, ever growing, and includes such products as thrombin and collagen (therapeutics), and trypsin and aprotinin (intermediates).

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A.S.Rishi (2001)

Table no.4 Therapeutic proteins produced in different plant host systems

Therapeutic protein and haemoglobin Human serum albumin Glucocerebrosidase interferon Protein C Epidermal growth factor Erythropoietin Trout growth factor Glutamate decarboxylase Human somatotorpin Calcitonin interferon Human granulocyte-macrophage Enkephalins Human homotrimeric collagen I tricosanthin Angiotensis-1- convering enzyme Host Tobacco Potato Tobacco Rice Tobacco Tobacco Tobaco Tobacco Tobacco Tobacco Potato Tobacco Tobacco Oilseed Tobacco Tobacco Tobacco/ tomato Potential use Blood substitute Blood substitute Gaucher disease Viral protection Anticoagulant Mitogen Mitogen Mitogen Diabetes Hypo pituitary dwarfism Paget disease, osteoporosis, Neutropenia Neutropenia Antihyperanalgesic by opiate activity Collagen HIV therapy hypertension

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A.S.Rishi (2001)

Table 5. Plant - derived pharmaceutical proteins that are closest to commercialization for the treatment of human diseases
Product Various single chain Fv antibody fragments CaroRx Class Antibody Indication Non-hodgkins lymphoma Company Large scale biology corp. Crop Viral vectors in tobacco Status Phase-I

Antibody

Dental caries

Planet biotechnology inc. Prodigene inc. Arntzen group

Transgenic tobacco Transgenic maize Transgenic potato Transgenic maize Transgenic potato Transgenic arabidopsis Transgenic maize Transgenic potato Viral vectors in spinach

Phase-II

E.Coli heat labile toxin

Vaccine

Diarrhoea

Phase I Phase I Phase II Phase I

Gastric lipase Hepatitis B virus surface antigen Human intrinsic factor Lactoferrin Norwalk virus capsid protein Rabies glycoprotein

Therapeutic enzyme Vaccine

Cystic fibrosis Hepatitis B

Meristem therapeutics Arntzen group

Dietary Dietary vaccine Vaccine

Vitamin B12 deficiency Gastrointestinal infections Norwalk virus infection Rabies

Cobento biotech AS Meristem therapeutics Arntzen group

Phase II Phase I Phase I Phase I

www.pharma-planta.org/EMBO%20paper.pdf

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Table no. 6 Some approved protein- based drugs

Drug
Aranesp

Company
Amgen

Indication
Anemia due to kidney failure, chemotherapy B cell chronic lymphocytic Pediatric oncology

Approved
May 1998

Campath Elitek

Llex oncology, Sanofi-synthelabo

May 2001 July 2002

Enbrel
Forteo LYMErix

Amgen,wyeth
Eli lilly Smithkline Beecham biologicals Scios Roche, inhale therapeutics Serono, pfizer Genentech IDEC

Rheumatoid arthritis
Osteoporosis Lyme disease prevention

Dec 2002
Nov 2001 Dec 1998

Natrecor Pegasys Rebif TNKase Zevalin

Congestive heart failure Chronic hepatitis c Multiple sclerosis Acute myocardial infarction B-cell non hodgkins lymphoma Anemia Infertility

August 2001 Oct 2002 Marich 2002 June2000 Feb 2002

Procrit Ovidrel

Ortho biotech Serono

Feb 2000 Sept 2000

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www.brucegoldfarb.com/FDL2.pdf

PLANTIBODIES

Several functional antibodies fragment antigen-binding (Fab) and single chain antibody fragments (ScFv) can be expressed in the leaves and seeds of plants without the loss of binding specificity. Expression of antibodies varies between different plant species, and a high level expression of scFvs was achieved in tobacco leaves, 7% of TSP. The first clinical trial using antibodies produced in plants was to prevent human tooth decay caused by streptococcus mutans.
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stefan schillberg (2002)

Figure 2. Antibody molecular farming

Antibody

Determining titer value

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PSSH1 PLANT EXPRESSION VECTOR CONSTRUCTS FOR THE ANTI

HCG RECOMBINANT ANTIBODIES

scFv : single chain variable fragment. CHS: chalcone synthase LPH : plant codon optimised leader peptide for heavy chain LPL : plant codon optimised leader peptide for light chain KDEL : signal for ER retention 3 UTR: untranslated region obtained for TMV S.R. Kathuria (2002)

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India

PROTEIN ANALYSIS OF ANTI HCG

ANTIBODY

Affinity purified plant expressed recombinant anti hCG antibody analyzed by SDS- PAGE

Individual expression of proteins as light and heavy chains

A: his 6 tag scFv, B: diabody, C: protein purified which gave pure antibody fragments
India

LC: light chain, HC: heavy chain, NI: non-infiltrated, LC+HC: light chain + heavy chain.
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S.R. Kathuria (2002)

Table no. 7 Recombinant antibodies produced in various plants

Recombinant antibodies Ig G1 Host plant system Tobacco Medical application First antibody expressed in plants; full length serum IgG produced by crossing plants that expressed heavy and light chains

Ig M
Ig A

Tobacco
Tobacco

First Ig M expressed in plants and protein targeted to chloroplast for accumulation

First secretory antibody expressed in plants by sequential crossing of four lines carrying individual components; at present the most advanced plant derived pharmaceutical protein First pharmaceutical protein produced in soybean

Ig G HSV HBV envelope protein scFv of IgG from mouse Bcell lymphoma scFvT84.66 against carcinoembryogenic antigen

Soybean

Tobacco Tobacco Cereals

First candidate expressed in plants ; third plant derived vaccine to reach clinical trials stage Treatment of hodgkins lymphoma Tumor associated marker antigen

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A.S.Rishi(2001)

Edible vaccines

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Vaccines

Vaccines work by priming the immune system to swiftly destroy specific disease causing agents before they can multiply enough to cause symptoms. This priming is achieved by presenting the immune system with whole viruses or bacteria that have been killed or attenuated. Classical vaccines pose a risk of causing diseases that they suppose to prevent.

To avoid that subunit vaccines were discovered but they are less effective and of high cost.
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Table no.8 Vaccine types, defination and immune response

Vaccine type Killed, inactivated Defination Pathogen is killed, usually through a chemical process such as formalin Pathogen is weakened by genetic manipulations such that growth in the host is limited and does not cause disease; other version of live vaccine is using an organism that is related to the pathogen, but grows poorly, naturally, in humans Well-defined part (or parts) of the organism is purified and used as an antigen (for example, proteins, polysaccharides, inactivated toxins) Immune response Evokes a robust immune response that mimics most of the responses seen during an infection Such vaccines evoke broad immune responses similar to that seen by the host infected with the natural pathogen examples Typhoid vaccine Salk polio vaccine Oral Sabin polio vaccine, Nasal influenza vaccine BCG vaccine

Live, attenuated

Sub unit acellular

Immune response is limited but may be robust; some forms (such as polysaccharides) may require the addition of other proteins (process called conjugation) to evoke a strong immune response Immune response can be modified and targeted by insertion of specific genetic sequences

Acellular pertussis vaccine Haemophilus influenzae type B (Hib) conjugate vaccine

recombinant

Defined genes are incorporated into plasmid vehicle to allow for the production of large quantities of well-defined proteins, which are then used as vaccines

Hepatitis B vaccine

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Sarah landry(2005)

Edible vaccines

Edible vaccines are sub- unit vaccines where the selected genes are introduced into the plants and the transgenic plant is then induced to manufacture the encoded protein.
The transgenic plants expressing the vaccine antigen, when eaten are expected to provide the antigenic stimulus that will generate an immune response in the host.

Edible vaccines are mucosal targeted vaccines where stimulation of both systematic and mucosal immune network takes place.
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4140

Transplant seedling to soil

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TARGETING AND EXPRESSION OF ANTIGENIC

PROTEINS

Efforts to enhance expression levels of transgene

coding

for

antigenic
targeting

proteins
sequences,

by
and

exploiting
enhancer

promoters,

elements have produced rather low quantities of the

antigen in plant tissues, but enough to induce

immune responses in feeding studies.

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Table no.9 Antigens along with their constructs produced in transgenic plants as candidate vaccine
Pathogen/disease Plant Promoter Antigen Targeting/enhancers/sigh nal peptides/terminator sequences SEKDEL SEKDEL + CTB leader

Vibrio cholera Cholera, enterotoxigenic E.coli, rotavirus Diabetes (autoimmune) E. coli heat labile enterotoxin B subunit (LT-B) Foot and mouth disease Hepatitis B

Potato Potato

mas P2 mas P1mas P2 mas P2 Ubi-1

Capsid protein 2L2I CTA2;CFA?- CTB; NSP4 Insulin LTB

Potato Corn

C terminus of CTB Codon optimised version of barley - amylase signal sequence 5 TEV 5 TEV leader, SEKDEL,

Arabidopsis Tobacco, potato, lupine, lettuce Soybean

35S 35S

Structural protein VP1 HbsAg

Herpes simplex virus 2

35S

Glycoprotein B

Tobacco extension signal peptide

Measles virus (MV)

Tobacco

35S

Haemagglutinin (H) protein

5 TEV leader + SEKDEL + signal peptide (SP) of tobacco prl a gene

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Schuyler S. Korban (2002)

Edible vaccine against hepatitis B

The DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin and lettuce cv Burpee Bibb expressing envelope surface protein. Mice and Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum Ig-G response to plant produced protein. 44

USA

J. Kapusta (1999)

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Schematic representation of pROK2S binary vector carrying the S gene of HBV.

LB: left border, RB: right border, NOSp: nopaline synthesis promoter, NOSt: nopatline synthase terminator, HbsAg: surface antigen of hepatitis B virus
Evaluation of HBsAG accumulation in transgenic lupin callus and lettuce lines. Individual transgenics are indicated on x-axis. Lupin and lettuce plants transformed with A. tumifaciens vector without HBsAg was used as control.

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USA J. Kapusta (1999)

Figure 9. Titer of antibodies in three individuals(1-3) immunized orally with transgenic lettuce harboring HBsAg

USA

J. Kapusta (1999)

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Advantages of edible vaccines

Low cost. Needle free shot. Less ethical issues. No refrigeration requirement. Occupational safety. Elicit mucosal as well as systemic immunity. Effective distribution in developing countries. Easy consumption by children. No purification required.
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Shows number of new biotech drugs and vaccines approved.

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Aziz Elbehri (2006)

NUMBER OF FIELD TEST PERMITS BY APHIS AS PHARMCEUTICAL, INDUSTRIAL OR NOVEL TRAITS.

Crop Corn Soybean Alfalfa Barley Rapeseed Tobacco Tomato Rice Safflower Wheat Sugarcane other 5 1 1 2 2 2 1 6 1 11 22 2 1 Industrial enzymes 11 10 2 Novel proteins 157 4 1 1 Pharma plants 63 16 1 1 1 14 1 8 Total 231 30 4 2 3 15 1 11 3 2 % 71.1 9.2 1.2 0.6 0.6 4.6 0.3 3.4 0.9 0.6 0.3 6.8
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Aziz Elbehri (2006)

Selected biotech firms of north America and Europe specializing in recombinant therapeutic proteins
Firms AltaGen bioscience Work Has transgenic platform to express therapeutic proteins in food crops. Focuses on biopharmaceuticals with proven therapeutic value, including hemoglobin, thrombin factor XIII, erythropoietin, interferon and growth hormone Made momclonal antibody remicade (rheumatoid arthritis), reopro and retavase for cardiac care arena. Working on to improve glycosylation of corn plants and monoclonal antibody Monoclonal Ab in corn (R 19 for RSV), HX8 for HSV Therapeutic proteins in tobacco projects in pipeline include production of Monoclonal Ab on HSV, human papiloma virus, HIV, Alzheimers disease, ulcerative colitis and hepatitis viruses Lead product is recombinant gastric lipase to treat cystic fibrosis

Centocer Monsanto protein technologies Dow AgroSciences CropTech Epicyte Meristem Therapeutics

Large Scale Biology

prodiGene Phytomedic

Best know for its work in proteomics, produces patient specific cancer vaccines in green house- raised transgenic tobacco plants.
Gm corn to produce vaccines, Ab, enzymes and other protein-based therapeutics. Company is developing a candidate vaccine for HIV Therapeutic proteins in genetically modified tobacco plants. Pipeline includes drug candidates to treat autoimmune disorders, diabetes, viral infection and cancer.

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www.brucegoldfarb.com/FDL2.pdf

BIOTECHNOLOGY

IN

INDIA

The first biotech based vaccine released in indian market was rDNA hepatitis B vaccine produced by shantha biotech Pvt.Ltd. Other biotech medication in market include recombinant insulin, human growth hormone, interferon, blood clotting factor VIII, renin and interleukin. There are 50 companies work in advanced biotech application.
60% industry ---- human health

30% industry ----- bioinformatics and genomics

10% industry ----- agril.biotech

Lead domestic players include reliance life sciences, Dr. 52 reddys laboratory, shantha biotech, panacea biotech and biocon.

SELECTED DOMESTIC COMPANIES AND ACTIVITIES

Company Dabur india Ltd. Reliance life sciences Biocon Shantha biotech Banglore genei Dr. Reddys laboratory Panacea Biotech Ltd. activity Genomic and proteomic profiling to detect molecular changes in cancer patients Active in areas of stem cell research, medical , plant and industrial biotechnology Produced recombinant insulin, pectin and cholesterol lowering drugs. Begun developing genetically engineered drugs Recombinant insulin and recombinant hepatitis B vaccine Tools for genomic research, modifying enzymes, polymerase enzyme etc The company has a licensing agreement with Novo Nordisk for diabetes therapeutic technology and is developing human therapeutic proteins through rDNA technology Produced a vaccine for anthrax developed jointly by the Centre for Biotechnology at Jawaharlal Nehru University and the Department of Biotechnology. The drug is expected to receive fast-track approval through the regulatory system

Avestha gar

focused application of plant molecular biology including genome sequencing (basmati rice), plant transformations, marker-aided selection and proteomics

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http://www.doir.wa.gov.au/documents/businessandindustry/biotechnology_in_india.pdf

RISK AND CONCERNS

1.

2. 3.

Environment contamination unintended harm to other organisms. eg. Monarch butterfly and caterpillars. reduced effectiveness of pesticides gene transfer to non target species Economic concerns patenting new GM plants varieties will raise price of seeds so high that small farmers will not be able to afford seeds. Health safety concerns allergenicity
http://www.csa.com/discoveryguides/discoveryguides-main.ph

1.

1.

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BREAKDOWN OF REGULATORY SYSTEM: PRODIGENE INCIDENT 2002

Few unfertilized stalks of corn for the previous years crops, engineered to express therapeutic proteins, contaminated soybean fields in lowa and nebraska. $500,000 fine + $3 contaminated soybean million to buy/destroy

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www.brucegoldfarb.com/FDL2.pdf

REGULATION OF GM PLANTS

Japan health testing of food is mandatory. India GEAC, SBCC, DLC. Brazil banned. Europe mandatory to label GM foods. US - three different regulatory bodies EPA (evaluates GM plants for environmental safety). USDA (evaluates whether plant is safe to grow). FDA (evaluates whether plant is safe to eat).
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2.
3.

http://www.csa.com/discoveryguides/discoveryguides-main.ph

SUGGESTED SAFEGUARDS FOR MOLECULAR FARMING

Sterility

Use male sterile plants

Physical differences

containmenent and segregation

Easily detectable by addition of 'reporter genes Complete disclosure of DNA sequences

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http://www.csa.com/discoveryguides/discoveryguides-main.ph

CONCLUSION

Molecular farming offers an alternative for recombinant therapeutic proteins. Government must proceed cautiously in this area to gain public acceptance. Transgenic plants can assemble and accumulate many complex valuable proteins which can be economically extracted or processed Strong promoters, targeting sequences and other transcriptional and/or translational sequences are optimized for various crops to get optimal production. Recombinant protein has showed high expression in plastids rather than nuclear genomes of transgenic plants.

Food as vehicles for production of edible vaccines and other therapeutic recombinant proteins is a novel field which should pay dividend for both human health and 58 agricultural sector.

FUTURE PROSPECTS

Plants expressing uniform expression levels of the desired antigen have to be identified in order to administer the correct dosage of vaccine. Ethylene inducible genes linked to fruit ripening would allow inducible expression of the antigen. Since ripening affects the color of many fruits it may be possible to develop a correlation between the color of the fruit and the level of antigen, to ensure an adequate dosage of the vaccine Secretion of recombinant proteins form roots and leaves will have to be evaluated for cost effectiveness of production and product stability Production of industrial enzymes in tree species, where high biomass is available, should be evaluated for commercial exploitation.
59 The emerging fields of genomics, proteomics and metabolomics will provide tools for molecular farming to cure several disorders.

Smoke or vaccine?
We all know that tobacco is an easy plant to both grow and manipulate. We also know that cigarettes are very good at transmitting harmful substances straight to a persons lungs. Why not we re-engineer tobacco for an enjoyable way to gain immunity?

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Thank you

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