Plant molecular farming for recombinant therapeutic

proteins

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CONTENTIntroductionDNA TransformationExpression technologyHost systemRecombinant proteins producedPlantibodiesEdible vaccineAcceptance of GM based drugs and firms involvedBiosafety concernsConclusionFuture prospects

What is Molecular farming?

Molecular farming, biofarming, greening of vaccinetechnology and plant molecular farming are expressionsfor the large scale production of recombinant proteinsin living cells or organisms.

Plant molecular farming is a novel approach to theproduction of pharmaceuticals, where valuablerecombinant proteins can be produced in transgenicplants on an industrial scale.

This can be considered as 3rd revolution.3

Methodology

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

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The first steps….

The first pharmaceutically relevant protein made in plantwas human growth hormone in 1986.

Since then many other human proteins have been producedincreasingly in an diverse range of crops.

First antibody was expressed in tobacco in 1989

In 1992 plants were used first time to produce anexperimental vaccine: hepatitis B virus surface antigen(HBV).

Range of recombinant proteins has extended to includeindustrial enzymes, technological proteins used inresearch, milk proteins, biopolymers and many more

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DNA transformation

Stable transformation into the nuclear genome isdone primarily using agrobacterium mediated

transformation or particle bombardment

method.

Transient transformation by transduction usingrecombinant viral vector.

DNA transformation to chloroplast.

Chloroplast based transformation

Several examples of chloroplast based molecularfarming have been reported in tobacco. These include

Production of human growth hormone at 8%TSP,human serum albumin at 11%TSP and cholera andtetanus toxin fragments at 25%TSP.

DISADVANTAGES: Inability to carry out post translationalmodification and horizontal gene transfer to bacteria isalso reported.

ALTERNATIVE APPROACH: Express protein from nucleargenome but introduce a chloroplast targeting sequence.e.g. Expression of camelid heavy chain antibody inpotatoes

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Jobling, S. A. et al., (2003)

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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)

Expression technology

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HIGH LEVEL TRANSGENE EXPRESSION.

1. Expression-construct design can help to achieve highyields by maximizing rate of transcription andtranslation.

2. Dicots --- strong and constitutive cauliflower mosaicvirus(CaMV35s) promoter.

3. Cereals --- maize ubiquitin-1(ubi1) promoter, intronmediated enhancement.

4. Regulated promoters can be used instead of constitutivepromoters.

5. Inducible promoters can also be used for time dependentexpression.

High level translation.

1. Translation rate can be optimized by ensuring removalof sequences from construct that cause instability tomRNA sequences.

2. Codon usage can be modify in some cases to maximizerate of protein synthesis and to eliminate introns.

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

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Protein targeting

UK Eva stoger et al., (2003)11

• Targeting of recombinant protein to oil bodies.e.g. oleosin fusion protein developed by semBiosysin which target recombinant protein is expressed inoilseed crop as fusion with oleosin.

• Targeting of recombinant protein to plasmamembrane.e.g. recombinant protein produced by fusing with tcell receptor membrane spanning domain.

• Targeting recombinant protein to exudates.

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Directing translational pathway

UK Eva stoger et al., (2003)11

• Sub cellular targeting can be used to increase yield.

• Secretary pathway is more suitable compartment forfolding and assembly than cytosolic.

• Recombinant protein pass through ER. In absence offurther signal, product is secreted in apoplast.

• Ab are less stable in apoplast. Ab protein can beretrieved in ER lumen using H/KDEL C-terminaltetra peptide tag. Ab produced by this are notmodified in golgi body.

Proteins Host plants

Tissue expression

Sub cellular targets

α – amylase Tobacco Leaves Apoplast

Avidin Corn Seeds Apoplast

Secretary

antibodies

Tobacco Leaves Apoplast

β – glucouronidase Brassica Seeds Oil bodies

Anti- oxazolone Tobacco Leaves ER

Xylanase Brassica Seeds Oil bodies

Anti-phytochrome Tobacco Leaves Cytosol

Anti β -1,4-

Endoglucanase

Potato Roots Cytosol

Hirudin Brassica Seeds Oil bodies

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Examples of recombinant proteins targeted to subcellular compartments in transgenic plants

Bulgaria kunka kamenarova et al., (2005)

Humanization of proteins

Plant derived recombinant protein tend to havecarbohydrate groups β(1→2)xylose and α(1→3)fucose.

It Lacks terminal galactose and sialic acid residueswhich are found in many mammalian glycoprotein.

Change in glycan structure may turn recombinantprotein immunogenic when administered tohuman.

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Strategies to humanize protein

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• The use of purified human β(1→4) galactosyltransferase enzyme for the in vitro modification ofplant derived recombinant protein.

• Expression of human β(1,4) galactosyl transferase intransgenic plants to produce recombinant Ab withgalactose extended glycans.

• Inhibition of fucosyl transferase and xylyl transferaseusing Ab, ribozyme, iRNA helps in removing plantspecific carbohydrates.

• Gene targeting by homologous recombination hasbeen used to produce recombinant proteins lackingplant specific glycans in moss phycomitrella patens.

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Host system

• Choice of host system affects overall cost,product quality , production timescale, scale upcapacity and biosafety.

• Various host system are used like bacteria, yeast,transgenic animals, plant cell cultures, transgenicplants.

• Plants are ideal host systems which are costeffective, rapidly scaled up, fewer ethical issuesand better public acceptance than transgenicanimals.

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Expression system

Advantages Disadvantages Cost per gram

Bacteria Established regulatory track; well-

understood genetics; cheap and easy to

grow

Proteins not usually secreted; contain

endotoxins; no posttranslational

modifications

Yeast Recognized as "safe;" long history of

use; fast; inexpensive; posttranslational

modifications

Overglycosylation can ruin bioactivity;

safety; potency; clearance; contains

immunogens/antigens

$50-100

Insect cells Posttranslational modifications;

properly folded proteins; fairly high

expression levels

Minimal regulatory track; slow growth;

expensive media; baculovirus infection

(extra step); mammalian virus can infect

cells

Mammalian

cells

Usually fold proteins properly; correct

posttranslation modifications; good

regulatory track record; only choice for

largest proteins

Expensive media; slow growth; may contain

allergens/contaminants; complicated

purification

$500-5,000

Transgenic

animals

Complex protein processing; very high

expression levels; easy scale up; low-

cost production

Little regulatory experience; potential for

viral contamination; long time scales;

$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

Comparison of host system

Aziz Elbehri (2006)

PLANT EXPRESSION HOST

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

Many factors need to be taken into consideration. Theyare

Yield of functional protein in given species.

Transformable capacity.

Biosafety concerns.

Storage and distribution of protein.

Cost of grain storage and distribution.

Cost of extraction and purification.18

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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.

Bulgaria kunka kamenarova et al., (2005)

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Leafy crops continued…

Alfa alfa and soybean give advantage of largedry biomass.

These crops also use atmospheric nitrogenthrough nitrogen fixation thus reducing cost offertilizers.

Lettuce is also used for edible vaccinesproduction.

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Cereals and legumes

Rice, wheat , pea, maize and soybean are used.

Maize gives high biomass yield , ease oftransformation, in vitro manipulation facilitiesand convenience of scale up.

Rice among cereals gives highest yield.

Disadvantage is of gene transfer via pollentransfer.

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Fruits and vegetables

The main benefit of fruit and vegetable is that theycan be consumed raw or particularly processedwhich makes them particularly suitable forsubunit edible vaccines.

Potatoes have been widely used for production ofplant derived vaccines and have beenadministered to humans in most of clinical trials.

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Tomatoes were used to produce the first plant-derived rabies vaccine and are more palatablethan than potatoes and offer high yield.

Bananas have been grown in developingcountries where vaccines are most needed. It canbe consumed raw or as puree by both adults andchildren.

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Alternative plant based production system

Plant cell suspension culture (derivation of hairyroots, shoot teratomas, immobilized cells, suspensioncell culture)

Requires simple, synthetic media, defined and sterileproduction conditions, inexpensive, carry out properglycosylation and folding of proteins.

Wide group of recombinant antibodies produced inBY2 tobacco and rice cell suspension culture includingfull size Ig, Fab Fragment, ScFv and fusion proteins.

Fischer et al.,.(1999)

Industrial proteins and enzymes.

Therapeutic and pharmaceutical proteins.

Plantibodies.

Plantigens.

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INDUSTRIAL PROTEINS AND ENZYMES

This group include hydrolyses encompassingboth glycosidases and proteases.

Avidin and β-glucouronidase (GUS) were the firstsuccessful commercial recombinant proteinproduced in corn and are marketed by sigma asresearch reagents.

All of these products are usually characterized bythe fact that they are needed in large amount anddon’t require high levels of purification.

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A.S.Rishi (2001)

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Industrial

enzyme

Potential

Use

Host

α- amylase Industry Tobacco

Phytase Industry Alfaalfa, tobacco

Manganese

peroxide

Industry Alfalafa, tobacco

β (1,4)xylanase industry Tobacco, canola

β (1,3)glucanase Industry Tobacco, barley

Avidin Research reagent Maize

Glucouronidase Research reagent Maize

Cellulase Industry Alfalafla, potato,

tobacco

industrial enzymes and proteins produced in different plant host system

A.S.Rishi (2001)

o Includes all proteins used directly as pharmaceuticalsalong with those proteins used in the making ofpharmaceuticals.

o The list of such proteins is long, ever growing, andincludes such products as thrombin and collagen(therapeutics), and trypsin and aprotinin(intermediates).

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A.S.Rishi (2001)

THERAPEUTIC AND PHARMACEUTICAL

PROTEINS

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Therapeutic protein Host Potential use

α and β haemoglobin Tobacco Blood substitute

Human serum albumin Potato Blood substitute

Glucocerebrosidase Tobacco Gaucher disease

α interferon Rice Viral protection

Protein C Tobacco Anticoagulant

Epidermal growth factor Tobacco Mitogen

Erythropoietin Tobaco Mitogen

Trout growth factor Tobacco Mitogen

Glutamate decarboxylase Tobacco Diabetes

Human somatotorpin Tobacco Hypo pituitary dwarfism

Calcitonin Potato Paget disease, osteoporosis,

β interferon Tobacco Neutropenia

Human granulocyte-macrophage Tobacco Neutropenia

Enkephalins Oilseed Antihyperanalgesic by opiate activity

Human homotrimeric collagen I Tobacco Collagen

α tricosanthin Tobacco HIV therapy

Angiotensis-1- convering enzyme Tobacco/ tomato hypertension

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

A.S.Rishi (2001)

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Product Class Indication Company Crop Status

Various single

chain Fv

antibody

fragments

Antibody Non-hodgkin’s

lymphoma

Large scale

biology corp.

Viral vectors

in tobacco

Phase-I

CaroRx Antibody Dental caries Planet

biotechnology

inc.

Transgenic

tobacco

Phase-II

E.Coli heat

labile toxin

Vaccine Diarrhoea Prodigene inc.

Arntzen group

Transgenic

maize

Transgenic

potato

Phase I

Phase I

Gastric lipase Therapeutic

enzyme

Cystic fibrosis Meristem

therapeutics

Transgenic

maize

Phase II

Hepatitis B

virus surface

antigen

Vaccine Hepatitis B Arntzen group Transgenic

potato

Phase I

Human intrinsic

factor

Dietary Vitamin B12

deficiency

Cobento biotech

AS

Transgenic

arabidopsis

Phase II

Lactoferrin Dietary Gastrointestinal

infections

Meristem

therapeutics

Transgenic

maize

Phase I

Norwalk virus

capsid protein

vaccine Norwalk virus

infection

Arntzen group Transgenic

potato

Phase I

Rabies

glycoprotein

Vaccine Rabies Viral vectors

in spinach

Phase I

Table 5. Plant - derived pharmaceutical proteins that are closest to commercialization for the

treatment of human diseases

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

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Drug Company Indication Approved

Aranesp Amgen Anemia due to kidney

failure, chemotherapy

May 1998

Campath Llex oncology, B cell chronic lymphocytic May 2001

Elitek Sanofi-synthelabo Pediatric oncology July 2002

Enbrel Amgen,wyeth Rheumatoid arthritis Dec 2002

Forteo Eli lilly Osteoporosis Nov 2001

LYMErix Smithkline Beecham

biologicals

Lyme disease prevention Dec 1998

Natrecor Scios Congestive heart failure August 2001

Pegasys Roche, inhale therapeutics Chronic hepatitis c Oct 2002

Rebif Serono, pfizer Multiple sclerosis Marich 2002

TNKase Genentech Acute myocardial infarction June2000

Zevalin IDEC B-cell non hodgkin’s

lymphoma

Feb 2002

Procrit Ortho biotech Anemia Feb 2000

Ovidrel Serono Infertility Sept 2000

Table no. 6 Some approved protein- based drugs

www.brucegoldfarb.com/FDL2.pdf

PLANTIBODIES

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stefan schillberg (2002)

Several functional antibodies fragment antigen-binding(Fab) and single chain antibody fragments (ScFv) canbe expressed in the leaves and seeds of plants withoutthe loss of binding specificity.

Expression of antibodies varies between different plantspecies, and a high level expression of scFvs wasachieved in tobacco leaves, 7% of TSP.

The first clinical trial using antibodies produced inplants was to prevent human tooth decay caused bystreptococcus mutans.

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Antibody

Determining titer value

Figure 2. Antibody molecular farming

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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

India S.R. Kathuria (2002)

PSSH1 PLANT EXPRESSION VECTOR CONSTRUCTS FOR

THE ANTI HCG RECOMBINANT ANTIBODIES

PROTEIN ANALYSIS OF ANTI HCG ANTIBODY

Affinity purified plant expressedrecombinant anti hCG antibodyanalyzed by SDS- PAGE

A: his 6 tag scFv, B: diabody, C:protein purified which gavepure antibody fragments

Individual expression of proteinsas light and heavy chains

LC: light chain, HC: heavy chain,NI: non-infiltrated, LC+HC: lightchain + heavy chain.

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India S.R. Kathuria (2002)

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Recombinant antibodies Host plant

system

Medical application

Ig G1 Tobacco First antibody expressed in plants; full length

serum IgG produced by crossing plants that

expressed heavy and light chains

Ig M Tobacco First Ig M expressed in plants and protein

targeted to chloroplast for accumulation

Ig A Tobacco First secretory antibody expressed in plants by

sequential crossing of four lines carrying

individual components; at present the most

advanced plant derived pharmaceutical protein

Ig G

HSV

Soybean First pharmaceutical protein produced in soybean

HBV envelope protein Tobacco First candidate expressed in plants ; third plant

derived vaccine to reach clinical trials stage

scFv of IgG from mouse B-

cell lymphoma

Tobacco Treatment of hodgkin’s lymphoma

scFvT84.66 against

carcinoembryogenic

antigen

Cereals Tumor associated marker antigen

Table no. 7 Recombinant antibodies produced in various plants

A.S.Rishi(2001)

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Edible vaccines

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•Vaccines

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Vaccines work by priming the immune system toswiftly destroy specific disease causing agents beforethey can multiply enough to cause symptoms.

This priming is achieved by presenting the immunesystem with whole viruses or bacteria that have beenkilled or attenuated.

Classical vaccines pose a risk of causing diseases thatthey suppose to prevent.

To avoid that subunit vaccines were discovered butthey are less effective and of high cost.

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Vaccine type Defination Immune response examples

Killed,

inactivated

Pathogen is killed, usually

through a chemical process

such as formalin

Evokes a robust immune

response that mimics most

of the responses seen

during an infection

Typhoid vaccine

Salk polio

vaccine

Live,

attenuated

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

Such vaccines evoke

broad immune responses

similar to that seen by the

host infected with the

natural pathogen

Oral Sabin polio

vaccine, Nasal

influenza vaccine

BCG vaccine

Sub unit

acellular

Well-defined part (or parts) of

the organism is purified and

used as an antigen (for

example, proteins,

polysaccharides, inactivated

toxins)

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

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

Immune response can be

modified and targeted by

insertion of specific

genetic sequences

Hepatitis B

vaccine

Table no.8 Vaccine types, defination and immune response

Sarah landry(2005)

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Edible vaccines

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• Edible vaccines are sub- unit vaccines where theselected genes are introduced into the plants andthe transgenic plant is then induced to manufacturethe encoded protein.

• The transgenic plants expressing the vaccineantigen, when eaten are expected to provide theantigenic stimulus that will generate an immuneresponse in the host.

• Edible vaccines are mucosal targeted vaccineswhere stimulation of both systematic and mucosalimmune network takes place.

4140

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Transplant seedling to soil

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TARGETING AND EXPRESSION OF ANTIGENIC

PROTEINS

Efforts to enhance expression levels of transgene

coding for antigenic proteins by exploiting

promoters, targeting sequences, and enhancer

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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Pathogen/disease Plant Promoter Antigen Targeting/enhancers/sigh

nal peptides/terminator

sequences

Vibrio cholera Potato mas P2 Capsid protein 2L2I SEKDEL

Cholera,

enterotoxigenic

E.coli, rotavirus

Potato mas P1-

mas P2

CTA2;CFA?- CTB;

NSP4

SEKDEL + CTB leader

Diabetes

(autoimmune)

Potato mas P2 Insulin C terminus of CTB

E. coli heat labile

enterotoxin B

subunit (LT-B)

Corn Ubi-1 LTB Codon optimised version

of barley α- amylase

signal sequence

Foot and mouth

disease

Arabidopsis 35S Structural protein

VP1

5‘ TEV

Hepatitis B Tobacco,

potato,

lupine,

lettuce

35S HbsAg 5‘ TEV leader, SEKDEL,

Herpes simplex

virus 2

Soybean 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

Table no.9 Antigens along with their constructs produced in transgenic

plants as candidate vaccine

Schuyler S. Korban (2002)

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Edible vaccine against hepatitis B

The DNA fragment encoding hepatitis B virus surfaceantigen was introduced into Agrobacteriumtumerifacience LBA4404 and used to obtain transgeniclupin and lettuce cv Burpee Bibb expressing envelopesurface protein.

Mice and Human volunteers, fed with transgeniclettuce plants expressing hepatitis B virus surfaceantigen, developed specific serum Ig-G response toplant produced protein.

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 HBsAGaccumulation in transgeniclupin callus and lettucelines.

Individual transgenics areindicated on x-axis.

Lupin and lettuce plantstransformed with A.tumifaciens vector withoutHBsAg was used ascontrol.

USA J. Kapusta (1999)46

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Figure 9. Titer of antibodies in three individuals(1-3)

immunized orally with transgenic lettuce harboring HBsAg

USA J. Kapusta (1999)46

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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.

Aziz Elbehri (2006)

NUMBER OF FIELD TEST PERMITS BY APHIS AS

PHARMCEUTICAL, INDUSTRIAL OR NOVEL TRAITS.

Crop Industrial

enzymes

Novel

proteins

Pharma

plants

Total %

Corn 11 157 63 231 71.1

Soybean 10 4 16 30 9.2

Alfalfa 2 1 1 4 1.2

Barley 1 1 2 0.6

Rapeseed 2 1 3 0.6

Tobacco 1 14 15 4.6

Tomato 1 1 0.3

Rice 1 2 8 11 3.4

Safflower 1 2 3 0.9

Wheat 2 2 0.6

Sugarcane 1 1 0.3

other 5 6 11 22 6.8 50

Aziz Elbehri (2006)

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Firms Work

AltaGen bioscience 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

Centocer Made momclonal antibody remicade (rheumatoid arthritis), reopro and retavase for

cardiac care arena.

Monsanto protein

technologies

Working on to improve glycosylation of corn plants and monoclonal antibody

Dow AgroSciences Monoclonal Ab in corn (R 19 for RSV), HX8 for HSV

CropTech Therapeutic proteins in tobacco

Epicyte projects in pipeline include production of Monoclonal Ab on HSV, human papiloma

virus, HIV, Alzheimer’s disease, ulcerative colitis and hepatitis viruses

Meristem

Therapeutics

Lead product is recombinant gastric lipase to treat cystic fibrosis

Large Scale

Biology

Best know for its work in proteomics, produces patient specific cancer vaccines in green

house- raised transgenic tobacco plants.

prodiGene Gm corn to produce vaccines, Ab, enzymes and other protein-based therapeutics.

Company is developing a candidate vaccine for HIV

Phytomedic Therapeutic proteins in genetically modified tobacco plants. Pipeline includes drug

candidates to treat autoimmune disorders, diabetes, viral infection and cancer.

Selected biotech firms of north America and Europe specializing in recombinant therapeutic proteins

www.brucegoldfarb.com/FDL2.pdf

BIOTECHNOLOGY IN INDIA

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The first biotech based vaccine released in indian marketwas rDNA hepatitis B vaccine produced by shanthabiotech Pvt.Ltd. Other biotech medication in marketinclude recombinant insulin, human growth hormone, αinterferon, blood clotting factor VIII, renin andinterleukin.

There are 50 companies work in advanced biotechapplication.

60% industry ---- human health

30% industry ----- bioinformatics and genomics

10% industry ----- agril.biotech

Lead domestic players include reliance life sciences, Dr.reddy’s laboratory, shantha biotech, panacea biotech andbiocon.

SELECTED DOMESTIC COMPANIES AND ACTIVITIES

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

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Company activity

Dabur india Ltd. Genomic and proteomic profiling to detect molecular changes in cancer

patients

Reliance life sciences Active in areas of stem cell research, medical , plant and industrial

biotechnology

Biocon Produced recombinant insulin, pectin and cholesterol lowering drugs.

Begun developing genetically engineered drugs

Shantha biotech Recombinant insulin and recombinant hepatitis B vaccine

Banglore genei Tools for genomic research, modifying enzymes, polymerase enzyme

etc

Dr. Reddy’s

laboratory

The company has a licensing agreement with Novo Nordisk for

diabetes therapeutic technology and is developing human therapeutic

proteins through rDNA technology

Panacea Biotech Ltd. 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

Environment contamination

1. unintended harm to other organisms. eg. Monarchbutterfly and caterpillars.

2. reduced effectiveness of pesticides

3. gene transfer to non target species

Economic concerns

1. patenting new GM plants varieties will raise price ofseeds so high that small farmers will not be able toafford seeds.

Health safety concerns

1. allergenicity 54

RISK AND CONCERNS

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

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www.brucegoldfarb.com/FDL2.pdf

Few unfertilized stalks of corn for the previousyear’s crops, engineered to express therapeuticproteins, contaminated soybean fields in lowa andnebraska.

$500,000 fine + $3 million to buy/destroycontaminated soybean

BREAKDOWN OF REGULATORY

SYSTEM: PRODIGENE INCIDENT 2002

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

1. EPA (evaluates GM plants for environmentalsafety).

2. USDA (evaluates whether plant is safe to grow).3. FDA (evaluates whether plant is safe to eat).

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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 recombinanttherapeutic proteins. Government must proceedcautiously in this area to gain public acceptance.

Transgenic plants can assemble and accumulate manycomplex valuable proteins which can be economicallyextracted or processed

Strong promoters, targeting sequences and othertranscriptional and/or translational sequences areoptimized for various crops to get optimal production.

Recombinant protein has showed high expression inplastids rather than nuclear genomes of transgenicplants.

Food as vehicles for production of edible vaccines andother therapeutic recombinant proteins is a novel fieldwhich should pay dividend for both human health andagricultural sector.

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FUTURE PROSPECTS Plants expressing uniform expression levels of the desired

antigen have to be identified in order to administer the correctdosage of vaccine.

Ethylene inducible genes linked to fruit ripening would allowinducible expression of the antigen. Since ripening affects thecolor of many fruits it may be possible to develop a correlationbetween the color of the fruit and the level of antigen, to ensurean adequate dosage of the vaccine

Secretion of recombinant proteins form roots and leaves willhave to be evaluated for cost effectiveness of production andproduct stability

Production of industrial enzymes in tree species, where highbiomass is available, should be evaluated for commercialexploitation.

The emerging fields of genomics, proteomics and metabolomicswill provide tools for molecular farming to cure severaldisorders.

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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 person’s lungs. Why not

we re-engineer tobacco for an enjoyable way to gain immunity?

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Thank you……