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Plant molecular farming for recombinant therapeutic proteins
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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.
4
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
6
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.
11
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.
26
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
32
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
36
•Vaccines
38
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)
45
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)
51
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
52
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
53
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
55
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).
56
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
57
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.
58
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……