Plants as Bioreactors

NISHA G V 3RD SEMESTER MSc BIOTECHNOLOGY

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Is a method used to integrate a foreign gene into plants for the production of commercial products Two types Medical: includes pharmaceutical products Non-Medical: includes Industrial Enzymes and Polymers

MOLECULAR FARMING

Industrial enzymes: laccase in transgenic maize, avidin in maize

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The first synthesis of a pharmaceutically relevant protein was human growth hormone in transgenic tobacco plants in 1986 In 1989 the first antibody was expressed in tobacco which showed that plants could assemble complex glycoprotein with several subunits
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WHY PLANTS?
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Plants are very flexible Crop plants can synthesize a wide variety of proteins that are free of mammalian toxins and pathogens Crop plants produce large amounts of biomass at low cost and require limited facilities Well suited for the therapeutic proteins
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production

of

safe
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low-cost

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HOW IS IT DONE?

Genetic enhancement is used to introduce and express genes coding for the high value proteins Through careful and skilled manipulation of genes Controlled expression of a specific gene which then expressed in a transgenic plant 4/22/12 66

Target protein can be expressed in a single plant tissue or during a specific developmental phase of the plants growth cycle Thus the production of any target protein 77

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PHARMACEUTICAL PROTEINS

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STRATEGIES FOR PROTEIN PRODUCTION

STABLE EXPRESSION

Strong, constitutive promoter (35S)

Bulk production of soluble proteins in leaves But yields may low

Targeted gene expression

Higher yields Storage organs helps to maintain biological integrity

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TRANSIENT EXPRESSION

HIRUDIN PRODUCTION IN Brassica napus

Anticoagulant peptide of 7 kDa found in salivary glands of the leech Hirudo medicinalis Inhibitor of thrombin Low immunogenicity, not require endogenous cofactors for activity and not interfere with other blood proteins Expressed in Brassica napus

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An endoprotease Factor Xa cleavage site was engineered between the C- terminus end of the oleosin and N- terminus end of the Hirudin Protein produced in oil bodies Purification

centrifugation of oil bodies treat them with endoprotease Factor Xa Re-centrifugation will be present in aqueous 1111 phase

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Glcocerebrosidase

Glucocerebroside into glucose and ceremide as aged red blood cells are removed by body Gauchiers disease Carole Cramer at Crop Tech Development corporation at Virginia developed transgenic tobacco

Human Serum Albumin

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35S promoter in tobacco

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Plant Transformation

The plant leaf disc is dipped in a solution of bacteria. The bacterial "Trojan Horse" infects the edges of the leaf disc and in the process integrates the pharmaceutical protein gone into the plant genome After infection the discs are placed on selection media that a flows only plant cells that carry the protein gene to survive and regenerate into plantlets. After about six weeks on selection media, a large number of plantlets that carry the pharmaceutical 1414 protein gene are visible at

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The plantlets are removed from the leaf disc and placed in clear plastic boxes that contain media that allows them to form roots The rooted plantlets are placed in pots and plants are allowed to grow and produce seed. This seed can then be used for large scale production of the 4/22/12 pharmaceutical protein

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Plantibodies

Plants can be used to produce monoclonal antibodies Tobacco, corn, potatoes, soy, alfalfa, rice Free from potential contamination of mammalian viruses GE Corn can produce up to 1 kg antibody/acre and can be stored at RT for up to 5 years
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AVICIDIN

IgG produced in corn plants showed anticancer activity side effects so product withdrawn ScFV based on idiotype of malignant B lymphocyte in mouse lymphoma cell line for non Hodgkin lymphoma

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CaroRX IgG/A in transgenic tobacco expression 4 4/22/12 separate transgene 1919

Full size monoclonal antibodies recently produced in transgenic plants

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Abs expressed in transgenic plants

Constant domains
Variable Light chain

Variable Heavy chain

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Recombinant hinge region 2121

Production Costs for Production cost in gr Antibodies costs $/ am hybrido 10

mas transgenic animals transgenic plants Source: Daniell et al. (2001) TIPS 6,
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00 1 0 1 0 0

E. coli & 4/22/12yeast

Tr. animals and animal cells

Transgenic 2222plants

Comparison of Mammalian and Plant-produced Antibodies

peptide sequence: identical correct cleavage of Ig-derived signal peptides kinetics & affinity: identical stability in seeds > 30 months antibody types: plant system more versatile (sIgA) post-translational processing: different

core glycan identical, terminal sugar different plus xylose & fucose antigenicity & clearance: (shorter half-life) apparently identical

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Transgenic scFv expressing tobacco plants (in the background) and control plants (in the foreground) 14 days after infection with different plant viruses 4/22/12 Source: AIPlanta Institute 2424

Plant produced Vaccines

Measles virus haemagglutinin Porcine TGEV Tetanus toxin Vacinia virus B5 Allergy vaccines HPV HIV gp41 and HIV-suppressing protein in spinach, Human vaccine for hepatitis B in potato Newcastle disease virus Rotavirus VP7
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PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF INFECTIOUS DISEASES

DISEASES Respiratory Syncytial Hepatitis B HIV Rabies Anthrax Diphtheria SARS Smallpox VACCINES PLANT Tobacco Lettuce Spinach Spinach Tobacco Tobacco Tomato Tobacco Tobacco Tomato Tobacco Tomato Tobacco ANTIBODY PLANT

PLANT-DERIVED BIOMEDICALS FOR TREATMENT OF CANCER

HUMAN DISEASES Colorectal Cancer Epithelial Tumors (EGF receptor) VACCINES ANTIBODY

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PMP Development - Highlights

Series of plant-derived vaccines from Arizona State University have completed clinical trials Prodigene has trialled two plant-derived vaccines LSBC pipeline of cancer vaccines prior to insolvency Guardian Bioscience coccidiosis vaccine, CFIA phase II ongoing Fraunhofer CMB, rabies vaccine trialled in humans DowAgro Newcastle disease vaccine, approved Feb 2006 Heberbiovac (Cuba) approved antibody for HepB vaccine purification 4/22/12 2727

EDIBLE VACCINES

Concept introduced by Charles Arntzen of Texas University, USA Hiatt and coworkers in 1989 First model system was tobacco Now produced in banana, tomato and potato How????

vaccines present in food shown to induce immune response Leave behind the memory cells High mucosal immunity
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EDIBLE VACCINES FOR HUMAN DISEASES

First published report on cell surface adhesion protein SpaA in transgenic tobacco plants Arnitzens group developed tobacco plants producing hepatitis B

HBsAg linked to 35S promoter 0.01% of soluble protein to VLPs

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Advantages: Administered Directly no purification required no hazards assoc. w/injections Production may be grown locally, where needed most no transportation costs

Naturally stored
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METABOLIC ENGINEERING OF CARBOHYDRATE

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Starch
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Chemical feed stocks: cereals, potatoes Alternation of ADP glucose pyrophosphorylase This enzyme in E.coli was mutated to alter its allosteric properties and transferred to potato Amylose to amylopectin ratio
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Food industry: GBSS1 down regulation produce amylose free starch Feed stock: SBE A and B down regulation
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Cyclodextrin
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Used in medical industry as solubilization of pharmaceuticals Made of glucopyranose seven membered ring of

Transgenic Potato tuber produced by transferring glycosyl tranferase gene from Klebsiella pneumoniae by fusing gene to plastid targetting sequence driven by patatin gene promoter Synthesized and stored in cellular vacuoles

Polyfructans
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Expressed chichory fructosyl transferase in 4/22/12 3333 onion

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BIOPLASTICS

Form of plastics derived from plant sources such as sweet potatoes, sugarcane, hemp oil, soy bean oil and corn starch Environmental friendly Biodegradable
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Plant Oil

COMPOSITION
Starch

Cellulose

Corn

Sugarcane

Potato

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 Starch based plastics: Bioplastics produced from classical chemical synthesis from bio based monomers Polylactic Acid (PLA) plastics: Bioplastics produced directly by natural or genetically modified organisms Polyamides 11: Polycaprolactones
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Classification of Bioplastics

Thermoset polymers Soy polyurethanes Unsaturated polyester resins Thermoplastic polymers Polylactic acid Modified starch polymers Thermoplastic polyesters Bio-polyolefins Polyhydroxy alkonates (PHA)
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Cellulose ethers

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Enzymes are used to break starch into glucose, which is fermented to lactic acid

This lactic acid is polymerized and converted into a plastic called polylactic acid, which can be used in the manufacture of products 4/22/12 3838 after being heated and shaped

Bioplastic Production in Switchgrass Plants

Metabolix, Inc. recently-completed greenhouse trials, switchgrass plants engineered using Metabolix multi-gene expression technology produced significant amounts of PHA bioplastics in leaf tissues This result is the first expression of a new functional multi-gene pathway in switchgrass
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Spring water bottle made from bioplastic

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COMPANIES

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Applications
Packaging
shopping bags Trays and containers

Catering products
Disposable crockery and cutlery

Gardening
flower pots
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Medical Products

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Bioplastics production in India

Jammu and Kashmir will be the first state in India to have a fully dedicated Bioplastics product manufacturing facility with an installed capacity of about 960 metric tones per year The J&K Agro Industries Ltd is going for a joint venture with EARTHSOUL for the manufacture of 100 per cent bio-degradable and compostable products. This would be Indias first integrated biopolymer facility

The facility would be manufacturing flower pots and trays for floriculture, carry bags for all shopping 4/22/12 4343 applications, outer packaging material for foodstuff

Industrial enzymes

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Phytase

Pytate into inositol and organic phosphate High quantities in seeds used as feeds to pigs and poultry animals Eutrophication Transgenic plant seed with this enzyme
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PRODUCTS CLOSE TO MARKET

PRODUCT Avidin collagen Lipase Lactoferrin COMPANY Prodigen Medicago, Prodigen Meristem therapeutics Ventria, Meristem therapeutics Ventria Ventria Large scale biological, Prodigen LOCATION Texas Canada, Texas California France PLANTS Maize Alfa alfa, Maize Maize Rice USE Imunodiagnostic Skin sealent Scar treatment Cystic fibrosis

Lysozyme Brazzein Aprotinin 4/22/12

California, France Texas California, Taxas

Maize, Rice Maize Maize, Tobacco

Natural defence protein Antiviral, antibacterial Natural protein sweetner 4747

GENETIC APPROACH

Expression construct is optimized for high yield Promoter and polyadenylation site derived from 19S and 35S of CaMV required in Franssinic strategy Ubiquitin-1 promoters for monocots Intron in the 5 untranslated region Tissue 4/22/12 specific promoters: Zein gene promoter, 4848

CHOICE OF PLANT PRODUCTION SYSTEM

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LEAFY CROPS
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Tobacco for pharmaceutical products

Gene transfer technology for in vitro regeneration Gene expression and high biomass yield Phyllosecretion and rhizosecretion

Alfa alfa, soyabean (Atm. N2 fixation) for rAbs

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FRUITS AND VEGETABLE CROPS

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Edible organs can be consumed uncooked and unprocessed Ideal for rSubunit vaccines, nutriceuticals and antibodies Potatoes for vaccine and tomatoes for rabies vaccine

FIBRE AND OIL CROPS

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Flax, cotton, oil seed rape reduction due to secondary revenues 5050

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BENEFITS

LIMITATIONS

Environment Low cost and Large scale production contamination

Capital and running cost

Food contamination are low

supply

Health safety concerns

Scalability PRODUCT AUTHENTICITY

Plants safer than microbes Ability to fold and assemble complex proteins Serum proteins, Secretary Abs

Stability and Safety

Only difference in post translational modification like glycan chain structure 4/22/12 5151

Current Challenges in Molecular Farming

yield of recombinant proteins real quantitative comparison protein stability (proteases) post-translational modification and downstream processing trials & regulatory approval

extraction clinical

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Regulatory Challenges
loci

of transgene insertion properties and levels

expression effects

of the transgene on the expression of flanking endogenous genes line banking to ensure product consistency with animal excreta, pesticides,

master

contamination

organic fertiliser
procedures

for detection and removal of weeds and

pests
cultivation
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variables
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Roadmap Plants for the Future

Plant production platforms

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Health food and quality

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Amino acids Oil Starch

Efficient agriculture
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Plant protection
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Vitamines Fatty acids Enzymes Bio-polymers Pigments Pharmaceutical products Fibers

Bt technology Herbicide resistance

Viruses Nematodes Fungi Insects

Stress resistance
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Cold Drought Salinization

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Planet Biotechnology Dental Caries: CaroRx

Current Pharm Companies

LEX System Lemna, (duckweed)

Colds due to Rhinovirus: RhinoRx Drug-induced Alopecia: DoxoRx

Rhizosecretion Biomass biorefinery based on switchgrass. Monoclonal antibodies Recombinant proetins Produce PHAs in green tissue plants for fuel generation.
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Kentucky Tobacco Research and Development Center Trangenic tobacco PMPs and non-protein substances (flavors and fragrances, medicinals, and natural insecticides)

Trangenic tobacco GeneWare

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Ventria Bioscience Prodigene Transgenic corn Trypsin Aprotinin

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Transgenic rice Lactoferrin Lysozyme

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Examples of Current Researchengineered Arabidopsis plants can Genetically

sequester arsenic from the soil (Dhankher et al. 2002 Nature Biotechnology)

Immunogenicity in human of an edible vaccine for hepatitis B (Thanavala et al., 2005. PNAS) Expression of single-chain antibodies in transgenic plants (Galeffi et al., 2005 Vaccine) Plant based HIV-1 vaccine candidate: Tat protein produced in spinach (Karasev et al. 2005 Vaccine) Plant-derived vaccines against diarrheal diseases 4/22/12 5858 (Tacket. 2005 Vaccine)

Risks and Concerns

Environment contamination Gene flow via pollen Non-target species near field sites Food supply contamination Accident gene flow Health safety concerns Non-target organ responses Side-effects Allergenicity
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Field Testing -permits -notifications

USD A

U.S. Regulatory System (existing regulations)

Food safety Feed safety

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EPA

Determination of non-regulated status

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Pesticide and herbicide registration

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Suggested Safeguards for molecular farming

1.

Physical differences E.g. purple maize, GFP Sterility Use male sterile plants and Terminator technology Easily detectable by addition of 'reporter genes' PCR markers (avoid antibiotic resistance markers)

1.

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4. Chloroplast expression system Increase yield Eliminates potential gene flow Technically difficult (Chlorogen Company) 5. Complete disclosure of DNA sequences
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REFERENCES

PLANT MOLECULAR BIOTECHNOLOGY S. MAHESH Biotechnology U satyanarayana Biotechnologies Pharming.ppt Influencing Agriculture: host Molecular and

Molecular farming in plants: expression technology. ppf Paul Christou and Rainer Fischer

systems

Richard M. Twyman, Eva Stoger, Stefan Schillberg,

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Action FA0804: Molecular farming: plants as a production platform for high value proteins.pdf Update on Agricultural Biotechnology Controversies Pharmaceutical Crops.pdf Allan Felsot Use of plant roots for phytoremediation and molecular farming.pdf DOLORESSA GLEBA PNAS Biopharmaceuticals derived from genetically modified plants.pdf D.A. GOLDSTEIN and J.A. THOMAS Molecular Farming: Plants as New Product Producers.pdf
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