1. Future directions for agricultural biotechnology
Dr. Kirstin Carroll
Outreach in Resource Biotechnology Program
Oregon State University

2. Lecture Outline What is molecular farming in plants? Why use plants?
What are the risks and concerns?
Current and evolving regulation

3. What is 'molecular farming in plants'?
The use of agricultural plants for the production of useful molecules for non food, feed or fiber applications.
Plants are already grown to produce valuable molecules, including many drugs.
Molecular farming is different because the plants are genetically engineered (GE) to produce the molecules we want them to.

4. What is GE?
Create recombinant DNA with gene from same or different organism
Transfer DNA to plant cell (use either Agrobacterium or ‘ballistic’ transformation)
Confirm introduced DNA and expression of foregin protein in plant
What is included in the recombinant DNA?
On/Off switch
Gene of interest
Marker gene
Environment contaminantion via gene flow
Contamination of food supply
Secondary metabolite – inctroduct allerginiicty or toxicity

5. Plant Products Plant derived pharmaceuticals (non-GE)
Over 120 pharmaceutical products currently in use are derived from plants. Mainly from tropical forest species
An example of recent plant pharmaceuticals is taxol, a secondary plant product derived from the bark or needles of the Pacific Yew tree. Taxol has been found to be effective against certain types of cancer. A synthetic form of taxol is being developed, with the goal of producing larger quantities of taxol and reducing damage or destruction to Pacific Yew trees.

6. Plant Products Plant-derived pharmaceuticals (non-GE)
2. Plant-made pharmaceuticals and industrial products (GE)
Industrial products proteins
enzymes
modified starches
fats
oils
waxes
plastics
Pharmaceuticals
recombinant human proteins
Therapeutic proteins
enzymes
Antibodies (plantibodies)
vaccines

7. Strategies for ‘Molecular Farming’
Plant gene expression strategies
Transient transformation
Stable transformation
Chloroplast transformation

8. Strategies for ‘Molecular Farming’
Plant gene expression strategies
2. Location of trans-gene expression?
Protein quantity and preservation
Whole plant
Target specific tissues (e.g. seed, root)
Whole plant – e.g. tobacco. Large amts of protein in the green matter of the plant – poor preservation though
Seed – high quanitites of protein are stored in the seed and root of most plants (soy, corn). Seeds also are attractive for long-term storage of the protein.

9. Strategies for ‘Molecular Farming’
Plant gene expression system
2. Location of trans-gene expression?
3. Selection of plant species and characteristics
Mode of reproduction – self/outcrossing
Yield, harvest, production, processing
Whole plant – e.g. tobacco. Large amts of protein in the green matter of the plant – poor preservation though
Seed – high quanitites of protein are stored in the seed and root of most plants (soy, corn). Seeds also are attractive for long-term storage of the protein.
Green matter – tobacco, alfalfa, duckweed
Seed – corn, rice, barley
Want plants that have been well-studied, transformable
Yield
Harvest
Production
Processing

10. Why use plants? Disadvantages Advantages Environment contamination
Food supply contamination
Health safety concerns
Advantages
Cost reduction
Stability
Safety

11. Examples of Industrial PMPs
Cellulase for production of alcohols
Avidin – medical diagnostics
b-glycoprotein – biomedical diagnostics
Plant-derived plastic:
Production of polyhydroxyalkanoate (PHA)
To date, more costly than fuel-based plastic

12. Examples of Industrial PMPs
High wax esters
Jojoba seeds - gene has been isolated and expressed in Arabidopsis (49-70% oil present as wax)
Astaxanthin
red pigment in shell-fish.
used in aquaculture
Compounds to increase flavor and fragrances

13. Plant-made Vaccines Edible vaccines Advantages: Administered Directly
no purification required
no hazards assoc. w/injections
Production
may be grown locally, where needed most no transportation costs
Naturally stored
Most vaccines consist primarily of killed or weakened version of a pathogen.

14. Plant-made Vaccines
Examples of edible vaccines ; pig vaccine in corn, HIV-suppressing protein in spinach, human vaccine for hepatitus B in potato.
Most vaccines consist primarily of killed or weakened version of a pathogen.

15. Plantibodies - Plants can be used to produce monoclonal antibodies
Tobacco, corn, potatoes, soy, alfalfa, rice
Free from potential contamination of mammalian viruses
Examples: cancer, dental caries, herpes simplex virus, respiratory syncytial virus
Most vaccines consist primarily of killed or weakened version of a pathogen.
**GE Corn can produce up to 1 kg antibody/acre and can be stored at RT for up to 5 years!
Humphreys DP et al. Curr Opin Drug Discover Dev 2001; 4:

16. Plant made Pharmaceuticals
Therapeutic proteins
Blood substitutes – human hemoglobin
Proteins to treat diseases
CF, HIV, Hypertension,
Hepatitis B…..many others
Most vaccines consist primarily of killed or weakened version of a pathogen.
**To date, no plant-produced pharmaceuticals are commercially available.

17. Current ‘Pharm’ Companies
Planet Biotechnology
Dental Caries: CaroRx™
Colds due to Rhinovirus: RhinoRx™
Drug-induced Alopecia: DoxoRx™
LEX System™
Lemna, (duckweed)
Rhizosecretion
Monoclonal antibodies (Drake et al., 2003)
Recombinant proetins (Gaume et al, 2003)
GeneWare® which involves growing a normal tobacco plant and then infecting it with a virus called Tobacco Mosaic Virus containing the foreign gene of interest. The virus has been tamed so that it does not spread or infect other plants. The foreign protein concentrates in the leaves. The leaves can be harvested and processed to isolate the therapeutic protein.
PLANET is focusing on the development of therapeutic and preventative drugs in four large, but significantly underserved medical markets:
Dental Caries: Tooth decay caused by bacterial infection results in approximately 70% of U.S. dental service expenditures, or approximately $50 billion annually, current "preventatives" notwithstanding. The combined treatable population in the U.S. and Europe is estimated at approximately 115 million people. For dental caries, Planet is developing CaroRx™.
Colds due to Rhinovirus: Rhinoviruses account for approximately half of all common colds and over 20 million doctors’ office visits a year. No readily available diagnostic exists to distinguish between rhinovirus-based colds and the myriad of other variants. Commonly prescribed antibiotics are completely ineffective in treating colds and available palliative preparations are generally ineffective after a single day of use. For rhinovirus colds, Planet is developing RhinoRx™.
Drug-induced Alopecia: Hair loss (alopecia) is one of the most disturbing side effects for cancer patients undergoing chemotherapy, and is commonly caused by doxorubicin and related anthracycline drugs. Over 250,000 patients in the U.S. receive anthracycline chemotherapy each year, and over 95% of these patients lose their hair. Currently, there is no way of preventing this side effect of chemotherapy. For chemotherapy drug-induced alopecia, Planet is developing DoxoRx™.
Biomass biorefinery based on switchgrass. Produce PHAs in green tissue plants for fuel generation.

18. Current ‘Pharm’ Companies
Kentucky Tobacco Research and Development Center
Trangenic tobacco
GeneWare®
Trangenic tobacco
PMPs and non-protein substances (flavors and fragrances, medicinals, and natural insecticides)
Controlled Pharming Ventures
In collaboration w/Purdue
Transgenic corn
Converted limestone mine facility

19. Current ‘Pharm’ Companies
Ventria Bioscience
Transgenic rice
Lactoferrin
Lysozyme
Prodigene
Transgenic corn
Trypsin and Aprotinin
Aprotinin – serine protease inhibitor. Purified from bovine lung tissue. Applications in cell culture, protein purification, diagnositc tests, and in cardiopulmonary bypass surgenery…inhibits proteases in wound care.
Trypsin –
Lactoferrin and lysozyme are human proteins found in breast milk and most epithelial surface secretions (tears, saliva, etc). Could be used to enhance human gastrointerstial health and for treatment of topical infections and inflammations. (has anti-viral, anti-fungal, antioxidant, anti-bacterial properties)

20. Examples of Current Research
Genetically engineered Arabidopsis plants can sequester arsenic from the soil. (Dhankher et al Nature Biotechnology)
Immunogenicity in human of an edible vaccine for hepatitis B (Thanavala et al., PNAS)
Expression of single-chain antibodies in transgenic plants. (Galeffi et al., 2005 Vaccine)
Arizona State University….have abandoned potato project and started using tobacco instead.
Plant based HIV-1 vaccine candidate: Tat protein produced in spinach. (Karasev et al Vaccine)
Plant-derived vaccines against diarrheal diseases. (Tacket Vaccine)

21. Risks and Concerns Environment contamination Gene flow via pollen
Non-target species near field sites e.g. butterflies, bees, etc
Food supply contamination
Accident, intentional, gene flow
Health safety concerns
Non-target organ responses
Side-effects
Allergenicity
USDA list - does not reveal the location of the crop or the type of protein being produced.
the first clinical trials involve pharmaceuticals, such as the anti-caries CaroRX, that are not ingested or injected
To avoid the consumer backlash seen in Europe and elsewhere against genetically modified crops, companies producing biopharmaceuticals are also eager to maintain a division in the public's thinking between crops grown for food and pharmacological purposes.

22. U.S. Regulatory System (existing regulations)
USDA
FDA
EPA
Field Testing
-permits
-notifications
Determination of non-regulated status
Food safety
Feed safety
Pesticide and herbicide registration

23. Breakdown of Regulatory System: Prodigene Incident 2002
2001 : Field trails of GE corn producing
pig vaccine were planted in IA and NB.
2002: USDA discovered “volunteer”
corn plants in fields in both IA and NE.
Soy was already planted in NE site.
$500,000 fine + $3 million to buy/destroy contaminated soy

24. USDA Response to Incident
Revised regulations so that they were distinct from commodity crops:
Designated equipment must be used.
At least 5 inspections/yr.
Pharm crops must be grown at least 1 mile away from any other fields and planted 28 days before/after surrounding crops

25. Current Evolving Regulations
FDA/USDA Guidance for Industry on Plant-Made Pharmaceuticals Regulations
November 2004: Draft Document
Other challenges:
Industrial hygiene and safety programs
Industrial hygiene and saftey: Must include: depends on activity of protein, route of exposure, inability to obtain relevant animal data because of high species-specificty…….need to ensure employee health. (Goldstein, 2005)

26. ‘Molecular farming’ in the US
Since 1995 ~ 300 biopharming plantings
USDA has received 16 applications for permits in the last 12 months.

27. ‘Molecular farming’ opposition
Concerns:
CONTAINMENT – opponents want a guarantee of 0% contamination of the food supply.
Full disclosure of field trials, crop, gene, location, etc.
Extensive regulatory framework

28. Suggested Safeguards for ‘molecular farming’
Physical differences
E.g. “purple” maize, GFP
Sterility
Use male sterile plants
Terminator technology?
Easily detectable by addition of 'reporter genes'
PCR markers
(avoid antibiotic resistance markers)

29. Suggested Safeguards for ‘molecular farming’
4. Chloroplast expression system
Increase yield
Eliminates potential gene flow
Technically difficult (Chlorogen Company)
5. Complete disclosure of DNA sequences
6. Legislate for administration.
Pblms with industrial espionage
Patent offices?
Health officials administer

30. Alternatives to ‘molecular farming’?
Use only traditional drug production systems microbial, yeast and fungi
mammalian cell culture
Use only fully contained production systems:
Plant cell cultures
Hydroponics (rhizosecretion)
Greenhouses
Use non-food crops Tobacco, Hemp/Cannabis
many of the newly engineered proteins in plants will occur anyway, naturally, in frequently eaten meat foodstuffs, and will pose little if any danger to the human food chain. Future legislation needs to be on a crop by crop, protein by protein basis

31. Economics
The expectation is for lower production costs however there is no evidence that pharming will produce cheaper, safe drugs.
There are unknown costs associated with containment, litigation and liability, production…..others?
Rice – silencing of genes expressing the allergenic protein (Tada et al, FEBS Lett 1996)
Attempts to do same thing in peanuts and wheat (Bannon et al, Int Arch Allergy Immunol 1999)