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

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

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.

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

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.

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

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

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.

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

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

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

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

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.

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.

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; 4:172-85.

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.

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.

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

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)

Examples of Current Research Genetically engineered Arabidopsis plants can 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) 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. 2005 Vaccine) Plant-derived vaccines against diarrheal diseases. (Tacket. 2005 Vaccine)

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.

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

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

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

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)

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

‘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

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)

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

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

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)