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

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

3. The use of agricultural plants for the production of useful molecules for non food, feed or fiber applications. (also called molecular farming, pharming, or biopharming) What is biopharming?

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

5. Plant Products Over 120 pharmaceutical products currently in use are derived from plants. Mainly from tropical forest species (e.g. Taxol from Yew trees) 1. Plant derived pharmaceuticals (non-GE)

6. Industrial products proteins enzymes modified starches fats oils waxes plastics Pharmaceuticals recombinant human proteins therapeutic proteins and pharmaceutical intermediates antibodies (plantibodies) vaccines 2. Plant-made pharmaceuticals (PMPs) and industrial products (PMIP) (GE) 1.Plant-derived pharmaceuticals (non-GE) Plant Products

7. Strategies for Biopharming 1.Plant gene expression strategies Transient transformation adv. – quick and easy production disadv. – small amount of product, processing pblms Stable transformation adv. – use for producing large quantities of protein, stability and storage disadv – gene flow - outcrossing w/native species Chloroplast transformation adv. – reduce gene flow through pollen disadv. – protein not stable for long periods of time therefore complications w/extraction/processing times

8. 1. Plant gene expression strategies Protein quantity and preservation Whole plant adv. - an obtain large amts of protein disadv. - problems w/preservation examples - tobacco, alfalfa, duckweed Target specific tissues (e.g. seed, root) adv. - high amts of protein in seed/root, long-term storage capability. examples: soy, corn, rice, barley 2. Location of transgene expression Strategies for Biopharming

9. 1. 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 Strategies for Biopharming

10. Advantages Cost reduction - scalability (e.g. Enbrel® ) - low/no inputs - low capital cost Stability - storage Safety - eukaroytic production system - free of animal viruses (e.g. BSE) Why use plants? Disadvantages Environment contamination - gene flow - wildlife exposure Food supply contamination - mistaken/intentional mixing w/human food Health safety concerns - Variable, case-specific

11. Avidin by Sigma transgenic corn traditionally isolated from chicken egg whites used in medical diagnostics GUS (  -glycuronidase) by Sigma transgenic corn traditionally isolated from bacterial sources (E.Coli) used as visual marker in research labs Trypsin by Sigma transgenic corn traditionally isolated from bovine pancreas variety of applications, including biopharmaceutical processing first large scale transgenic plant product Worldwide market = US$120 million in 2004 Industrial products on the market

12. Industrial products close to market

13. Plant- made vaccines (edible vaccines) Plant-made antibodies (plantibodies) Plant-made therapeutic proteins and intermediates Unlike PMIPs, no PMPS are currently available on the market Plant-made Pharmaceuticals (PMPs)

14. 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 no need for refrigeration or special storage Plant-made Vaccines

15. Examples of edible vaccines under development: pig vaccine in corn HIV-suppressing protein in spinach human vaccine for hepatitis B in potato Plant-made Vaccines

16. Plantibodies - monoclonal antibodies produced in plants Plants used include tobacco, corn, potatoes, soy, alfalfa, and rice Free from potential contamination of mammalian viruses Examples: cancer, dental caries, herpes simplex virus, respiratory syncytial virus Plantibodies

17. **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. Dental Caries MAb– expected to reach the market soon MAb directed against genital herpes – estimated to reach market within 5 years (Horn et al, 2004)

18. Therapeutic proteins and intermediates Blood substitutes – human hemoglobin Proteins to treat diseases such as CF, HIV, Hypertension, Hepatitis B…..many others Plant made Pharmaceuticals

19. **To date, there are no plant-produced pharmaceuticals commercially available Plant made Pharmaceuticals Patient advocacy groups: American Autoimmune Related Diseases Association Arthritis Foundation Cystic Fibrosis Foundation

20. Current ‘Pharm’ Companies

21. LEX System™ Lemna (duckweed) trangenic tobacco PMPs and non-protein substances (flavors and fragrances, medicinals, and natural insecticides) Kentucky Tobacco Research and Development Center Controlled Pharming Ventures collaboration w/Purdue transgenic corn converted limestone mine facility

22. Rhizosecretion Monoclonal antibodies (Drake et al., 2003) Recombinant proteins (Gaume et al, 2003) biomass biorefinery based on switchgrass. used to produce PHAs in green tissue plants for fuel generation. Current ‘Pharm’ Companies

23. 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) Examples of Current Research 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. (Tacket. 2005 Vaccine)

24. 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 Risks and Concerns

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

26. Breakdown of Regulatory System: Prodigene Incident 2002 2001 : Field trails of GE corn producing pig vaccine were planted in IA and NE. 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

27. 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

28. FDA/USDA Guidance for Industry on Plant-Made Pharmaceuticals Regulations November 2004: Draft Document Other challenges: Industrial hygiene and safety programs – these will depend on the activity of the protein, route of exposure. Difficulty in obtaining relevant data because of high species-specificity. (Goldstein, 2005) Current Evolving Regulations

29. www.ucsusa.org Biopharming field trials in the US Since 1995 ~ 300 biopharming plantings The USDA receives/reviews applications for permits for biopharm trials.

30. www.ucsusa.org Biopharming field trials in the US http://go.ucsusa.org/food_and_environment/pharm/index.php?s_keyword=XX US Pharma Crop Database

31. Biopharming in Colorado

32. Biopharming in N.Carolina

33. The 2005 Oregon Biopharm Bill

34. Main concern is containment. Opponents want: a guarantee of 0% contamination of the food supply. full disclosure of field trials, crop, gene, location, etc. an extensive regulatory framework Biopharm opposition

35. 1. Physical differences e.g. “purple” maize, GFP 2. Sterility male sterile plants terminator technology 3. Easily detectable by addition of 'reporter genes‘ e.g. PCR markers Suggested Safeguards for biopharm operations

36. 4.Use chloroplast expression system will help increase yield will eliminate potential gene flow via pollen disadv. = technically difficult (Chlorogen Company) 5. Complete disclosure of DNA sequences 6. Legislate for administration Suggested Safeguards for biopharm operations

37. 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 Alternatives to biopharming?

38. The expectation is for lower production costs however there is no evidence that pharming will produce cheaper, safe drugs. Moreover, there are unknown costs associated with containment, litigation and liability, production…..others? Economics

39. Future directions for agricultural biotechnology? Public perception of risk Regulation Science has developed genetically enhanced crops and has/can develop plant-made industrial and pharmaceuticals crops. The extent to which these crops will be further developed for commercial and/or humanitarian use will ultimately depend on…..

40. Discussion Questions: Do you think nutritionally enhanced plants should be developed even though there are oral supplements available? Why or why not? Do you support the development of pharm crops? Do you feel that the potential benefits of pharm crops are worth the potential risks? What are your thoughts on using food vs. non- food crops as “phactories” for pharmaceutical or industrial protein production?

41. Linkage Discussion Questions: In the lecture on sustainability, Proebsting painted a picture that all of conventional ag is so out of whack in its water/energy/soil effects that biotech’s benefits are, by implication, irrelevant. Is that what he meant? Do you agree or disagree with this basic view? Why or why not? In the lecture on organic ag, Stone showed the many ways in which farmers can work to improve soil quality and reduce energy use. The list of “excluded practices” aside, in what ways are the goals of organic ag the same or different from conventional and other sustainable forms of ag? The first generation of GMO crops are often cited as having benefits for farmers and seed companies but not for consumers/public. In what ways is this true or false?

42. Potrykus painted a picture of a regulatory system so out of whack that GMO crops with huge potential benefits for the poor and ill are held up to the same or a greater degree as are crops whose main beneficiaries might be agribusiness or the developed world. Do you agree? What should a smart system look like? How would it compare to the system in use for conventionally bred crops? Genetic pollution is often cited as unmanageable and thus a reason not to completely exclude biotech crops in entire countries or states. But toxicology teaches us that “the dose makes the poison” (thus, by analogy its not a pollutant in consequence unless it is above a given threshold). Should adventitious presence be called pollution/contamination at all? When? How should it be dealt with by society?