Future directions for agricultural biotechnology

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. What is biopharming? 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)

4. What is biopharming? 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.

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

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

7. Strategies for Biopharming • 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. Strategies for Biopharming • Plant gene expression strategies 2. Location of transgene expression • 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

9. Strategies for Biopharming • 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

10. Why use plants? 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) Disadvantages Environment contamination - gene flow - wildlife exposure Food supply contamination - mistaken/intentional mixing w/human food Health safety concerns - Variable, case-specific

11. Industrial products on the market • Avidin by Sigma • transgenic corn • traditionally isolated from chicken egg whites • used in medical diagnostics • GUS (b-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

12. Industrial products close to market

13. Plant-made Pharmaceuticals (PMPs) • 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

14. 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 • no need for refrigeration or special storage

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

16. Plantibodies • 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

17. Plantibodies 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) **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.

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

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

20. Current ‘Pharm’ Companies

21. Current ‘Pharm’ Companies • LEX System™ • Lemna (duckweed) Kentucky Tobacco Research and Development Center • trangenic tobacco • PMPs and non-protein substances (flavors and fragrances, medicinals, and natural insecticides) • 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) Current ‘Pharm’ Companies • biomass biorefinery • based on switchgrass. • used to produce PHAs in green tissue plants for fuel generation.

23. 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) • 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. 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

25. 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

26. Breakdown of Regulatory System: Prodigene Incident 2002 2001 : Field trails of GE corn producing pig vaccinewere 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. Current Evolving Regulations • 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)

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

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

31. Biopharming in Colorado

32. Biopharming in N.Carolina

33. The 2005 Oregon Biopharm Bill

34. Biopharm opposition • 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

35. Suggested Safeguards for biopharm operations • 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

36. Suggested Safeguards for biopharm operations • 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 • Legislate for administration

37. Alternatives to biopharming? • 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

38. Economics 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?

39. Future directions for agricultural biotechnology? 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 commercialand/or humanitarianuse will ultimately depend on….. Public perception of risk Regulation

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?