Future directions for agricultural biotechnology

Dr. Kirstin Carroll

Outreach in Biotechnology Program

Oregon State University

Lecture Outline

• What is biopharming?

• Why use plants?

• Current and evolving regulation

• What are the risks and concerns?

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?

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?

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)

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

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

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

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

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

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

Industrial products close to market

• 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)

Edible vaccinesAdvantages:

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

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

• 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

Plantibodies

**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)

Therapeutic proteins and intermediates

• Blood substitutes – human hemoglobin

• Proteins to treat diseases such as CF, HIV, Hypertension, Hepatitis B…..many others

Plant made Pharmaceuticals

**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

Current ‘Pharm’ Companies

Current ‘Pharm’ Companies

•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

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

• 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)

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

U.S. Regulatory System (existing regulations)

Field Testing-permits-notifications

Determination of non-regulated status

Food safety

Feed safety

Pesticide and herbicide registration

USDA FDA EPA

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

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

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

www.ucsusa.org

Biopharming field trials in the US

Since 1995 ~ 300 biopharming plantings

The USDA receives/reviews applications for permits for biopharm trials.

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

Biopharming in Colorado

Biopharming in N.Carolina

The 2005 Oregon Biopharm Bill

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

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

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

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?

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

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…..

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?

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? 

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?