Future directions for agricultural biotechnology

Nutritionally enhanced and “phactory” crops

Dr. Kirstin Carroll

Project ManagerBiotech Outreach

Oregon State University

Future directions for agricultural biotechnology

• Nutritionally enhanced crops

• Plant production systems – “Phactories”

Golden Rice Humanitarian Project

Ingo Potrykus and Peter Beyer

Vit A deficiency~250 million worldwide are at risk (WHO)

GE Rice

Golden Rice Humanitarian Project

Ingo Potrykus and Peter Beyer

1990’s. All public funding Peter Beyer – Univ. of Freiburg. Igno Potrykus – Univ. of Switzerland, Zurich

Engineered rice to produce β-carotene in endosperm

8 years to produce

Results first published in Science in 2000

Ye et al, 2000. Engineering the provitamin A biosynthetic pathway into rice endosperm. Science 287: 303-305

Golden Rice Humanitarian Project Goal : Prevent Vit-A deficiency in the poor and disadvantaged of developing countries

– make technology available free of charge and restrictions to farmers of developing countries

- To do this, care was taken to use only public funding

• Rockefeller Foundation (1993-1996)• European Community Biotech Program (FAIR CT96,

1996-1999)Swiss Federal Office for Education and Science

• Swiss Federal Institute of Technology (1993-1996).• European Union

Challenges for Golden Rice

“freedom to operate for humanitarian use”

70 IPRs and TPRs belonging to 32 different companies and universities

- received free licenses for all intellectual and technical property involved.

1. IPRs and TPRs

2. Definition of “subsistence farmer” and “humanitarian use”

Challenges for Golden Rice

“freedom to operate for humanitarian use”

2. Definition of “subsistence farmer” and “humanitarian use”

Syngenta (was Zeneca Company (Fernhurst, UK))

– received an exclusive license for commercial use

- supports the humanitarian use of golden rice

-humanitarian < $10,000 income from golden rice

Challenges for Golden Rice Radical GMO Opposition

www.greenpeace.org

“Fools Gold”

Initial arguments against GE and Golden Rice were untrue…….

Responses to GMO Opposition to Golden Rice

Golden rice has not been developed by or for industry.

It presents a sustainable, cost-free solution, not requiring other resources.

Industry does not benefit from it.

Those who benefit are the poor and disadvantaged.

It is given free of charge and restrictions to subsistence farmers.

It does not create any new dependencies.

Ingo Potrykus 2001, Plant Physiol 125: 1157-1161

Responses to GMO Opposition to Golden Rice

It will be grown without any additional inputs.

It can be resown every year from the saved harvest.

It does not reduce agricultural biodiversity.

It does not affect natural biodiversity.

There is, so far, no conceptual negative effect on the environment.

There is, so far, no conceivable risk to consumer health.

Ingo Potrykus 2001, Plant Physiol 125: 1157-1161

Challenges for Golden Rice Radical GMO Opposition

Opposition arguments now focus on nutritional aspects…

“useless…3.75kg/day would have to be consumed”

Golden Rice as the “Trojan Horse”

www.greenpeace.org

Challenges for Golden Rice

Deregulation

Extensive regulatory restriction on GE plants

- Time

Challenges for Golden Rice

Deregulation

Extensive regulatory restriction on GE plants

- Time

“What is more important to our society – a strict regulatory framework for minor and mostly hypothetical risks, or the life and health of numerous underprivileged human beings?

Ingo Potrykus2003, Nutr. Reviews Vol 61 (6): S101-104

Golden Rice Humanitarian Project Progress Report - 2003

Golden Indica and Japonica Rice Lines Amenable to Deregulation

Hoa et al, 2003. Plant Physiology. Vol 133. 161-169

Improvements to Golden Rice:Agrobacterium tumefaciens-mediated transformation

Confirmed transformation of desired gene(s) e.g. selected against beyond-border transfer

Replaced antibiotic selection marker

Selected transformants with single integrations

Hoa et al, 2003. Plant Physiology. Vol 133. 161-169

Golden Rice Humanitarian Project Progress Report - 2003

Golden Indica and Japonica Rice Lines Amenable to DeregulationHoa et al, 2003. Plant Physiology. Vol 133. 161-169

Three Rice Lines were transformed:

Japonica Taipei 309 – lab workhorse

Elite Indica cultivars of Southeast Asia IR64 MTL250

Golden Rice Humanitarian Project Progress Report - 2003

Variable and stable expression in transformed lines

T3 generation

Hoa et al, 2003

Golden Rice Humanitarian Project Progress Report - 2003

New improved Golden Rice is now expected to conform with regulatory requirements

This allows for follow-up studies which could not be done before:

Nutritional

Risk Assessment

Hoa et al, 2003. Plant Physiology. Vol 133. 161-169

Other nutritionally enhanced cropsHealthier oils

soy, canolaDupontCalgene

Increased vitaminsVit E corn (10-15 fold increase)

Vit C - isolation of strawberry gene and overexpression in Arabidopsis (2 fold increase)

Vit E –Soy transformed with gene isolated from Arabidopsis (5 fold increase in seeds)

Increased ironCorn – 49% increase in iron contentRice – Golden rice

Other nutritionally enhanced crops, cont’d

Tobacco – nicotine reduced tobacco.

Reduced AllergensGE Rice used to prevent hay fever

GE Soybean with reduced allergenic protein levels in seed

Terpenoids(interest for their anticarcinogenic properties)-isolation of genes from lemon were expressed in tobacco

Increased ProteinGE Potato – 35-45% more proteinGE Soy – higher protein tofu products

Other nutritionally enhanced crops, cont’d

AntioxidantsCloned genes from tomato and tobacco –overexpression in tomato showed increased antioxidant activity

FolateTransformed tomatoes w/mammalian enzyme –increased folate in ripe fruit

Phytonutrients – substances for improving health/preventing disease

Soy – isoflavones

Tomatoes – lycopene, 2.5 fold increase (anti-oxidant)

Other nutritionally enhanced crops, cont’d

Most of these products are still in development stage

“proof of concept”

These products may be coming down commercial pipeline……

But the pipeline is blocked…Dramatic decline of field trials

of transgenic crops

International: OECD field trial data,

1986 through 2000

Field Trials of USA fruits and vegetables (California Agriculture 2004, Redenbaugh & McHughen)

Plant Production Systems “Phactories”

www.ucsusa.org

Plant Production Systems “Phactories”

1. Industrial products

proteinsenzymesmodified starchesfatsoilswaxesplastics

2. Pharmaceuticals (pharm crops)

enzymesantibodies (plantibodies)vaccines

www.ucsusa.org

Plant Production Systems “Phactories”

Advantages

Cost reduction

Stability

Safety

Disadvantages

Contaminationenvironmentfood supply

Health Safety

www.ucsusa.org

Industrial Products

www.ucsusa.org

Cellulase for production of alcohols

Avidin – medical diagnostics

β-glycoprotein – biomedical diagnostics

Plant-derived plastic –polyhydroxyalkanoate (PHA)MonsantoTo date, more costly than fuel-based plastic

Industrial Products

www.ucsusa.org

High wax esters

Jojoba seeds - gene has been isolated and expressed in Arabidopsis (49-70% oil present as wax)

Astaxanthinred pigment in shell-fish.

used in aquaculture

Compounds to increase flavor and fragrances

www.ucsusa.org

Pharmaceutical Products – Pharm Crops

Edible vaccines

Plant-produced antibodies

Human proteins

www.ucsusa.org

Pharm Crops

Edible vaccines

Advantages:Administered Directly

no purification required

no hazards assoc. w/injections

Productionmay be grown locally, where needed most

no transportation costs

Naturally stored

Pharm Crops

www.ucsusa.org

Edible vaccines

1998 Trial with a transgenic potato containing a vaccine

2000 GE Potatoes containing hepatitis B antigen

2000 GE Corn w/pig vaccine

2001 GE spinach expressing HIV-suppressing proteins

Other vaccines being developed in banana, potato, sweet potato and tomato

Pharm Crops

www.ucsusa.org

‘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

**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.

www.ucsusa.org

Pharm Crops

Therapeutic proteins

Blood substitutes – human hemoglobin

Proteins to treat diseasesCF, HIV, Hypertension, Hepatitis B…..many others

**To date, no plant-produced pharmaceuticals are commercially available.

Recombinant Protein Production Comparison

Transgenic plants

Plant viruses Bacteria

Mammalian cell cultures

Transgenic animals

Cost/storage Cheap/RT Cheap/-20C Cheap/-20C Cheap/-20C Expensive

Distribution Easy Easy Feasible Difficult Difficult

Gene size Not limited Limited Unknown Limited Limited

Glycosylation Correct? Correct? Absent Correct? Correct?

Multimericprotein assembly (SigA) Yes No No No No

Production cost Low Low Medium High High

Production scale Worldwide Worldwide Limited Limited Limited

Production vehicle Yes Yes Yes Yes Yes

Goldstein and Thomas, 2004. Q J Med 97:705-716

Recombinant Protein Production Comparison

Transgenic plants

Plant viruses Bacteria

Mammalian cell cultures

Transgenic animals

Propagation Easy Feasible Easy Hard FeasibleProtein folding accuracy High? High? Low High HighProtein homogeneity High? Medium Low Medium HighProtein yield High V. High Medium Medium-High High

Public perception of risk High High Low Medium HighSafety High High Low Medium HighScale-up costs Low Low High High HighTherapeutic risk Unknown Unknown Yes Yes YesTime required Medium Low Low High High

Goldstein and Thomas, 2004. Q J Med 97:705-716

Considerations for pharm crop production

1. How will the gene be expressed? Plant expression strategies:

1. Transient transformation

2. Stable transformation

3. Chloroplast transformation

Considerations for pharm crop production

2. Where will the gene be expressed?

1. Whole plantAdv - Large amts of protein

ex. tobaccoDisadv – poor preservation

2. Specific tissues e.g. seed, root

ex. High protein seed (corn, soy)

Adv – ability to store product

Considerations for pharm crop production

3. What plant species will be used?

Green matter – tobacco, alfalfa, duckweed

Seed – corn, rice, barley

Considerations for pharm crop production

3. What plant species will be used?

Green matter – tobacco, alfalfa, duckweed

Seed – corn, rice, barley

Main Q?: Use of food crops or non-food crops for production?

Argument for food crops = wealth of knowledge concerning growing, planting, processing, etc.

Production, safety and efficacy considerationsContainment

Liability- currently unclear- unintended contamination, theft, intentional misuse

Regulatory issuesUSDA oversees pharm crop research permits

Since 1995 ~ 300 biopharming plantings

FDA – specific regulatory procedures being developed

Permanent FDA regulation is likely

Examples of current FDA/ORA contamination allowances:• 50 g cornmeal (CPG 7104.02, Sec 578.200)

• < 1 whole insect, or• < 50 insect fragments, or• < 2 rodent hairs, or• < 1 rodent excreta fragments

• 100 g tomato paste (CPG 7114.29, Sec 585.890)• 29 fly eggs, or• 14 fly eggs + 1 maggot, or• < 2 maggots

The question of contamination

? What should the allowances be for GE crops ?

Production, safety and efficacy considerations

FDA Draft Guidance Document:

"Guidance for Industry: Recommendations for the Early Food Safety Evaluation of New Non-Pesticidal Proteins Produced by New Plant Varieties Intended for Food Use."

To make see draft document or make comments, visit: http://www.cfsan.fda.gov/~dms/bioprgui.html

Production, safety and efficacy considerationsSafety and efficacy

1. Field site monitoring and security

2. Product safety

1. Non-target organ responses2. Side-effects3. Allergenicity4. Non-target species near field sites e.g.

butterflies, bees, etc5. Contamination of human food supply e.g.

accident, gene flow

Production, safety and efficacy considerations

Economics

The expectation is for lower production costs however there is no evidence that pharming will produce cheaper, safe drugs.

What about costs associated with containment, litigation and liability, production…..others?

Alternatives to pharming?

Current method of laboratory-based drug production

Plant cell cultures

Hydroponics

CropTech Corporation – plant does not express the protein until it is injured when it is shredded for processing

Pharm crop opposition

Main Concern = CONTAINMENT

Opponents want guarantee of 0% contamination

- full disclosure = field trials, crop, gene, location, etc.

- an extensive regulatory framework

Pharming operations in the US

www.ucsusa.org

Prodigene – currently holding field trials.

? ?

Ventria – hybrid rice. Declined in CAMoved to Missouri

Monsanto – pull out of all pharm crop operations

Since 1995 ~ 300 biopharming plantings

USDA has received 16 applications for permits in the last 12 mos

Prodigene Incident 2002

GE corn producing pig vaccine – trails planted 2001

Soy was planted in same field in 2002

USDA discovered “volunteer” corn plants in soy field

Soy was harvest before “volunteers” were removed.

$500,000 fine + $3 million to buy contaminated soy

Prodigene Incident 2002

USDA Response to Incident:

Revised regulations so that they were distinct from commodity crops:

1. Designated equipment must be used.

2. At least 5 inspections/yr.

3. Pharm crops must be grown at least 1 mile away from any other fields and planted 28 days before/after surrounding crops

Future directions for agricultural biotechnology ??

Science has developed genetically enhanced crops. The extent to which these crops will be further developed for humanitarian and/or commercial use will depend on…..

Public perception of risk

Regulatory process

Discussion Questions:

1. Do you think nutritionally enhanced plants should be developed even though there are oral supplements available? Why or why not?

2. Do you support the development of pharm crops. e.g. Do you feel that the potential benefits of pharm crops are worth the potential risks?

3. What are your thoughts on using food vs. non-food crops as “phactories”?