Future Direction Molecular Farming

8/3/2019 Future Direction Molecular Farming

http://slidepdf.com/reader/full/future-direction-molecular-farming 1/32

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



The use of agricultural plants for the production ofuseful molecules for non food, feed or fiberapplications.

Plants are already grown to produce valuablemolecules, including many drugs.

Molecular farming is different because the plants aregenetically engineered (GE) to produce themolecules we want them to.

What is 'molecular

farming in plants'?



What is GE?

Create recombinant DNA with gene from same or different organismTransfer 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 interestMarker gene

Environment contaminantion via gene flowContamination of food supplySecondary metabolite – inctroduct allerginiicty or toxicity



Plant Products

• Over 120 pharmaceutical products currently in

use are derived from plants. Mainly from tropicalforest species

1. Plant derived pharmaceuticals (non-GE)



Industrial productsproteinsenzymes

modified starchesfatsoilswaxes

plastics

Pharmaceuticalsrecombinant human proteinsTherapeutic proteins

enzymesAntibodies (plantibodies)vaccines

2. Plant-made pharmaceuticals andindustrial products (GE)

1.Plant-derived pharmaceuticals (non-GE)

Plant Products



Strategies for „Molecular Farming‟

1.Plant gene expression strategies

• Transient transformation

• Stable transformation

• Chloroplast transformation




1. Plant gene expression strategies

Protein quantity and preservation

• Whole plant

• Target specific tissues (e.g. seed, root)

2. Location of trans-gene expression?




1. Plant gene expression system2. Location of trans-gene expression?

3. Selection of plant species and characteristics

• Mode of reproduction – self/outcrossing

• Yield, harvest, production, processing



Advantages

Cost reduction

Stability

Safety

Why use plants?

Disadvantages

Environment contamination

Food supply contamination

Health safety concerns



Cellulase for production of alcohols

Avidin – medical diagnostics

-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 andexpressed in Arabidopsis (49-70% oil present as

wax)

Astaxanthinred pigment in shell-fish.

used in aquaculture

Compounds to increase flavor and fragrances

Examples of Industrial PMPs



Edible vaccinesAdvantages:

Administered Directlyno purification required

no hazards assoc. w/injections

Productionmay be grown locally, where needed

most no transportation costs

Naturally stored

Plant-made Vaccines



Examples of edible vaccines ; pig vaccine in corn,HIV-suppressing protein in spinach, humanvaccine for hepatitus B in potato.

Plant-made Vaccines



- Plants can be used to produce monoclonalantibodies- 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/acreand can be stored at RT for up to 5 years!Humphreys DP et al. Curr Opin Drug Discover Dev 2001; 4:172-85.

Plantibodies



Therapeutic proteins

Blood substitutes – human hemoglobin

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

**To date, no plant-produced pharmaceuticalsare commercially available.

Plant made Pharmaceuticals



Rhizosecretion• Monoclonal antibodies

(Drake et al., 2003)• Recombinant proetins

(Gaume et al, 2003)

LEX System™

Lemna , (duckweed)

Dental Caries: CaroRx™ Colds due to Rhinovirus: RhinoRx™ Drug-induced Alopecia: DoxoRx™

Planet Biotechnology

Biomass biorefinerybased on switchgrass.Produce PHAs in greentissue plants for fuelgeneration.

Current „Pharm‟ Companies



Trangenic tobaccoPMPs and non-proteinsubstances (flavors and

fragrances, medicinals,and natural insecticides)

Kentucky Tobacco Research

and Development Center Trangenic tobaccoGeneWare®


Controlled PharmingVenturesIn collaboration w/PurdueTransgenic corn

Converted limestone mine facility



Transgenic corn

Trypsin andAprotinin

Prodigene


Ventria BioscienceTransgenic riceLactoferrinLysozyme



• 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 transgenicplants. (Galeffi et al., 2005 Vaccine )

• Plant based HIV-1 vaccine candidate: Tatprotein produced in spinach. (Karasev et al. 2005

Vaccine)

• Plant-derived vaccines against diarrheal diseases.(Tacket. 2005 Vaccine )



Environment contaminationGene flow via pollenNon-target species near field sites e.g.

butterflies, bees, etc

Food supply contaminationAccident, intentional, gene flow

Health safety concernsNon-target organ responsesSide-effectsAllergenicity

Risks and Concerns



U.S. Regulatory System

(existing regulations)

Field Testing-permits-notifications

Determination ofnon-regulatedstatus

Food safetyFeed safety

Pesticide andherbicideregistration

USDA FDA EPA



Breakdown of RegulatorySystem: Prodigene Incident 2002

2001 : Field trails of GE corn producingpig 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/destroycontaminated soy



USDA Response to Incident

Revised regulations so that they were distinctfrom commodity crops:

• Designated equipment must be used.

• At least 5 inspections/yr.

• Pharm crops must be grown at least 1 mileaway from any other fields and planted 28days before/after surrounding crops



FDA/USDA Guidance for Industry on Plant-MadePharmaceuticals Regulations

November 2004: Draft Document

Other challenges:Industrial hygiene and safety programs

Current Evolving Regulations



www.ucsusa.org

„Molecular farming‟ in the US

Since 1995 ~ 300 biopharming plantings

USDA has received 16 applications for permits in

the last 12 months.



Concerns:

• CONTAINMENT – opponents want a guarantee of0% contamination of the food supply.

• Full disclosure of field trials, crop, gene, location,etc.

• Extensive regulatory framework

„Molecular farming‟ opposition

S t d S f d f



1.Physical differencesE.g. “purple” maize, GFP

2.SterilityUse male sterile plantsTerminator technology?

3.Easily detectable by addition of 'reporter genes'PCR markers(avoid antibiotic resistance markers)

Suggested Safeguards for„molecular farming‟

S t d S f d f



4. Chloroplast expression systemIncrease yieldEliminates potential gene flowTechnically difficult (Chlorogen Company)

5. Complete disclosure of DNA sequences

6. Legislate for administration.

Suggested Safeguards for„molecular farming‟



Use only traditional drug productionsystems microbial, yeast and fungi

mammalian cell culture

Use only fully contained production systems:Plant cell culturesHydroponics (rhizosecretion)Greenhouses

Use non-food cropsTobacco, Hemp/Cannabis

Alternatives to „molecular farming‟?



The expectation is for lower production costshowever there is no evidence that pharming willproduce cheaper, safe drugs.

There are unknown costs associated withcontainment, litigation and liability,production…..others?

Economics

Future Direction Molecular Farming

Documents

Transcript of Future Direction Molecular Farming