'molecular farming in plants'? - Oregon State University

workkinkajouBiotechnology

Dec 5, 2012 (4 years and 10 months ago)

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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 of
useful molecules for non food, feed or fiber
applications.


Plants are already grown to produce valuable
molecules, including many drugs.


Molecular farming is different because the plants are
genetically engineered (GE) to produce the
molecules we want them to.

What is 'molecular
farming in plants'?

What is GE?

Create recombinant DNA with gene from same or different organism

Transfer 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 interest

Marker gene


Environment contaminantion via gene flow

Contamination of food supply



Secondary metabolite


inctroduct allerginiicty or toxicity


Plant Products


Over 120 pharmaceutical products currently in
use are derived from plants. Mainly from tropical
forest species

1.

Plant derived pharmaceuticals (non
-
GE)

Industrial products

proteins


enzymes


modified starches


fats


oils


waxes


plastics

Pharmaceuticals

recombinant human proteins

Therapeutic proteins

enzymes

Antibodies (plantibodies)

vaccines


2. Plant
-
made pharmaceuticals and
industrial 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

Strategies for ‘Molecular Farming’

1.

Plant gene expression strategies

Protein quantity and preservation



Whole plant



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

2. Location of trans
-
gene expression?

Strategies for ‘Molecular Farming’

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

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


b
-
杬g捯灲潴敩渠


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 and
expressed in Arabidopsis (49
-
70% oil present as
wax)



Astaxanthin




red pigment in shell
-
fish.







used in aquaculture



Compounds to increase flavor and fragrances

Examples of Industrial PMPs

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

Plant
-
made Vaccines

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

Plant
-
made Vaccines

-

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.





Plantibodies

Therapeutic proteins



Blood substitutes


human hemoglobin



Proteins to treat diseases



CF, HIV, Hypertension,



Hepatitis B…..many others





**To date, no plant
-
produced pharmaceuticals
are 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 biorefinery
based on switchgrass.
Produce PHAs in green
tissue plants for fuel
generation.

Current ‘Pharm’ Companies

Trangenic tobacco

PMPs and non
-
protein
substances (flavors and
fragrances, medicinals,
and natural insecticides)

Kentucky Tobacco Research
and Development Center

Trangenic tobacco

GeneWare®

Current ‘Pharm’ Companies

Controlled Pharming
Ventures

In collaboration w/Purdue

Transgenic corn

Converted limestone mine facility

Transgenic corn

Trypsin and
Aprotinin

Prodigene

Current ‘Pharm’ Companies

Ventria Bioscience

Transgenic rice

Lactoferrin

Lysozyme


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 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/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

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 of
0% contamination of the food supply.



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



Extensive regulatory framework




‘Molecular farming’ opposition

1.
Physical differences

E.g. “purple” maize, GFP


2.
Sterility

Use male sterile plants

Terminator technology?


3.
Easily detectable by addition of 'reporter genes'

PCR markers

(avoid antibiotic resistance markers)

Suggested Safeguards for
‘molecular farming’


4. Chloroplast expression system

Increase yield

Eliminates potential gene flow

Technically difficult (Chlorogen Company)


5. Complete disclosure of DNA sequences


6. Legislate for administration.


Suggested Safeguards for
‘molecular farming’

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 ‘molecular farming’?

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


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

Economics