Molecular farming

Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

Definition:

“ The use of whole organisms, organs, tissues or

cells, or cell cultures, as bio-reactors for the

production of commercially valuable products via

recombinant DNA techniques.”


A brief history of molecular farming 1986

1989

1990

1992

1992

1995

First plant - derived recombinant therapeutic protein-human GH in tobacco & sunflower. (A. Barta, D. Thompson et al.)

First plant - derived recombinant antibody –full-sized IgG in tobacco. (A. Hiatt, K. Bowdish)

First native human protein produced in plants –human serum albumin in tobacco & potato. (P. C. Sijmons et al.)

First plant derived vaccine candidate –hepatitis B virus surface antigen in tobacco. (H. S. Meson, D. M. Lam)

First plant derived industrial enzyme –α-amylase in tobacco. (J.Pen, L. Molendijk et al.)

Secretory IgA produced in tobacco. (J. K. Ma, A. Hiatt, M. Hein et al.)


Continued…. 1996

1997

1997

2000

2003

2003

First plant derived protein polymer –

artificial elastin in tobacco. (X. Zhang, D. W. Urry, H. Daniel)

First clinical trial using recombinant bacterial

antigen delivered in a transgenic potato. (C. O. Tacket et al.)

Commercial production of avidin in maize. (E. E. Hood et al.)

Human GH produced in tobacco chloroplast. (J. M. Staub et al.)

Expression and assembly of a functional antibody

in algae. (S. P. Mayfield, S. E. Franklin et al.)

Commercial production of bovine trypsin in maize.(S. L. Woodard et al.)


Molecular Farming Strategy Clone a gene of interest

Transform the host platform species

Grow the host species, recover biomass

Process biomass

Purify product of interest

Deliver product of interest


Molecular Farming Hosts Bacteria

Yeasts, (single celled fungi)

Unicellular algae

Mammalian, insect, plant, and filamentous fungal cell cultures

Whole plants, ( corn, barley, rice, duckweed, moss protonema)

Whole animals, (insects, birds, fish, mammals)


Do not produce glycosylated full – sized antibodies.

Contaminating endotoxin difficult to remove.

Recombinant proteins often form inclusion bodies.

Labour- and cost – intensive refolding in vitro necessary.

Lower scalability

Preferred for the production of small, aglycosylated proteins

like Insulin, interferon-β.

Bacteria :


Limited by legal and ethical restriction

Require expensive equipment & media

Delicate nature of mammalian cells

Human pathogens and oncogenes

Scaling up problems

Animal Based Systems:


Plant Molecular Farming Significantly lower production cost than with transgenic

animals, fermentation or bioreactors.

Infrastructure & expertise already exists for the planting,

harvesting & processing of plant material.

Plants contain no known human pathogens (such as prions,

virions, etc.) that could contaminate the final product.

Higher plants generally synthesize proteins from eukaryotes

with correct folding, glycosylation & activity.


Continued……

Plant cells can direct proteins to environments that reduce

degradation and therefore increase stability.

Low ethical concerns.

Easier purification (homologs don’t pose any purification

challenge, e.g. serum proteins or antibodies).

Versatile (production of a broad diversity of proteins).

× Take more time to develop

× Transgene & protein pollution


Cost of Production: Antibodies

Animal cell culture $ 333/g

Transgenic milk $ 100/g

Yeast cell culture $ 100/g

Milled corn endosperm $ 0.2/g

Enriched corn fraction $ 0.6/g

Extracted corn fraction $ 2.1/g

Moderate purity $ 3.3/g

Rx purity $ 20 -200/g

(Rainer Fischer; Stefan Schillberg)


Expression systems for PMF

Transgenic plants

Plant - cell - suspension culture

Transplastomic plants

Transient expression system

Hydroponic cultures


Transgenic plants: Foreign DNA incorporated into the nuclear genome using

Agrobacterium tumefaciens

Particle bombardment

Most common

Long term non-refrigerated storage

Scalability

More ‘gene to protein’ time

Biosafety concerns


Plant cell suspension culture

Culture derived from

transgenic explants

Transformation after desegregation

Recombinant protein localization depends on

Presence of targeting / leader peptides in the recombinant protein

Permeability of plant cell wall for macromolecules

Containment & production under GMP procedure

Low scale up capacity


Su-May Yu; Institute of Molecular Biology Academia Sinica Nankang, TaipeiPresented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

Transplastomic plants: DNA introduced into chloroplast genome

High transgene copy number

No gene silencing

Recombinant protein accumulate in chloroplast

Natural transgene containment

Long term storage not possible

Long development time

Limited use for production of therapeutic glycoproteins


Transient expression system

Approaches: Biolistic delivery of ‘naked DNA’

Usually reaches only a few cells

Can be used for a rapid test for protein expression

Agroinfiltration

Delivery of Agrobacterium in intact leaf tissue by vacuum

infiltration

Targets many more cells in a leaf

Infection with modified viral vectors


Virus infected plants

Gene of interest is cloned into the genome of a viral plant pathogen

Infectious recombinant viral transcripts are used to infect plants

Rapid & systemic infection

High level production soon after inoculation

Genetic modification of plant is entirely avoided


Over view of transient-gene-expression approaches in plants

R. Fischer and others(1999)


Hydroponic culture A signal peptide is attached to the recombinant protein directing it to

the secretory pathway

Protein can be recovered from the root exudates (Rhizosecretion) or

leaf guttation fluid (Phylosecretion)

Technology being developed by the US biotechnology company

Phytomedics Inc.

Purification is easier

Reduced fear of unintentional environmental release

Expensive to operate hydroponic facilities


Comparison of different production systems for expression of recombinant proteins

S. Biemelt;U. Sonnewald (2004)


Downstream processing & analysis of recombinant proteins from plants

R. Fischer and others(1999)Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

Choice of host species

Depends on:

Protein to be produced & its desired application

Transformation efficiency

Overall production cost

Containment


Comparison of various plant expression host speciesFeatures/crop

Organ Yield Storage/proteinstability

Transform-ation

Productioncosts

Specialty

Tobacco Leaf High Limited Well established

Good Nonfood/feed

Alfalfa Leaf High Limited Established Good Homogenous N glycosylation, use atmospheric N2

Wheat Seed Good Optimal Inefficient Optimal

Maize Seed High Optimal Established Optimal

Pea Seed Good Optimal Limited Good

Rapeseed Seed Good Optimal Established Optimal Fusion with oleosin for easy purification

Potato Tuber Good Good Well established

Good

Banana Fruit Good Good Inefficient Good Can be eaten raw

S. Biemelt;U. Sonnewald (2004)Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

Classes of proteins within molecular farming

Parental therapeutics and pharmaceutical intermediates

Industrial proteins and enzymes

Monoclonal antibodies

Antigens for edible vaccines

Biopolymers


Therapeutic proteins produced in different plant hosts system

Therapeutic protein Potential use hostα- and β- haemoglobin Blood substitute Tobacco

Human serum albumin(HAS) Blood substitute Potato

α-tricosanthin HIV Therapy tobacco

α- interferon Viral protection anticancer Rice

Epidermal growth factor, Erythropoietin, Tuber growth factor

Mitogen Tobacco

Hirudin Anticoagulant Canola

Protein C Anticoagulant Tobacco

Glutamate decarboxylase Diabetes Tobacco

Human somatotropin Hypopituitary dwarfism Tobacco

Calcitonin Paget disease, osteoporosis, parathyroid gland carcinoma

potatoA.S. Rishi; N.D.Nelson; A.Goyal (2001)


Industrial enzymes & proteins produced in different plant host system

Industrial enzymes Potential use Host α- amylase Industry Tobacco

Phytase Industry Alfalfa, Tobacco

Cellulase Industry Alfalfa, Tobacco, potato

Manganese peroxidase Industry Alfalfa, Tobacco

β- (1,4) xylanase Industry Tobacco, Canola

β-(1,3-1,4)glucanase Industry Tobacco, Barley

Avidin Research reagent Maize

Glucuronidase Research reagent Maize

A.S. Rishi; N.D.Nelson; A.Goyal (2001)


Antibodies produced in different plant host system

Potential use Antigen used Host

HSV-2 Glycoprotein B of HSV SoybeanColon cancer Colon cancer antigen Tobacco Dental care (tooth decay)

S. mutans antigen Tobacco

Hodgkin’s lymphoma ScFv of IgG from mouse B-cell lyphoma

Tobacco

Tumor associated marker antigen

ScFv84.64 against carcinoembryogenic antigen

Cereals

Research Human creatine kinase Arabidopsis Phytoremediation Atrazine Tobacco Plant protection Nematode antigen Tobacco

A.S. Rishi; N.D.Nelson; A.Goyal (2001)Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

Antigen Casual organism/Disease Host system

Rabies virus glycoprotein Rabies Tomato, tobacco, spinach

Capsid protein epitope Mink enteritis virus Cowpea

Spike protein Piglet diarrhea Tobacco

CT-B toxin Cholera Potato

LT-B toxin Travelers diarrhea Potato

Hepatitis B surface antigen Hepatitis B Tobacco, Potato

Human cytomegalovirus glycoprotein B

Human cytomegalovirus Tobacco

Norwalk virus antigen Gastrointestinal distress Tobacco, Potato

Foot & mouth disease antigen Foot and mouth disease Cowpea

Malarial antigens Malaria Tobacco

Gp 41 peptide HIV-1 Cowpea

Hemagglutinin Influenza Tobacco

c-Myc cancer Tobacco

Vaccines produced in different plant host systems

A.S. Rishi; N.D.Nelson; A.Goyal (2001)Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

Products on the market

Avidin

Hood et al. (1997) reported the production of chicken egg white avidin in

transgenic corn

β-Glucuronidase

First reported to be produced commercially in transgenic corn (Kusnadi et

al. 1998, Witcher et al. 1998)

Trypsin (TrypZeanTM)

First large scale protein product from transgenic plant technology


PMF products & producers

M.E.Horn; S.L.Wooddard; J.A.Howard (2004)Presented in Credit Seminar (Division of Agricultural Physics, IARI, New Delhi) by Nirmal Kumar

PMF LeadersCanada Medicago (Québec): alfalfa leaves for hemoglobin production. Sembiosys Genetics (Calgary): safflower for production of a fat-

fighting peptide and somatotrophin. Plantigen (Ontario): trials of several plants for protein production.United States AtlaGen Bioscience (Morgan Hill, CA and Richland, WA): potato

leaves. Ventria Bioscience: potato tubers. API: rice and other plants. CropTech: tobacco leaves for production of uronidase, irunosidase,

glucocerebrosidase (for Gaucher’s disease) and vaccines. Dow AgroSciences: corn for production of vaccines and antibodies to

prevent certain animal diseases. IPT (Monsanto): corn for antibody and somatotrophin production. Epicyte Pharmaceutical (San Diego): corn and rice seeds.


Continued: Phytomedics: (Dayton, NJ): tobacco and tomatoes. Prodigene (College Station, TX): corn seeds for production of

laccase, avidin, betaglucoronidase and aprotinin. Monsanto Protein Technologies: corn.

Germany Planton: potato tubers. Greenovation: corn for production of factor IX for hemophilia B

treatment. MPB Cologne: potato tubers, canola seedsFrance Meristem Therapeutics (Clermont-Ferrand): corn seeds, tobacco

leaves for production of hemoglobin, gastric lipase, collagen, beta interferon, lactoferrin and albumin.

Switzerland Syngenta: antibodies and others.


Biosafety issues in molecular farming


Challenges

Gene and protein pollution

Applied to all transgenic plants.

Product safety

Applied to all pharmaceutical product.


Transgene pollution – the problems

Transgene pollution is the spread of transgenes beyond the

intended genetically-modified species by natural gene flow

mechanisms.

Two classes of transgene pollution:

The possible spread of primary transgenes.

The possible spread of superfluous DNA sequences.


Transgene pollution – the mechanismsVertical gene transfer

Vertical gene transfer is the movement of DNA between plants that are

at least partially sexually compatible.

Most prevalent form of transgene pollution.

Occurs predominantly via the dispersal of transgenic pollen.

Also by seed dispersal.

herbicide resistance genes have introgressed from transgenic oil seed

rape to its weedy cousin Brassica campestris by hybridization

(Mikkelsen et al.1996).


Horizontal gene transfer

Horizontal gene transfer is the movement of genes between species

that are not sexually compatible and may belong to very different

taxonomic groups.

The process is common in bacteria, resulting in the transfer of

plasmid-borne antibiotic resistance traits.

Agrobacterium tumefaciens and related species represent a special

case.

Antibiotic resistance markers and transgenes encoding

pharmaceutical proteins could be acquired by human pathogens.


what not to look for in a platform crop formolecular farming

Abundant pollen production

Abundant seed production

Small, easily dispersed seeds

Important food/feed crop

Widely planted throughout the world

Often grown as open-pollinated varieties

Spontaneously mates with wild relatives

High frequency of gene flow by outcrossing


Transgene pollution – possible solutions

Minimum required genetic modification.

Elimination of non-essential genetic information.

Containment of essential transgenes.

Alternative production systems

transient expression.

plant suspension cultures in sealed, sterile reactor vessels (Fischer

et al., 1999a; Doran, 2000).


Containment of essential transgenes

Physical or artificial

Maintained in green house

Concealing flowers/fruits in plastic bags in field

Isolation

Barrier crops

Biological containment


Biological containment methods to prevent transgene pollution

Use of self-pollinating crops, exploitation of cleistogamy Asynchronous flowering times, atypical growing seasons Use of crops lacking wild relatives that are compatible for

hybridization Strengthening of hybridization barriers between compatible species Apomixis Interference with flower development Male sterility (interference with pollen development) Seed sterility (by ‘terminator technology’) Maternal inheritance (plastid transformation) Transgene integration on incompatible genomes Transgenic mitigation Conditional transgene excision


Protein pollution – the problems Direct consumption (e.g. the ingestion of toxins by aphids, and knock-

on effects to ladybirds and birds further up the food chain)

By simple exposure to the plant (e.g. the effects of pollen on butterflies

and moths)

From the exudation of recombinant protein into the rhizosphere or

leaf guttation fluid (most likely to affect microorganisms)

By the consumption of dead and decaying plant material by

saprophytes

Waste plant material


Protein pollution – possible solutions To a certain extent by physical containment Controlling gene expression

Restricting expression to particular tissue To bring the transgene under inducible control as has been shown

for recombinant ‘glucocerebrosidase’ (Cramer et al., 1999).

Controlling protein accumulation and activity protein can also be targeted to a specific intracellular compartment Recombinant proteins can also be produced as inactive precursors

that have to be processed by proteolytic cleavage before they attain full biological activity.

used for the expression of hirudin


Product safety

The purified protein may be contaminated with toxic substances from

the plant or applied to the plant, e.g. plant derived metabolites,

allergens, field chemicals (e.g. herbicides, pesticides, fungicides),

fertilizers, dung and manure.

The product itself, due to intrinsic properties, may be harmful.


Conclusion:

Use of virus infected plants is best approach for molecular farming

Molecular farming provides an opportunity for the economical and

large-scale production of pharmaceuticals, industrial enzymes and

technical proteins that are currently produced at great expense and in

small quantities.

We must ensure that these benefits are not outweighed by risks to

human health and the environment


Molecular farming

Education

Transcript of Molecular farming