By Lorenza Daroda

Plant molecular farming

Genetically engineered crops have been one of the most rapidly adopted
technologies in the history of agriculture, and recently transgenic crops
have been successfully exploited for the expression of pharmaceuticals,
industrial enzymes, functional proteins, fine chemicals, and other important
metabolites, representing the growing business of plant molecular farming.
In the past 30 years it has been possible to transform plants to improve
their agronomic qualities and enhance their nutritional value. More recently
it has also been possible to use plants as innovative factories for the
production of biopharmaceuticals such as drugs, antibodies, hormones,
enzymes, blood proteins, and vaccines. The number of small companies,
universities, and research centers that are at present investing in this
field is in fact increasing rapidly worldwide, as it is recognized that
plant made-pharmaceuticals, PMPs, can offer numerous benefits, including
inexpensive production and biological safety.

A wide range of host systems has been examined for large-scale production of
recombinant proteins. Compared to the traditional bacterial and yeast
cell-cultures, plants, seeds, and cultured plant cells represent one of the
most economical and promising systems for the biochemical, veterinary, and
pharmaceutical industries. In fact, several plant systems have already been
tested, including aquatic plants, food and non-food crops, leafy crops,
fruits, vegetables, and seeds, mostly from legumes and cereals, revealing
important benefits in rapid growth, high yields, and ease of
transformation1. Nonetheless, some concerns have been raised over the
potential impacts of these so-called ?pharma crops? upon the environment and
human health. Of particular concern has been the accidental dispersal of
their bioactive products, especially when food and feed crops such as maize,
potato, rice, and safflower are used for PMP production.

Environmental concerns
New health and environmental risks?i.e., the undesired exposure of
pharmaceuticals to the public, intra-species and inter-species gene flow,
and contamination of water and livestock feed?could result from the open
field cultivation of pharma crops that are designed as high yield production
systems and that produce products not intended for human or animal
consumption as food or feed. Excessive concentration of toxins or other
pharmaceutical products could theoretically cause ?protein pollution? of the
soil environment, representing a hazard for soil and rhizosphere microbial
communities as well as for non-target organisms2.

It should be noted, however, that stringent regulatory requirements for
correct management and rigorous confinement are applied to the genetically
engineered plant chosen as the production system3. Moreover, the magnitude
of the hazard would depend on several complex factors, including the nature
of the substances, quality and safety of the medical product, and whether it
is a chemical, toxin, industrial enzyme, allergen, hormone, vaccine, or
antibody. Even though most proteins are digestible and not toxic, they could
conceivably be harmful in higher doses or if they persist and accumulate in
the soil. Therefore when assessing the environmental risk of plant
expression systems, a broad range of other factors have to be considered
beyond the potential hazards of the novel protein substance, e.g., host
plant species, cultivation method, land qualities, and how the plants are
monitored. The potential for risks in PMP production should be kept in
perspective, considering also that the land necessary for the production of
large amounts of plant protein is relatively small compared to other
cultivations and that, if geographical isolation and temporal and biological
containment procedures are adopted, any resulting risk will be confined to
the actual site of PMP production.

Several research groups have investigated innovative and combined approaches
for the assessment of environmental risks related to PMP cultivation or
explored the ecological implications and changes arising from plant
molecular farming technologies2. Documented studies on the possible effects
on soil ecosystem, microbial communities, and non-target organisms of
genetically engineered plants expressing different traits, including
bio-molecules for industrial purposes or PMPs, have been recently reported4.
Soils from laboratories, glasshouses, and open field cultures of different
genetically engineered plants expressing different traits were examined and
compared. In some cases, when soils associated with control and genetically
engineered plants were analyzed, differences were identified among microbial
communities, but these differences were not related to any effect, since no
effects on the rhizosphere were found. Comparable dissimilarities and
variability have been reported in other studies5, suggesting that further
and dedicated investigation is needed for a complete understanding of root
exudation mechanisms in genetically engineered plants.

Root expression investigation
As noted previously, it seems important to study root exudation in plants
producing pharmaceuticals, in particular for those agriculture crops?i.e.,
tobacco, maize, and potato?that are meant to be grown in open fields
worldwide. Moreover, additional research should be directed at crops
engineered to produce PMPs via a secretory pathway, since it represents an
appealing, innovative, and low-cost platform for the production of
recombinant pharmaceutical proteins, thanks to its intrinsic ability to
manage the correct protein folding, modification, transport, and sorting.

Therefore, data obtained from past model studies on first generation
genetically engineered plants, such as Bt crops, seem insufficient to assess
the present risks and production potential associated with plant molecular
farming technology. Such assessments need to be performed on a case-by-case
basis, and in some circumstances, would demand a comprehensive
plant-specific or tissue-specific analysis, since recent reports indicate
that protein sorting and trafficking in plants is a very complex and
unpredictable mechanism6. Therefore additional studies on root exudation
might be required for the complete understanding and the valuable and safe
exploitation of a plant?s secretory pathway with respect to the environment
and in particular for field-grown pharma crops. For these reasons, we
investigated the root expression of two different recombinant single-chain
antibodies?scFvB9 and scFvH10?in the root tissue of genetically engineered
Nicotiana plants. The intra/intercellular expression and rhizosecretion of
the antibodies were examined in plants grown in sterile hydroponic
conditions.

Both recombinant antibodies were targeted to the plant secretory pathway,
however the scFvH10 construct carried a C-terminal KDEL signal sequence for
endoplasmic reticulum (ER) retention. It is known that proteins targeted to
the plant secretory pathway can be accumulated in cell ER by fusing a KDEL
tetrapeptide sequence to the C-terminus of the normallysecreted protein.
Among all signal sequences, the KDEL tag is widely used for plant-antibody
targeting and accumulation, giving rise to significant increases in protein
production yields.

We examined the functionality of KDEL in different plant tissues, not only
in leaves and seeds, which most previous reports have addressed6. We
verified whether the secretion into the intercellular fluids of leaves,
stems, and roots was prevented for the KDEL-tagged antibody. Root exudates
of hydroponic cultures of Nicotiana plants were examined to verify if any
KDEL-tagged ScFvH10 was secreted into the culture medium. The data reported
in our work show that the KDELtagged recombinant single-chain antibody in
transgenic tobacco roots is not rhizosecreted nor released by a
diffusion-based process into the environment. We showed that, in tobacco
plants, KDEL-tagged scFvH10 dispersal from the plant?s secretory pathway via
root exudates or leakage deriving from damage to plant root tissues does not
occur. To our knowledge, it was the first time that exudation of a
KDEL-tagged antibody was evaluated in root tissues. Thereof we conclude that
KDEL tag signal sequences might be used to prevent exudation of bioactive
proteins in the environment, particularly for crops producing PMPs, yet
further experiments will be conducted to verify that KDEL
secretion-inhibiting sequences are an appropriate tobacco biosafety system.

For experimental details, see the manuscript recently published in
Environmental Biosafety Research7.

References
1. Basaran P, Rodriguez-Cerezo E. 2008. Plant molecular farming:
opportunities and challenges. Crit. Rev. Biotechnol. 28, No.3. p.153-72
2. Shama L, Peterson R. 2008. Assessing risks of plant-based pharmaceuticals
II: non?target organism exposure. Human and Ecological Risk Assessment 14,
p.194-204
3. Spök A, Tyyman R, Fischer R, Ma J, Sparrow P. 2008. Evolution of a
regulatory framework for pharmaceuticals derived from genetically modified
plants. Trends in Biotechnology 26, No 9. p.506-517
4. Wolt JD, Wang K. 2007. Risk assessment for plant-made pharmaceuticals.
CAB reviews: perspectives in agriculture, veterinary science, nutrition and
natural resources vol.2, p.1-9
5. Sabharwal N, Icoz I, Saxena D, Stotzky G. 2007. Release of the
recombinant proteins, human serum albumin, beta-glucuronidase, glycoprotein
B from human cytomegalovirus, and green fluorescent protein, in root
exudates from transgenic tobacco and their effects on microbes and enzymatic
activities in soil. Plant Physiol. Biochem. 45, No.6-7 p.464-9
6. Rademacher T, Sack M, Arcalis E, Stadlann J, Balzer S, Altmann F,
Quendler H, Stiegler G, Kunert R, Fischer R, Stoger E. 2008. Recombinant
antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and
contains predominantly single-GlcNAc N-glycans. Plant Biotechnology 6, No.2.
p.189-201
7. Pizzuti F, Daroda L. 2008. Investigating recombinant protein exudation
from roots of transgenic tobacco. Environ. Biosafety Res. DOI:
10.1051/ebr:2008020
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