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Plants can be used as production systems for pharmaceutical active proteins.
However, there is one big disadvantage - a particular step in the protein
production differs in plants and humans. Researchers in Germany have now
been able to change this step in the moss species Physcomitrella patens,
August 2005 by Katharina Schoebi, Checkbiotech.

Plants are a promising system for the production of pharmaceutical
proteins. Compared to bacteria or mammalian production systems, they have
many advantages: they perform protein modifications similar to those of
mammals, the risk of contamination by toxins or viruses is minimised and
there is high capacity to scale up the production volumes.

Despite the advantages, an important production step varies in plants and
humans - protein glycosylation, that is the binding of sugar chains to the
rest of the protein. Human derived proteins have a terminal galactose, where
plants lack it. Another difference is plant derived proteins have xylose
attached to their proteins, where as human proteins do not. Further
complicating the situation, the binding of fucose differs between plants and
humans as well.

Regardless of the advantages, the differences often render plant-derived
proteins unsuitable for the use in humans, because they can cause immune
responses, such as the production of antibodies against the plant-derived
protein or an allergic reaction.

Profitable moss

With these facts in mind, a research team headed by Dr. Eva Decker, from the
Faculty of Biology at the University of Freiburg in Germany in cooperation
with greenovation Biotech , genetically engineered the moss Physcomitrella
patens so that it produces proteins with humanized glycosylation profiles.
The researchers published their work in the journal Plant Biology.

The researchers chose to use moss as a production system, because moss does
not have the previously mentioned disadvantages of other plant species, such
as the lack of containment during field production. Their cultivation in
bioreactors provides controlled conditions in strict biological containment,
the possibility to release the proteins into the medium has major advantages
for product cleansing, In addition, the genome of Physcomitrella patens is
quite well mapped out. ?Sequencing of the moss genome at the Joint Genome
Institute in California was initiated by an international collaboration and
will be finished by the end of this year,? Dr. Decker told Checkbiotech.

Physcomitrella is the only known plant that can be genetically engineered by
targeted gene disruption via the so-called homologous recombination - a
technique allowing the destruction of unwanted gene functions. Thus,
allergic reactions in humans caused by plant-specific glycosylation can be
avoided by a systematic switch-off of the responsible genes. However, also a
well directed introduction of genes of another organism to the moss genome
is possible.

Human glycosylation patterns

In their experiments, the researchers introduced in the moss the genes that
encode the enzymes, which catalyze the binding of galactose and disrupted
those genes responsible for the binding of fucose and xylose. As a result,
the plants produced proteins with human glycosylation patterns.

Besides these genetic transformations, however, the plants showed no
deviations in growth, development and morphology when compared to
non-transformed moss. Also the secretion capacity remained at the same
level, which was recently measured as 5.8 milligram product per gram dry
weight for a functional immunoglobulin molecule (Gorr and Jost,
Bioprocessing J., July/August 2005). Thus, the protein modifications did not
effect the moss? metabolism and normal growth.

For the moment, the researchers do not plan on carrying out the same
experiments with other moss varieties on a large scale. ?The experiments
described in our publication served as a proof of concept for the
feasibility of targeted knockout of plant-specific enzymes combined with the
introduction of necessary human genes,? Dr. Decker said. Transgenic moss
used as production lines for specific protein products with humanised
glycosylation profiles for commercial use will be created separately by
greenovation.

Application in human medicine

Checkbiotech learned from Dr. Decker, that Physcomitrella is the only plant
so far in which the humanization of the plant glycosylation pattern is
possible by genetic transformation. ?I believe that moss will succeed as one
of a very few plant species to be used as an alternative to mammalian cell
lines or transgenic animals for the production of recombinant
pharmaceuticals,? Dr. Decker said. ?However, when exactly this time point
will be, is not decided by technical development, but rather by economical
evaluations, or the duration of clinical trials.?

Since Physcomitrella can be grown in bioreactors in a very simple medium -
just water and some minerals - the culture conditions can be modified
without problems, thus satisfying the needs of any protein that is secreted
to the culture medium.

In contrast to most other plants grown in liquid or suspension cultures, Dr.
Decker and her team did not use cells lines, rather a differentiated tissue.
This guarantees highest genetic stability and ?the moss can be proliferated
vegetatively and maintained in its juvenile stage by regular mechanical
disruption,? Dr. Decker told Checkbiotech.

Focus on protein secretion

The researchers are now focussing on the secretion of the protein into the
growth medium. ?Costs for further processes will be reduced as the
concentration of additional proteins in the medium is rather low compared to
within the cells, and as harvesting the protein of interest can be done
without destroying the plants,? Dr. Decker told Checkbiotech. On the other
hand, many proteins of interest are also naturally secreted from the cells
in which they were synthesized.

The researchers are now working on optimizing the secretion system by
analyzing protein secretion signals as well as engineering the moss cell
wall for an improved protein release. To ensure their research reaches the
final stage, they are looking for further financial support/funding.

Katharina Schoebi is a biologist and a Science Writer for Checkbiotech.
Contact her at katharina.schoebi@bluewin.ch.

E. L. Decker et al. Glyco-Engineering of Moss Lacking Plant-Specific Sugar
Residues. Plant Biology 7 (2005) pp. 292-299

Link:
[www.thieme-connect.com]-
2005-837653

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