By Katharina Schoebi, Checkbiotech

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.

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.

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

Link: http://www.thieme-connect.com/ejournals/abstract/plantbiology/doi/10.1055/s-2005-837653