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Using mosses to produce medicines

Human disease can be treated with substances from traditional medicinal plants or with molecular pharming products. Molecular pharming uses genetic engineering techniques to insert genes into plants or animals that would otherwise not express these genes. These transgenics can then be used to produce therapeutic proteins such as antibodies. Dr. Eva Decker from the University of Freiburg and her team have now successfully produced a key protein of the human immune system in the moss Physcomitrella patens. Recombinant factor H might be used at some stage in the future for the therapy of atypical haemolytic uremic syndrome, a rare, life-threatening disease that affects kidney function. This is the common goal of Freiburg University Medical Centre and a company called Greenovation.

Atypical haemolytic uremic syndrome (aHUS) is a life-threatening, progressive disease that usually develops in early childhood, and when left untreated, leads to chronic kidney failure or death in around 60 percent of those affected. In most cases, the disease is caused by a defective complement system, which destroys the body's endothelial cells. The complement system is part of the innate immune system. Approximately 50 percent of all aHUS cases are inherited, and are due to mutations of the complement regulatory protein factor H that are passed on to the next generation. The disease is characterised by an excessive activation of complement, meaning that defence reactions are directed against the body's own structures, primarily in the kidney. Although such mutations are relatively rare, aHUS patients are generally condemned to lifelong treatment. Current treatment methods primarily involve the regular exchange of blood plasma using donor blood along with kidney transplants. However, these interventions only control rather than cure the disease.

Factor H as regulator

Eva Decker © Christina Dages

The complement system, which is part of the humoral immune response, induces a number of inflammatory reactions when stimulated by one of several triggers. The complement system is made up of around 30 or so proteins, including serum proteins and integral cell membrane proteins, whose mission it is to identify and eliminate microorganisms. "This part of the innate immune system is permanently subliminally active, which means that it provides immediate defence against infection without a disease needing to be established or antigen-recognising antibodies formed," says Dr. Eva Decker, a researcher in the Department of Plant Biotechnology at the University of Freiburg led by Prof. Dr. Ralf Reski.

The main task of the complement system is to make invading pathogens more susceptible to phagocytosis. This molecular mechanism, which is known as opsonisation, works as follows: complement factors bind to the surface of invading pathogens, thereby making the pathogens attractive to phagocytes. Pathogen stimulation triggers an amplifying activation cascade, which activates a protein complex that induces pore formation. This eventually leads to cell lysis and cell death. The formation of cell-killing membrane attack complexes (MAC) on the surface of invading pathogens is primarily triggered by the conversion of the complement component C3 into C3b. "The problem is that when a system designed to destroy cells is permanently active, regulators that protect the body against attack by its own complement system are also required," says Decker. Factor H is one such regulator and it ensures that the complement system is directed towards pathogens rather than own tissue. As long as C3b is in a free state or bound to body cells, factor H and factor I are able to inactivate C3b. In contrast, C3b remains active when bound on the surface of pathogens, thus triggering further steps involved in the formation of MAC. In healthy people, factor H is constitutively synthesised, which means it is always present, ensuring that the immune system does not attack own body cells.

Excessive activation in the presence of factor H

The antibody eculizumab and factor H target different components of the activation cascade of the complement system. The activation cascade consists of several protein factors (C3-C9), and this targeting prevents the establishment of the cell-killing membrane attack complex (MAC). © Eva Decker, modified from Howes et al. 2006, Review of Ophthalmology

Different mutations might prevent the formation of factor H. The lack of this regulatory protein leads to an uncontrolled, hyperactive complement system. The result is that the body's own cells are also attacked, leading to the deposition of intermediary products, especially on the basement membrane of the renal corpuscles. "The absence of factor H leads to permanent activation of the complement system, resulting in tissue damage with varied consequences," says Decker. One of these consequences is aHUS. The monoclonal antibody eculizumab, one of the most expensive medicines in the world, is already approved for aHUS treatment. Eculizumab blocks the cascade, thus preventing MAC from being formed. Decker and her team are trying to come up with an alternative treatment. In cooperation with Dr. Karsten Häffner from the Children's Hospital in Freiburg and Greenovation Biotech GmbH, Decker and her group of researchers are assessing an approach whereby the reaction cascade of the complement cascade is corrected by providing factor H. Dr. Decker comments: "Eculizumab interferes with the cascade later than factor H does, namely at C5 rather than C3. By then, all intermediary products that can lead to renal damage have accumulated." Decker therefore considers it far more sensible to interfere further upstream of the cascade, namely at the C3 site where a natural mechanism has failed.

Moss as an effective glycoprotein producer

Im Moos-Bioreaktor arbeitet Physcomitrella im Dienst der Wissenschaft und bald vielleicht auch für die Arzneimittelproduktion. © Eva Decker, Universität Freiburg

In 2010, Decker and her colleagues were the first to successfully produce the complement factor H in the moss Physcomitrella. They now want to examine whether the human glycoprotein is suitable for the medicinal treatment of aHUS. This will be the subject of a two-year project. Although the moss Physcomitrella patens does not normally produce or need factor H, transgenic Physcomitrella is able to express a full-length human factor and produce this glycoprotein from the transferred gene. However, the glycoprotein can only exert its proper function with specific carbohydrates attached to it. The glycosylation reaction must therefore ensure that the resulting glycosylation patterns will not lead to immune reactions in the patient. Glyco-optimising the recombinant protein version is a huge challenge, as the reaction can lead to different results in different organisms. Bacteria are unable to carry out post-translational glycosylation reactions; humans and plants have complex glycosylation patterns that differ considerably between the two: plants form xylose and fucose, two carbohydrates that humans do not have or which are linked differently to proteins. Physcomitrella incorporates fucose and xylose into recombinant factor H at different places from that of human factor H. Decker managed to glyco-optimise factor H using a genetic trick: "As we did not want fucose and xylose in factor H, we simply silenced the enzymes that attach them to proteins." Compared to the glycosylation reaction in humans, the reaction in moss is far more homogenic. This might be of advantage, as it helps the researchers achieve a high purity of the pharmacologically active substance.

The experiments the researchers will carry out over the next few years will show whether recombinant factor H is likely to be suitable for clinical application. Its suitability will initially be tested in vitro and subsequently in vivo in mice. Whatever the result, the approach is quite interesting. Instead of treating aHUS with complex and expensive therapies such as PE or the antibody eclizumab, the researchers foresee factor H being administered on demand in the same way as insulin.

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