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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.

Glossary

  • Amino acids are the subunits of proteins; twenty different amino acids are universally found in proteins.
  • Antigens are foreign substances that stimulate the immune system to produce antibodies.
  • Antibodies are blood proteins (immunoglobulins) which are produced by the B lymphocytes in response to disease. They recognise foreign substances that have entered the body (e.g. bacteria) and help the body fight against a particular disease and develop an immunity to that disease.
  • Bacteria are microscopically small, unicellular organisms belonging to the prokaryotes.
  • Enzymes are catalysts in the living cells. They allow the execution of chemical transformations of the metabolism at body temperature.
  • A gene is a hereditary unit which has effects on the traits and thus on the phenotype of an organism. Part on the DNA which contains genetic information for the synthesis of a protein or functional RNA (e.g. tRNA).
  • The genome is entire genetic material of an organism. Each cell of an organism contains the entire genetic material in its nucleus.
  • Genetic engineering is a term describing different molecular biological techniques. It allows the recombination of DNA fragments of different origin, their proliferation and expression in suitable host cells.
  • A glycoprotein is a protein linked to a polysaccharide chain (N-acetylhexosamine, galactose, mannose, glucose).
  • Insulin is a hormone produced in the ß cells of the islands of Langerhans of the pancreas and leads to the reduction of the blood glucose level. People suffering from diabetes lack this hormone.
  • Mutations are any kind of alteration of genetic information (e.g., the exchange of a base; translocation of a DNA section; introduction of additional bases; loss of bases or entire DNA sections). Mutations occur constantly in nature (induced by UV rays, natural radioactivity) and are the basis of evolution.
  • A protein is a high-molecular complex made up of amino acids. The proteins perform a wide variety of activities in the cells and represent more than 50% of organic mass.
  • The immune system is the body’s defence system that protects the organism against dangerous pathogens. Furthermore, it destroys abnormal body cells. Those defence mechanisms are build up by a complex interaction of several organs, different cell types and chemical molecules.
  • The endothelium is a cell layer on the inside of lymphatic and blood vessels.
  • Glycosylation is the addition of carbohydrate to an organic molecule, mostly proteins.
  • Endothelial cells line the inner surface of lymphatic and blood vessels. They build a semi-permeable barrier between the vessle lumen and the tissue.

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.

Glossary

  • Antibodies are blood proteins (immunoglobulins) which are produced by the B lymphocytes in response to disease. They recognise foreign substances that have entered the body (e.g. bacteria) and help the body fight against a particular disease and develop an immunity to that disease.
  • Bacteria are microscopically small, unicellular organisms belonging to the prokaryotes.
  • Biotechnology is the study of all processes involving life cells or enzymes for the transformation and production of certain substances.
  • A gene is a hereditary unit which has effects on the traits and thus on the phenotype of an organism. Part on the DNA which contains genetic information for the synthesis of a protein or functional RNA (e.g. tRNA).
  • In a biological context, insertion often means the introduction of DNA fragments into another DNA molecule.
  • Being lytic is the feature of a bacteriophage leading to the destruction (lysis) of the host cell upon infection.
  • Monoclonal antibodies are structurally identical antibodies which hence have an identical antigen-binding site.
  • There are two definitions for the term organism: a) Any biological unit which is capable of reproduction and which is autonomous, i.e. that is able to exist without foreign help (microorganisms, fungi, plants, animals including humans). b) Definition from the Gentechnikgesetz (German Genetic Engineering Law): “Any biological unit which is capable of reproducing or transferring genetic material.“ This definition also includes viruses and viroids. In consequence, any genetic engineering work involving these kinds of particles is regulated by the Genetic Engineering Law.
  • Pathogenity is the ability to cause a disease. One differentiates between human, animal, and plant pathogens which specifically cause a disease in either humans, animals or plants.
  • Recombination is the process in which DNA is recombined. As a natural process, recombination takes place in sexual reproduction during meiosis. In vitro recombination involves the joining of DNA molecules of different origin using recombinant DNA technologies.
  • Genetic sequences are successions of the bases adenine, thymine, guanine, and cytosine on the DNA (or uracil instead of thymine in the case of RNA).
  • Transgenic organisms are organisms (microorganisms, animals, plants), into which foreign material has been introduced using genetic engineering methods and which is inherited from one generation to the other. Transgenic organisms are thus genetically-modified organisms.
  • Expression means the biosynthesis of a gene product. Usually, DNA is transcribed into mRNA and subsequently translated into proteins.
  • The serum is the fluid part of the blood that remains after separation of the blood cells by centrifugation of a blood sample. The serum mainly consists of water and proteins, the so-called serum proteins.
  • Molecular means: at the level of molecules.
  • Pharmacology is the study of interactions between drugs and organisms. There are two methods of evaluation: The pharmacokinetics describes the uptake, distribution, metabolism and excretion of an active substance. The pharmacodynamics describes the effects of a drug in the organism.
  • Polyehylene (abbrevation: PE) is the polymer of ethylene. It belongs to the thermoplastic synthetics.
  • Glycosylation is the addition of carbohydrate to an organic molecule, mostly proteins.
  • Engrafting cells, tissue or organs from a donor into a recipient is called transplantation. There exist several types of transplantation which are classified in three categories: function, location and derivation. Engrafting an organ of a different species into a recipient is called xenogenic transplantation, whereas an allogenic transplantation describes the tranfer of grafts from a donor into a recipient being a member of the same species. If donor and recipient are the same individual, the transplantion is called autogenic. Furthermore there exists a syngenic transplantation which is characterized by grafts originating from the uniovular twin. Furthermore, engrafting artificial material is called alloplastic transplantation. To suppress the natural immune response being raised by the transplanted organs, recipients have to be administered immunosuppressants. Since 1997 in Germany the legitimacy of organ donation is regulated by the Transplantation Law. In the case of brain death, an organ donor card has to be available or relatives have to agree with the organ donation. Today, the most frequently transplanted organs are heart, liver, kidney and cornea.
  • Endothelial cells line the inner surface of lymphatic and blood vessels. They build a semi-permeable barrier between the vessle lumen and the tissue.
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