Molecular Biotechnology

The Molecular Biotechnology Unit develops and establishes cellular and molecular assay systems. The methods comprise cell-based assays, analysis of gene expression, immunological methods, protein biochemistry, complex cell culture models as well as animal experiments. The unit further provides service for cellular compound screening such as efficacy, toxicity and transport. In addition, a main focus is on establishing and validating animal models for the investigation of the enzyme functions.

Biomarkers to predict vascular reocclusion following surgery

Arteriosclerotic changes in the body’s vessels are the primary cause of poor tissue perfusion. Depending on the perfusion area in question, this can lead to heart attacks and strokes. If, however, the muscles are not supplied with enough oxygen, many patients also experience impaired mobility, for instance due to pain in their legs. In advanced stages, the narrowed segment tends to either be circumvented by means of a bypass or the vessel is enlarged in the catheter laboratory using a balloon. For a high number of patients, however, this intervention is immediately followed by the treated vessel occluding once again, which in turn results in symptoms reemerging. An easily accessible biomarker capable of indicating respective negative processes at an early stage is therefore of great medical interest. The goal would be to more closely examine relevant high-risk patients with a view to intervening earlier and thus improving therapy results.

Researchers at Fraunhofer IZI have already identified a promising biomarker in previous studies – an enzyme (glutaminyl cyclase) whose activity is able to give a prognosis as to the healing process of the treated blood vessel. But these previous studies were initially only based on a small patient population. This biomarker can now be validated using a larger group of patients, graded according to concomitant diseases emerging in parallel such as type II diabetes mellitus or hyperlipidemia.

The Clinic for Vascular Surgery at the St. Elisabeth and St. Barbara Hospital in Halle (Saale), Germany, is an important partner here. Blood samples will be taken from patients at regular intervals before the operation and up to one year afterwards. These samples will then be sent to the Fraunhofer Institute for examination, as part of which a biochemical analysis of enzyme activity will be conducted in what will, to begin with, still be a relatively complex laboratory process. At the same time a simplified immunological test based on the amount of enzymes will be developed. A user-friendly and cost-efficient test would contribute to increased patient safety and make for a more targeted therapy for high-risk patients.

EFRE

Viromer carrier systems for the genebased treatment of inflammatory diseases

Broadly speaking, gene therapies seek to replace defective genes by either introducing healthy copies of the gene or inactivating defective gene products. In order to do this, corresponding gene sequences are delivered into the target cells which then regulate the production of the respective proteins at the translational level. Transporting the regulatory nucleic acids to the target cells is, however, no easy feat. Free nucleic acids are not usually absorbed by cells and are exposed to enzymes (nucleases) in biological fluids, which quickly break them down. Hence the need for molecular carrier systems to guide the nucleic acids to the point of action, delivering them into the target cells before releasing them.

So-called viromers present a highly promising solution here. Like other transporters, viromers form nanoparticles ranging between 100–500 nm in size and have nucleic acids packed inside them. Investigations carried out by Lipocalyx GmbH (www.viromer-transfection.com) have demonstrated high transfection efficiencies among immune cells, especially monocytes / macrophages and dendritic cells, when using the viromer technology.

The project will investigate the therapeutic potential of viromers as a carrier system, focusing on whether disease-relevant inflammatory mediators can be influenced by the transfection of regulatory DNA and / or RNA molecules. Using a model system for inflammatory joint diseases such as that for rheumatoid arthritis, viromers are to be identified that can be enhanced together with nucleic acids which specifically regulate inflammation, with the ultimate goal of developing a gene therapy.

It is generally expected that the development of new carrier systems for gene-based treatment concepts will close the gap between successful testing in vitro and efficacy in the disease model.

EFRE

Determination of pharmacokinetic parameters of small molecules

A comprehensive characterization of physico-chemical, cell-biological and pharmacokinetic properties of small molecules are prerequisite for their preclinical development. This process is required for the application of efficacious, safe and well-tolerated molecules in human subjects later during clinical development. Important steps during preclinics are investigations on liberation, absorption, distribution, metabolism and excretion (L-ADME parameters) in animal models. Here, information on exposure, bioavailability and terminal half-life will be collected. These data serve as decision points for selecting preclinical candidates or are used for optimization, e.g. bioavailability of an already selected candidate, by formulation development. The Department of Drug Design and Target Validation at Fraunhofer IZI develops new molecular therapies for neurodegenerative and inflammatory disorders. The department’s strategy includes identifying novel drug target and testing novel therapies. For characterizing new small molecule classes, a catheter-based rat model for analyzing pharmacokinetics of such compounds has been established by the Molecular Biotechnology unit. The model is comprised of surgical application of a catheter in the jugular vein (V. jugularis) and in the carotid artery (A. carotis communis), respectively. Using this method, it is possible to obtain complete compound profiles from a single animal, which avoids inter-individual variations, e.g. when using mice. In addition, a close collaboration with the Drug Design and Analytical Chemistry unit enables rapid determination of compounds concentrations in blood samples by LC-MS. The applied method is being used successfully within the Department of Drug Design and Target Validation, e.g. for own projects, such as the development of novel inhibitors for alternative beta-secretases or the development of novel inhibitors for the treatment of periodontitis. It is also requested and used by partners from industry and academia.

Pathological role of superantigens derived from expression of human endogenous retroviruses

Multiple Sclerosis (MS) is a progressive and chronic inflammatory disorder of the central nervous system. MS is characterized by a pathological demyelination of axons. Depending on the site of inflammation symptoms vary greatly and include paralysis or numbness of extremities, impaired vision and speech, dizziness, cognitive impairment, and fatigue. An underlying cause for the development of MS has not been identified so far. A widely accepted theory of MS pathology focusses on auto-reactive T-cells acting against axonal myelin. This simplistic view is challenged by a number of clinical observations arguing against a sole autoimmunological process.

First, newly emerging MS lesions are devoid of immune cells and second, MRI imaging suggests alterations in affected areas before the appearance of signs of inflammation. Furthermore, relapses during disease progression do not correlate with the disability patients face in the later stages of MS and standard therapy using immune modulatory and/or immune suppressive drugs has no influence on primary or secondary progredient forms of MS. Consequently, MS consists of 2 arms, 1) an inflammatory part (relapses) and 2) a slowly progressive neurodegenerative part.

The less understood 2nd arm might be correlated with the expression of human endogenous retroviruses (HERVs). HERVs are remnants of an ancient retroviral infection integrating viral DNA into the human genome. A number of HERVs still possess open reading frames, e.g. coding for the former viral envelope protein. These proteins could show superantigenic properties.

Therefore, the project focusses on the immunological properties of HERV-derived viral envelope proteins and the development of humanized monoclonal antibodies as alternative therapy for the treatment of MS.

Novel humanized animal models for studying the pathogenesis of Alzheimer’s disease

Alzheimer’s disease (AD) is characterized by a progressive loss of neurons and is accompanied by an impairment of learning and memory in elderly individuals. Besides major efforts in academic and industrial research, modeling the course of the disease in animals is challenging. A number of established animal models, especially mouse models of AD usually enable investigations only on certain pathological aspects. This is mainly attributed to the overexpression of Alzheimer-related genes in those models frequently including rare mutations devoid in patients with sporadic AD. We have selected 4 key proteins responsible for the development of AD and replaced them in mice by their human counterparts under control of the natural murine promoters. Fully humanization at these AD-specific loci by crossbreeding builds a platform to test new drugs against Alzheimer’s disease.

Rabbit model of in-stent restenosis for drug development

© Fraunhofer IZI

Coronary arteries narrowed by atherosclerotic changes are the underlying cause of angina pectoris and myocardial infarction. To re-open an occluded vessel, percutaneous transluminal angioplasty (PTA) with and without stent application is performed. Frequently, the operated vessel segment is re-occluded by hyperproliferation of smooth muscle cells in combination with invasion of monocytes forming a neointima. The process is called in-stent restenosis (ISR) if occurring after stent application.

The Molecular Biotechnology Unit has an established in vivo model of ISR available. Stent application in atherosclerotic New Zealand white rabbits is a powerful tool to investigate novel drug candidates, biomaterials and medicinal products.

Compound Screening in vitro

Activation of a GPCR by different ligands
© Fraunhofer IZI

Activation of a GPCR by different ligands

Efficacy

Permanent cell cultures are used for efficacy testing of novel compounds. Assays specific for the investigated target proteins are applied. Aim is the selection of the most potent compounds.

 

Determination of cytotoxicity for 2 compounds (A and B) using a human hepatocyte cell line
© Fraunhofer IZI

Determination of cytotoxicity for 2 compounds (A and B) using a human hepatocyte cell line

Toxicity

Cytotoxic and proliferative effects of novel compounds are screened on mammalian cell lines. > 100 cell lines are available for screening. Due to their pivotal role in drug metabolism, all compounds are tested on permanent human hepatocytes.

Apparent permeability coefficient of selected molecules
© Fraunhofer IZI

Apparent permeability coefficient of selected molecules

Transport

For the prediction of bioavailability in animal models, compounds are tested on an epithelial cell-derived transport model. The applied mammalian cells are human colorectal adenocarcinoma cell line, CaCo-2. Analysis is performed by LC-MS. The apparent permeability coefficient (Papp) is used to estimate bioavailability.

Cell culture of Iba1-positive primary murine microglia cells
© Fraunhofer IZI

Cell culture of Iba1-positive primary murine microglia cells

Cell culture of GFAP-positive primary murine glia cells
© Fraunhofer IZI

Cell culture of GFAP-positive primary murine glia cells

Primary cell models

The Molecular Biotechnology Unit has a longstanding expertise in experimental research using primary cells of the central nervous system.

For the prediction of bioavailability in animal models, compounds are tested on an epithelial cell-derived transport model. The applied mammalian cells are human colorectal adenocarcinoma cell line, CaCo-2. Analysis is performed by LC-MS. The apparent permeability coefficient (Papp) is used to estimate bioavailability.

Phenotyping transgenic mouse models

8 x PhenoMaster-System (TSE-Systems)
© Fraunhofer IZI

8 x PhenoMaster-System (TSE-Systems)

IntelliCage (NewBehavior) for automated behavior screening in groups
© Fraunhofer IZI

IntelliCage (NewBehavior) for automated behavior screening in groups

Phenotyping of transgenic mouse models follows the SHIRPA protocol. It is divided into primary, secondary and tertiary screen. Comprehensive test batteries for evaluating animal health and specific functions, e.g. motor coordination, cognition and emotion are available. Focus is on automated home cage observation to minimize the contact of humans with the laboratory mice.

 

Histology

Hematoxylin/Oil-Red staining of a plaque in the arterial wall of an atherosclerotic rabbit
© Fraunhofer IZI

Hematoxylin/Oil-Red staining of a plaque in the arterial wall of an atherosclerotic rabbit

Thioflavin S-positive deposits (green) in hippocampus of an Alzheimer mouse model
© Fraunhofer IZI

Thioflavin S-positive deposits (green) in hippocampus of an Alzheimer mouse model

To investigate disease-relevant pathologic alterations, a number of cutting and staining techniques are available.

 

Pharmacology

Concentration of a compound in the circulation after oral exposure
© Fraunhofer IZI

Concentration of a compound in the circulation after oral exposure

The determination of pharmacological parameters, e.g. bioavailability and plasma half-life, is done in-house using catheterized rats. Experiments are performed after extensive testing of compounds in isolated cell cultures and after allowance by the local animal well-fare committee. For measuring compound levels in body fluids, LC-MS-based methods are available.

  • Cynis H, Frost JL, Crehan H , Lemere CA. Immunotherapy targeting pyroglutamate-3 Aβ: prospects and challenges. Mol Neurodegener. 2016 Jun 30;11(1):48. DOI dx.doi.org/10.1186/s13024-016-0115-2
  • Cynis H, Funkelstein L, Toneff T, Mosier C, Ziegler M, Koch B, Demuth HU, Hook V. Pyroglutamate-Amyloid-beta and Glutaminyl Cyclase Are Colocalized with Amyloid-beta in Secretory Vesicles and Undergo Activity-Dependent, Regulated Secretion. Neurodegenerative Diseases. 2014; 14(2):85-97. DOI http://dx.doi.org/10.1159/000358430
  • Höfling C, Indrischek H, Höpcke T, Waniek A, Cynis H, Koch B, Schilling S, Morawski M, Demuth HU, Roßner S, Hartlage-Rübsamen M. Mouse strain and brain region-specific expression of the glutaminyl cyclases QC and isoQC. International Journal of Developmental Neuroscience. 2014 Aug; 36:64-73. DOI http://dx.doi.org/10.1016/j.ijdevneu.2014.05.008
  • Morawski M, Schilling S, Kreuzberger M, Waniek A, Jäger C, Koch B, Cynis H, Kehlen A, Arendt T, Hartlage-Rübsamen M, Demuth HU, Roßner S. Glutaminyl cyclase in human cortex: correlation with (pGlu)-amyloid-beta load and cognitive decline in Alzheimer‘s disease. Journal of Alzheimer‘s Disease. 2014;39(2):385-400. DOI dx.doi.org/10.3233/JAD-131535.
  • Cynis H, Kehlen A, Haegele M, Hoffmann T, Heiser U, Fujii M, Shibazaki Y, Yoneyama H, Schilling S, Demuth HU. Inhibition of Glutaminyl Cyclase alleviates CCL2-mediated inflammation of non-alcoholic fatty liver disease. Int. J. Exp. Pathol. 94 (2013), 3:217-225. DOI dx.doi.org/10.1111/iep.12020
  • Nussbaum J, Schilling S, Cynis H, Silva A, Swanson E, Wangsanut T, Tayler K, Wiltgen B, Hatami A, Rönicke R, Reymann K, Hutter-Paier B, Alexandru A, Jagla W, Graubner S, Glabe CG, Demuth HU, Bloom GS. Prion-like behavior and tau-dependent cytotoxicity of pyroglutamylated amyloid peptides. Nature 485 (2012), 7400:651-655. DOI dx.doi.org/10.1038/nature11060
  • Cynis H, Hoffmann T, Friedrich D, Kehlen A, Gans K, Kleinschmidt M, Rahfeld JU, Wolf R, Wermann M, Stephan A, Haegele M, Sedlmeier R, Graubner S, Jagla W, Müller A, Eichentopf R, Heiser U, Seifert F, Quax PH, de Vries MR, Hesse I, Trautwein D, Wollert U, Berg S, Freyse EJ, Schilling S, Demuth HU. The isoenzyme of Glutaminyl Cyclase is an important regulator of monocycte infiltration under inflammatory conditions. EMBO Mol. Med. 3 (2011), 9:545-558. DOI dx.doi.org/10.1002/emmm.201100158
  • Wirths O, Breyhan H, Cynis H, Schilling S, Demuth HU, Bayer TA. Intraneuronal pyroglutamate-Abeta 3-42 triggers neurodegeneration and lethal neurological deficits in a transgenic mouse model. Acta Neuropathol. 118 (2009), 4:487-496. DOI dx.doi.org/10.1007/s00401-009-0557-5
  • Cynis H, Scheel E, Saido T, Schilling S, Demuth HU. Amyloidogenic processing of amyloid precursor protein: evidence of a pivotal role of Glutaminyl Cyclase in generation of pyroglutamate-modified amyloid-beta. Biochemistry 47 (2008), 28:7405-7413. DOI dx.doi.org/10.1021/bi800250p
  • Schilling S, Zeitschel U, Hoffmann T, Heiser U, Francke M, Kehlen A, Holzer M, Hutter-Paier B, Prokesch M, Windisch M, Jagla W, Schlenzig D, Lindner C, Rudolph T, Reuter G, Cynis H, Montag D, Demuth HU, Rossner S. Glutaminyl Cyclase inhibition attenuates pyroglutamate Abeta and Alzheimer’s disease like pathology. Nat. Med. 14 (2008), 10:1106-1111. DOI dx.doi.org/10.1038/nm.1872
  • Cynis H, Rahfeld JU, Stephan A, Kehlen A, Koch B, Wermann M, Demuth HU, Schilling S. Isolation of an isoenzyme of human Glutaminyl Cyclase: Retention in the Golgi complex suggests involvement in the protein maturation machinery. J. Mol. Biol. 379 (2008), 5:966-980. DOI dx.doi.org/10.1016/j.jmb.2008.03.078
  • Cynis H, Schilling S, Bodnar M, Hoffmann T, Heiser U, Saido TC, Demuth HU. Inhibition of Glutaminyl Cyclase alters pyroglutamate formation in mammalian cells. Biochim. Biophys. Acta 1764 (2006), 10:1618-1625.