Synthetic Biology (SB)

Synthesis of single cell, single molecule RNA probes for the detection of enogenous microRNAs in human cancer to dentify metastatically relevant cance cell fractions in heterogenous tumors

Regine Kleber — Mon, 30 Jul 2018 11:16:37 +0000

Synthesis of single cell, single molecule RNA probes for the detection of enogenous microRNAs in human cancer to dentify metastatically relevant cance cell fractions in heterogenous tumors Regine Kleber Mon, 07/30/2018 - 13:16

It is estimated that metastasis is the main reason in about 90% of cancer deaths. Therefore, a better understanding of the key molecular players in determining and regulating this process is urgently needed. Previous studies in fixed cells in culture have shown, that certain microRNA (miRNA) molecules are in significantly higher numbers in exosomes of highly metastatic cells in comparison to low metastatic cells [1]. However, in real patient tissues, issues such as the cell heterogeneity in the primary tumor and the definition of metastatic clones are a major challenge in the context of metastasis.

In this project, we used human colon tissue sections, which are relevant samples for routine diagnosis in pathology, to study the chromatin nanostructure of different cells types and the presence and distribution of five metastatically relevant miRNAs (miR-21, miR-31, mi-R210, miR-135b and miR-218).

DNA-sequences complementary to the above-mentioned metastatically relevant miRNA were synthesized and modified so that a fluorescent dye was incorporated in the middle of the sequence. Human formalin-fixed paraffin-embedded (FFPE) colon tissues were sliced into 10mm thick sections and fluorescent in situ hybridization (FISH) with the different miRNA probes was performed at normal and colon cancer tissue sections followed by a subsequent nuclear counterstain with Sytox Orange (SO).

Confocal images allowed the quantification of the different miRNAs in human tissue samples. Pro-metastatic miRNAs were found in significantly higher numbers of molecules in epithelial cells of tumor samples in comparison with their normal counterparts. More variability and a strong patient-associated quantification was observed for miR-218, an anti-metastatic miRNA.

We successfully performed, for the first time, single molecule localization microscopy (SMLM) for the chromatin nanostructure and human microRNAs at the single molecule level in routine patient tissues. Preliminary results showed the distribution of miR-21 in the periphery and the inside of a tumor cell nucleus. Vesicle-like structures outside the nuclear region of sizes ranging from 110 to 240 nm appear to be loaded with this miRNA.

Research bridges

Advanced Imaging Platform (AIP)

Synthetic Biology (SB)

Start date

01/01/2018

12/31/2018

Project management

Medical Faculty Mannheim

Heidelberg University

Experimental Surgery, Cancer Metastasis

Institute for Organic Chemistry

Karlsruhe Institute of Technology

Wagenknecht Group

Modular shining architectures for cellular imaging

Regine Kleber — Mon, 12 Nov 2018 16:06:46 +0000

Modular shining architectures for cellular imaging Regine Kleber Mon, 11/12/2018 - 17:06

In this project, we will design and develop a modular approach to address target-specifically accumulating conjugates consisting of bright fluorescent dyes and target-affine peptides. For this, we will create fluorescent dyes with bright fluorescence in the optimal optical imaging window (targeting the IR range) and derivatize them with functional groups to obtain substances which can be efficiently introduced in various bioactive molecules such as - but not limited to - receptor-affine peptides. On the other side, we will synthesize receptor-specific peptides being able to efficiently address our model receptor system (gastrin-releasing peptide receptor) and chemically modify them to achieve a high stability and appropriate hydrophilicity (depending on the respective fluorescent dye used) to obtain fluorescent drugs with high potential for prospective in vivo imaging.

For this purpose, chemical developments have to be carried out to establish an approach yielding substances with high future potential. The developed model ligands will be characterized regarding their optical properties as well as their hydrophilicity and in vitro stability.

This project complements the activities of the Bräse group within the GRK 2939 - the Wängler group has profound expertise in particular in in vitro evaluations and tumor mouse models which is not present in this depth in the GRK.

In addition, the Wängler group adds radiochemistry expertise lacking in Karlsruhe and its GRK.

Research bridges

Advanced Imaging Platform (AIP)

Synthetic Biology (SB)

Start date

01/01/2019

12/31/2019

Project management

Institut für Klinische Radiologie und Nuklearmedizin

Heidelberg University

CV Carmen Wängler

Director Research Bridge SB

Institute of Organic Chemistry (IOC)

Karlsruhe Institute of Technology

Research Group Bräse

Modulation of epithelial-to-mesenchymal transition (EMT) with small molecules

Regine Kleber — Mon, 12 Nov 2018 16:17:19 +0000

Modulation of epithelial-to-mesenchymal transition (EMT) with small molecules Regine Kleber Mon, 11/12/2018 - 17:17

EMT is a widespread and important biological process that relies on a genetically induced reduction of coordinated cell behavior. This reduction allows individual cells inside an epithelium to change their properties (i.e. enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and increased production of extracellular matrix components) and leave it as individual, mesenchymal cells. Understanding the regulation of cell behavior during EMT is crucial for understanding core developmental processes like muscle and heart development, and it has recently become a focus in the investigation of new strategies against cancer and cancer resistance.

The Lemke lab has identified two extreme modes of EMT-related processes in the midge C. riparius and the fruit fly D. melanogaster. In Chironomus, levels of cell coordination were low: future muscle cells leave the epithelium and translocate to the inside of the embryo using canonical EMT. In Drosophila, levels of cell coordination were high and EMT was delayed: cells remained in an epithelium, collectively folded into the inside of the embryo, and only then gradually separated. Genetic investigations have shown that two genes act as a switch to evoke canonical EMT in Drosophila, and delayed EMT in Chironomus, rendering this pair of species as an in vivo test tube to reveal factors that are necessary and/or sufficient to strengthen epithelial integrity and suppress EMT. To identify factors beyond the known genetic players, we will identify and investigate small molecules that trigger changes in cell and tissue behavior. The initial compounds will be obtained by natural product isolation and selection of promising core structures from the ComPlat library. By comparing genetic with compound-induced changes, the mechanism of EMT suppression will be further dissected. More generally, we aim to identify previously proposed but still unknown biological targets that regulate the coordination of cell behavior.

Research bridges

Synthetic Biology (SB)

Start date

01/01/2019

12/31/2019

Project management

Centre for Organismal Studies Heidelberg (COS)

Heidelberg University

Junior Research Group Lemke

Institute of Organic Chemistry (IOC)

Karlsruhe Institute of Technology

ComPlat @KIT

3D bioprinting of a blood-retina-barrier to elucidate the role of mesenchymal stromal/stem cells as potential therapy in diabetic retinopathy (3D-BRB)

Regine Kleber — Mon, 12 Nov 2018 16:18:40 +0000

3D bioprinting of a blood-retina-barrier to elucidate the role of mesenchymal stromal/stem cells as potential therapy in diabetic retinopathy (3D-BRB) Regine Kleber Mon, 11/12/2018 - 17:18

Pericyte loss is the earliest morphological sign of diabetic retinopathy. During the course of the disease the retinal blood vessels face a loss in the stabilizing pericytes which leads to destabilization of the blood vessel, haemorrhage, and due to local hypoxia to the angiogenesis of new capillaries leading to blindness. It has been hypothesized that diabetic retinopathy can be delayed or prevented through restoration of pericytes and pericyte function by adipose tissue-derived stem/stromal cells (ASC). According to surface and internal marker validation, ASCs share pericyte characteristics, and may have a therapeutic benefit in injured micro vessels in the eye. In combination with endothelial cells (ECs), ASCs are able to establish functional vessels in vivo. However, the factors that coordinate EC-ASC communications to promote migration of these cells toward one another, and their heterotypic assembly into vascular structures are not well defined. To understand the mechanisms of EC-ASC interaction, we will team up to develop a sophisticated in vitro 3D model by 3D bio printing of retinal blood vessels from EC in which the migration and homing and differentiation of the ASCs can be monitored over a long period of time. There are preclinical data that prove the homing of ASCs towards these blood vessels when injected intravitreally. Eventually they migrate, adhere to and interact with EC, differentiate to take over the function of the formerly lost pericytes. We would like to understand, what are the secreted or presented factors of the EC-layer or other involved cells responsible for homing of ASCs and how the interact with the endothelium affects pericyte-like differentiation and subsequently control of endothelial cells. In addition the 3D systems offers a unique chance to study the influence of ASC/pericyte-like ASC on additional cell types, especially immune and inflammatory cells such as regulatory T cells and microglia in the context of diabetic retinopathy.

Research bridges

Synthetic Biology (SB)

Start date

01/01/2019

12/31/2019

Project management

Medical Faculty Mannheim / Experimental Cell Therapy

Heidelberg University

Research Group Bieback

Institute of Functional Interfaces (IFG)

Karlsruhe Institute of Technology

Research Group Schepers

Development of novel compound classes to establish the marine sea anemone Aiptasia as a laboratory system for coral symbiosis

Maria Garcia Hernandez — Thu, 07 Mar 2019 12:38:32 +0000

Development of novel compound classes to establish the marine sea anemone Aiptasia as a laboratory system for coral symbiosis Maria Garcia H… Thu, 03/07/2019 - 13:38

This collaborative HeiKa project between Prof. Stefan Bräse (KIT) und Dr. Annika Guse (COS Heidelberg) aimed to advance our current understanding of the mechanisms underlying intracellular coral-algal-symbiosis. Coral reef ecosystems are of immense ecological and economic importance. However, the molecular principles underlying coral symbiosis are poorly understood, mostly because corals are not suitable as experimental systems. In this project, we followed an interdisciplinary approach by combining organic chemistry, cell and organismal biology to further develop Aiptasia, a marine see anemone as an emerging model system for corals. Together, we screened a chemical compound library to identify biologically relevant effectors and started to synthesize gorgosterol, a peculiar sterol derivative, that is thought to play a key role for coral symbiosis as the basis for functional studies. The initiated experiments are still ongoing and additional ideas have been generated. Accordingly, both PI´s will continue to collaborate and exchange expertise to advance the common goal of uncovering how sterols contribute to establishment and maintenance of coral-algal-symbiosis.

Research bridges

Synthetic Biology (SB)

Project management

Centre for Organismal Studies

University of Heidelberg

Contact

Director Research Bridge SB

Institute of Organic Chemistry (IOC)

Karlsruhe Institute of Technology

Research Group Bräse

Engineering the three-dimensional environment of eukaryotic cells: a combined experimental and modeling approach

Maria Garcia Hernandez — Thu, 07 Mar 2019 12:47:19 +0000

Engineering the three-dimensional environment of eukaryotic cells: a combined experimental and modeling approach Maria Garcia H… Thu, 03/07/2019 - 13:47

Cell shape is an important indicator for essential processes such as cell migration or division. Cell shape is strongly determined by the organization of the actin cytoskeleton, which in turn responds dynamically to a large range of extracellular cues. Quantitative analysis of cell shape helps to reveal the physical processes underlying shape determination, but it is notoriously difficult to conduct it in three dimensions (3D). The Bastmeyer group used direct laser writing to create 3D open scaffolds for adhesion of connective tissue cells through well-defined adhesion sites. Due to actomyosin contractility in the cell contour, characteristic invaginations lined by actin bundles form between adjacent adhesion sites as previously described for cells on 2D substrates. Using quantitative image processing and mathematical modeling, the Schwarz group demonstrated that the resulting shapes in 3D can be described by the tension-elasticity model. Our results have been written up as short report and are currently under review by Biophysical Journal. They suggests that shapes are determined not only by contractile forces, but also by elastic stress in the peripheral actin bundles. Intracellular forces are mainly generated by non-muscle myosin II (NMM II) motors assembled into minfilaments and can be sustained either by the NMM II motors themselves or by crosslinks like alpha-actinin. It is largely unknown how pN-forces generated by single minifilaments are translated into nN-forces measured at single cell-matrix adhesions and and how these forces are divided up between motor and crosslinking activity. We currently quantify the dynamics of NMM II minifilaments associated with peripheral actin bundles to clarify this important point. Our combined experimental and theoretical approach will help to derive a more detailed understanding how individual contractile units generate forces on the cellular level and contribute to defined cell shapes. We also plan to use our improved understanding of cell forces and shapes in 3D for engineering cell function through design of extracellular cues.

Research bridges

Synthetic Biology (SB)

Project management

Institute for Theoretical Physics

University of Heidelberg

contact

Zoological Institute

KIT

Contact

A proof-of-concept approach for high-throughput screening of protective vs. harmful antibody profiles in infectious diseases

Maria Garcia Hernandez — Thu, 07 Mar 2019 12:51:39 +0000

A proof-of-concept approach for high-throughput screening of protective vs. harmful antibody profiles in infectious diseases Maria Garcia H… Thu, 03/07/2019 - 13:51

In this project, we have expanded our successful collaboration on malaria and flavivirus research on the basis of a new technological array platform. We analyzed interactions of human serum antibodies with linear peptides for their value in diagnostic applications and understanding disease mechanisms.

The goal of this project was to apply systematic partial and whole proteome approaches to characterize antibody profiles of infectious diseases where antibodies are implicated in protection or enhancing disease – but at the same time not well characterized. Therefore, we exploited our novel peptide array technologies to generate arrays containing the whole proteome of different pathogens (or large parts thereof) as overlapping linear peptides. Then, we analyzed our well-characterized patient serum collection with these arrays to obtain the antibody interaction profiles. With this information, we could find important pathogen-specific epitopes with diagnostic and immunogenic value. Furthermore, we compared and quantified the antibody response between different related pathogens (dengue virus and zika virus) to gain insight into possible immune mechanisms.

Three tropical infectious diseases, malaria, dengue fever, and zika served as examples, but the proof-of-principle results will be applicable for a wide range of diseases, where heterogeneous antibody populations are implicated in the clinical symptoms or severity. Furthermore, we could establish the single amino acid fingerprinting approach, to acquire in depth knowledge on binding epitopes and their specificity.

Dengue virus (DENV) infections are currently on the rise in many countries, causing a global pandemic, which could potentially also threaten parts of Europe in the context of climate change. The disease can take a severe course where the underlying pathophysiology is still poorly understood – yet, most researchers agree that immunological mechanisms, and especially enhancement by antibodies, seem to play an important role.

Zika virus (ZIKV) has recently emerged in the Americas causing an epidemic, which lead WHO to declare a ‘Public Health Emergency of International Concern’ for the clusters of microcephaly and neurological disease associated with Zika Infections. An immunological interaction after previous exposure to Dengue is potentially associated with a higher risk of severe congenital complications after Zika virus infection. In adults, the auto-immune mediated ‘Guillain-Barré-Syndrome’ was the most important complication seen after Zika virus infections. The detailed pathomechanism is unknown, but antibodies need to be examined if they contribute to this complication.

In malaria, a decrease in the transmission is associated with a decrease of other infectious diseases, notably bacteremia and sepsis. These indirect beneficial effects of anti-malaria control interventions are potentially ascribed to an immune-modulatory effect of acute malaria, which is not well understood.

With these proof-of-principle results, we can expand our successful collaboration to investigate other pathogens and analyze the role of elicited antibodies in immunity and immune escape mechanisms.

Research bridges

Synthetic Biology (SB)

Project management

Section of Tropical Medicine

Heidelberg University Hospital

Contact

Institute of Microstructure Technology (IMT)

Karlsruhe Institute of Technology

3D bioprinting of heart tissue for transplantation - Effects of materials and endothelial factors on stem cell maturation to functional myocardial tissue

Maria Garcia Hernandez — Tue, 10 Dec 2019 14:46:14 +0000

3D bioprinting of heart tissue for transplantation - Effects of materials and endothelial factors on stem cell maturation to functional myocardial tissue Maria Garcia H… Tue, 12/10/2019 - 15:46

Heart disease is the cause of most deaths worldwide. Although some patients can be treated by heart transplantation, thousands of patients suffer due to a lack of donor organs.

Therefore, it is important in the field of regenerative medicine to develop new therapies in order to meet the emergency. In this project, artificial heart tissue will be produced by 3D bioprinting. In a proof of principle study a prototype murine heart tissue will be produced in 3D in order to test its functionality, cell communication and tissue differentiation in vivo and ex vivo. In a second step, the structure will be prepared to be scaled to a size of a human heart.

Different cardiovascular cell types, differentiated from mouse embryonic stem cells (mESCs) will be printed in individually adapted hydrogels with different printing processes and spatial resolutions to produce an artificial heart that comes as close as possible to a murine heart in terms of shape, topography and functionality. Vascularization will be achieved by printing of sacrificial hydrogel structures to form a pre-formed vascular network, which will be subsequently colonized with endothelial cells. During and after the printing process, the artificial blood vessels will be perfused with a newly developed microperfusion system in order to supply the printed organ with oxygen and nutrients ex vivo. The printed organs will be further subjected to structural and functional testing in vitro and in vivo after transplantation in mice. Here, the role of endothelial-derived signals through direct cellular contacts or angiocrine signals on cardiomyocyte proliferation, survival and maturation will be investigated. Within this project, we will closely collaborate with the Tal Dvir’s group that has achieved a major breakthrough in the field of 3D bioprinting.

Research bridges

Medical Technology for Health (MTH)

Synthetic Biology (SB)

Start date

01/01/2020

12/31/2020

Project management

Vaskuläre Biologie und Medizin

Medizinische Fakultät Mannheim

Gergana Dobreva

Institute of Functional Interfaces (IFG)

Karlsruhe Institute of Technology

Research Group Schepers

CRISPR/Cas based tissue engineering

Maria Garcia Hernandez — Tue, 10 Dec 2019 14:48:15 +0000

CRISPR/Cas based tissue engineering Maria Garcia H… Tue, 12/10/2019 - 15:48

Application of CRISPR/Cas based technologies are revolutionizing biology and are laying ground for various new approaches of synthetic biology. In its simplest form the Cas9 nuclease is guided by a single guide RNA (sgRNA) to a specific site in the genome, where it introduces a double strand break (DSBs). Thus, the technology is used to change eukaryotic genomes in a programmed way. Here, we want to establish a completely new application of the system: CRISPR/Cas based tissue engineering. The Puchta group at the KIT was able to show that the Cas9 nuclease from Staphylococcus aureus (SaCas9) is a much more efficient tool for DSB induction in plants than the widely used enzyme from Streptococcus pyogenes (SpCas9). A surprising discovery was that if a single sgRNA is used to target SaCas9 at highly repetitive sequences in the genome, like the rDNA genes or centromeric repeats, the repair machinery is overwhelmed and cell death can be induced. Thus, SaCas9 should be useable as a novel tool for killing defined classes of cells (“CRISPR-Kill”). The aim of the suggested project is to test if CRISPR-Kill is able to eliminate specific stem cell types out of more complex organisms or organs by modulating its expression. Thus, artificial developmental switches or new combinations of cell types for biochemical synthesis should come into reach. The potential of the technology to reprogram root and shoot development in plants should be elucidated based on the unique expertise of the Wolf group at the UHD. The group has setup sophisticated tools for microscopically monitoring microscopically root and shoot development in combination with cell type specific expression systems. Depending on the promoters used for SaCas9 expression, it should be possible to eliminate different stem cell types at will during growth and study developmental and biochemical consequences. The general potential of the technology reaches far beyond plants and might be useful for the production of synthetic organs.

Research bridges

Synthetic Biology (SB)

Start date

01/01/2020

12/31/2020

Project management

Centre for Organsimal Studies (COS)

Universität Heidelberg

Sebastian Wolf

Botanisches Institut

Karlsruher Institut für Technologie

Holger Puchta

High-throughput compound screening in 3D glioma tumorsphere models

Maria Garcia Hernandez — Tue, 10 Dec 2019 14:50:22 +0000

High-throughput compound screening in 3D glioma tumorsphere models Maria Garcia H… Tue, 12/10/2019 - 15:50

Gliomas are the most common primary brain tumors in adults and consist of both higher-grade and lower-grade tumors. Lower-grade gliomas (LGGs), although slow evolving, ultimately progress into aggressive and largely incurable higher-grade gliomas. Molecular characterization of gliomas has revealed the uniform co-occurrence of isocitrate dehydrogenase (IDH) mutations in a majority of LGGs. Standard of care for IDH mutated LGGs include maximal safe surgical resection followed by adjuvant chemotherapy and radiotherapy. Salvage options for progressive disease are limited, and there is a need to develop effective therapeutic agents for glioma patients to extend overall survival and improve quality of life. To date, screening based drug discovery efforts in IDH mutated gliomas are lacking due to paucity of preclinical models. Over the last year, through collaborative efforts, we collected several patient-derived glioma tumorsphere lines and patient-derived xenograft models harbouring endogenous IDH1 mutations at the University of Heidelberg. In this joint project, we are planning to utilize a highly novel miniaturized Droplet Microarray platform developed at the Karlsruhe Institute of Technology for high throughput screening of patient-derived glioma tumorsphere lines with FDA-approved anti-cancer compound library. The overall goal of the project is to reveal potential therapeutic targets for treatment of IDH mutated LGGs. We plan to accomplish our objective by pursuing the following 2 specific aims.

In Aim 1, we will utilize our 3D IDH mutated tumorsphere models to screen a library of FDAapproved compounds on Droplet Microarray.

In Aim 2, we will prioritize candidates from Aim 1 and validate in additional tumorsphere models.

Research bridges

Synthetic Biology (SB)

Start date

01/01/2020

12/31/2020

Project management

Neuro-Onkologie

Universitätsklinikum Heidelberg

Sevin Turcan

Institut für Toxikologie und Genetik

Karlsruher Institut für Technologie

Anna Popova