ECRT Projekte

Single-domain antibodies, also called nanobodies, represent a novel class of antibodies, which due to their small size, can rapidly penetrate large tissue samples and therefore facilitate detailed 2- and 3-dimensional histological stainings. However, due to inefficient nanobody fluorescence-labelling tools available, stainings still require time-consuming protocols using fluorescently-labeled secondary IgG antibodies, limiting the advantages of nanobody application.

This project aims to develop an innovative labelling approach for nanobodies and other proteins using click-chemistry, non-canonical amino acids (ncAA) as well as codon reassignment. For this purpose, a TAG codon is introduced at the 3’ end of the nanobody DNA sequence by site-directed mutagenesis and the DNA plasmid is transformed into a bacteria strain that does not possess any intrinsic TAG codons. By transforming an additional plasmid encoding an orthogonal aminoacyl-tRNA synthetase/tRNA pair, the host’s translational machinery is able to incorporate ncAAs into the polypeptide chain of the nanobody. Using ncAA derivatives suitable for click-chemistry, site-specific labelling of the nanobody with azide derivatives of fluorescent dyes through click-chemistry technology is performed.

If successful, this project will not only improve the visualization of entire tissue samples by direct nanobody labelling but also be potentially applicable as a general protein-labeling strategy in biochemistry.

Team: Ina Büschlen, Nils Rouven Hansmeier, Samira Picht

Funding: Einstein Kickbox - Young Scientists

Inflammatory demyelinating polyneuropathies (IDPs) are a heterogeneous group of neuropathies that share a common hallmark of autoimmune-mediated demyelination, where the general pathophysiology is believed to be a combination of macrophage-mediated demyelination and T cell activation. This cell-mediated autoimmunity is suspected to be facilitated by autoantibodies of the humoral immune system found in the sera of IDP patients. Clinical presentation and underlying molecular pathology have diverse manifestations, leading to difficulties in differential diagnosis. Together with insufficient current standard of treatment and limitations of current animal models, there is a strong unmet medical need for a model that can better reproduce the conditions of the disease. Thus, using human induced pluripotent stem cells (iPSCs), we aim to establish a fully human co-culture of peripheral sensory neurons myelinated by Schwann cells to model and study IDP. Schwann cells and sensory neurons will be differentiated from human iPSCs and cultured separately. Upon maturation of iPSC-DSN, iPSC-SCs will be added to the iPSC-DSN and cultivated together for myelination. Immunofluorescence will be conducted for morphological characterization while voltage-sensitive dye imaging will be conducted for functional validation. Once assembled, the co-culture model will be incubated with patient sera in a serum-based cell binding assay to validate the presence and binding targets of autoantibodies.

Team: Lois Hew, Smilla Maierhof, Christian Schinke

Funding: Einstein Kickbox - Young Scientists

CRISPR-Cas9 gene editing offers novel ways of developing genetically enhanced cellular- and tissue-based therapies to enhance regenerative processes in chronic diseases. Non‑viral gene editing, commonly performed using double-stranded (ds)DNA templates, is effective in primary human T cells, but restricted by significant dose dependent dsDNA toxicity. Other cell types, such as natural killer cells, innate lymphocytes, monocytes and mesenchymal stem cells express intracellular DNA sensors at higher levels than conventional T cells. These sensitive cell types do not tolerate transfection with high concentration of dsDNA that is needed for effective reprogramming. Single-stranded (ss)DNA would be the optimal alternative for safer and less toxic gene transfer in sensitive cell types. In contrast to dsDNA, we expect a significantly reduced risk for undesired integration of our DNA template and limited toxicity, as there are fewer ssDNA-specific innate immune receptors. We aim to establish DNA-template modifications, which increase gene editing efficacy by overcoming poor delivery of the ssDNA into the cell nucleus. In a collaborative team of scientists investigating the immune, musculoskeletal, and cardiovascular systems, we are establishing this novel ssDNA based knock-in platform to integrate therapeutically relevant genetic cargo in precise locations of multiple DNA-sensitive cell types.

Team: Viktor Glaser, Weijie Du, Nina Stelzer, Clemens Franke, Timo Nazari-Shafti

Funding: Einstein Kickbox - Young Scientists

About 1 million dental implants are placed per year in Germany, while 22% of all implanted patients are affected by peri-implantitis. Peri-implantitis is an inflammatory disease in the surrounding tissues of dental implants characterized by progressive loss of supporting bone that can result in implant failure. The presence of a biofilm seems critical for the pathogenesis of peri-implantitis, thus, plaque removal is essential to halt the progression of the disease. During the inflammatory phase of peri-implantitis, granulation tissue is locally formed. Once the plaque is removed by implant surface debridement the inflammation is resolved, however, the presence of the granulation tissue persists and osseointegration is not recovered. Importantly, the peri-implantitis granulation tissue still expresses typical bone matrix molecules, although, in a reduced amount, which can indicate a native osteogenic potential, that might be modulated and used to reinstate osseointegration. To avoid peri-implantitis recurrence bone regeneration post-peri-implantitis is of great clinical interest. In the present project, we aim to test the osteogenic potential of peri-implantitis granulation tissue, for it could be used as the foundation for peri-implant bone regeneration. We envision that in the future topical stimulation in the probing area around implants directly on the granulation tissue and on the implant surface could be used as a chair-side protocol for re-osseointegration.

Team: Ana Prates Soares, Edoardo de Vasconcelos Emim, Heilwig Fischer

Funding: Einstein Kickbox - Young Scientists

Despite best medical care, motor recovery after stroke is often incomplete. Basic research in adult mice has shown that substantial degrees of spontaneous recovery after cortical stroke rely on the rewiring of spinal circuits, that attract new synaptic innervation from multiple supraspinal brain areas. Pharmacogenetic experiments in rats have also confirmed, that the plastic reorganization of corticospinal projections from the non-affected hemisphere onto spinal circuits is causally implicated in the improvement of motor function. In our ECRT Kickbox Project, we intend to further stimulate the rewiring of corticospinal neurons in mice by amplifying their post-stroke growth capacity through the use of viral tools recently developed in our laboratory. In order to vigorously boost the sprouting of bulbospinal fibers, we will use viral tracing techniques to selectively activate signalling pathways imperative to cellular growth, whose recent upregulation resulted in successful axon regeneration followed by significant motor function recovery in mouse spinal cord injury model. In parallel, we plan to document the effects of enhanced corticospinal sprouting on motor performance through extensive kinematic testing across distinctive movement domains relevant to upper limb dexterity and gait.

Team: Matej Skrobot, Rafael De Sa

Funding: Einstein Kickbox - Young Scientists

The spotlight attracted by mass use of mRNA for SARS-CoV2 vaccines has shed light on two practical limitations of mRNA drugs: their short-lived effect and demanding storage conditions.

mRNA vaccines need to be administered in a series of shots but have poor stability at ambient conditions, posing a high financial and logistic burden. Furthermore, therapeutic efficacy of mRNA is often limited by its short-lived effects. The aim of our project is to develop a hydrogel-based implantable drug depot for sustained delivery of mRNA drugs.

Such a construct would consist of an implantable, biocompatible hydrogel scaffold loaded with mRNA coding for a protein of interest. To neutralize its negative charge and facilitate cell entry, mRNA needs to be complexed using cationic lipids or polymers. Although a few studies have demonstrated release of mRNA-particles from scaffolds, little attention has been paid to the physicochemical interface between nucleic acid, transfection particle and scaffold polymers. We want to understand and optimize this interface with regard to mRNA degradation, transfection particle stability and release kinetics.

Team: Norman Drzeniek, Manfred Gossen, Hans-Dieter Volk

Funding: Einstein Kickbox - Advanced Scientists

Patients treated with cytotoxic chemotherapy frequently report a decline in cognitive abilities, such as short-term memory loss, reduced multitasking ability, or deficits in language. These cognitive side effects can greatly influence the quality of life in cancer survivors for extended periods of time. Although several mechanisms of action have been proposed, satisfactory treatment and prevention strategies remain to be identified. In this project we want to investigate whether induced pluripotent stem cell derived brain organoids can serve as a human model system for the study of chemotherapy induced central nervous system toxicity. The advantage of this approach in comparison to clinical or animal studies is that patient derived brain organoids can be used to directly and systematically compare the response between susceptible and non-susceptible patients. As a first step towards this goal, we want to develop a workflow for cell type composition analysis and for the measurement of toxic responses in brain organoids. We will then measure the effects to paclitaxel exposure. Paclitaxel is a highly neurotoxic chemotherapeutic agent and existing in vivo and in vitro data with this drug allow assessing the model’s suitability.

Team: Sophie Scholz, Karen Lewis, Lina Hellwig, Petra Loge, Petra Hühnchen

Funding: Einstein Kickbox - Young Scientists

Team: David Bode, Madeleine Schorr, Cristian Sotomayor-Flores

Funding: Einstein Kickbox - Young Scientists

Despite stunning clinical effects in some, most patients still do not respond to single-agent anti-cancer immunotherapy due to inaccessibility of tumors for immune cells or induction of acquired resistance. Therefore, developing suitable screening platforms to identify reasonable combination strategies is highly warranted.

We recently performed phenotypic analysis of blood samples from patients with colorectal cancer treated with cetuximab followed by a combined maintenance therapy containing the anti-PD-L1 mAb avelumab. We detected a fast drop in the number of peripheral NK cells in the majority of patients accompanied by a differentiated increase in NK cell activation. Hence, our preliminary findings suggest a role of NK cells for the clinical response to IgG1 mAbs. Though ADCC by IgG1 mAbs is well-established, current studies lack information about the importance of immune composition or migration, immune dysregulation or tissue morphology, which lowers their clinical significance.

Within the new project, we want to test our hypothesis of antibody-mediated NK cell tumor infiltration and its role in tissue regeneration. Therefore, we will establish a highly sophisticated ex vivo model using co-cultivation of precision tissue slices and autologous PBMCs within a Boyden chamber approach. Our results and the novel method might improve the understanding of the role of NK cells during anti-cancer therapy and serve as a screening platform to guide personalized medicine.

Team: Phillip Schiele, Stefan Kolling, Christien Beez

Funding: Einstein Kickbox - Young Scientists

Osteopetrosis is a genetic disorder leading to increased bone density due to impaired bone resorption by osteoclasts. The Kornak lab (Institute of Human Genetics – Universitätsmedizin Göttingen) analyzed an osteopetrosis patient who did not show any known osteopetrosis related gene mutations. Instead, they found a large homozygous in-frame deletion of the LAMA3 gene, resulting in a shortened but presumably functional LAMA3 protein. Preliminary data from Salaheddine Ali (Mundlos lab at MPI for Molecular Genetics) suggest that the LAMA3 gene carries an enhancer region affecting osteoclast development or function. To investigate the possible novel link of LAMA3 and osteopetrosis, we plan to mimic this deletion in monocytes using CRISPR/Cas gene editing. A Cas9 enzyme pair with two distinct sgRNAs will introduce double-strand breaks at two target sites, and the flanked 10kb DNA sequence will be excised. To improve the rate of large excisions in primary monocytes, we plan to use inhibitors of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), which decelerates non-homologous end joining. Furthermore, we plan to co-deliver microhomology-based ssDNA oligos to improve excision efficiency. We will differentiate the monocytes, carrying the patient specific LAMA3 deletion, into mature osteoclasts to assess their morphology and function.

In this proof of concept study, we aim to decipher the effects of a novel gene mutation on osteopetrosis and establish an efficient CRISPR-Cas platform for large genomic excisions in myeloid cells. If successful, our study provides the foundation to investigate the contribution of large gene regulatory elements during monocyte differentiation, a key progenitor cell type important in immune responses as well as bone regeneration.

Team: Nina Stelzer, Viktor Glaser, Tomislav Kostevc

Funding: Einstein Kickbox - Young Scientists

Team: Nurcan Hastar, Erik Brauer, Petra Knaus, Harald Stachelscheid

Funding: Einstein Kickbox - Advanced Scientists

Aging is associated with a functional decline of the immune system, which is linked to the inferior immune response of elderly people to infections and vaccinations. Naïve T cells (TN) harbor the greatest T cell receptor diversity and hence, are important for reactivity against new infections and vaccines. In humans however, the generation of new TN clones ceases as early as puberty. To maintain a high number of CD4+ TN, the cells must undergo activation-independent proliferation, known as homeostatic proliferation. CD4+ TN which lack the surface marker CD31 (CD31-), are enriched for cells which have undergone several rounds of homeostatic proliferation compared to CD31 expressing TN (CD31+). Indeed, the number of CD31+ CD4+ TN decreases with age as well as the telomere length in TN, demonstrating that cells from older individuals experienced a higher number of homeostatic proliferation events. In our preliminary data we could show that already in individuals <= 30 years, CD31+ and CD31- CD4+ TN show significant epigenetic differences on the level of DNA methylation.
With the support of the Einstein funding we will further characterize potential differences in transcriptional regulation in CD31+ and CD31- TN, which we hypothesize to increase with accumulation of homeostatic proliferation events.

Team: Dania Hamo, Andreas Magg, Mai Phan

Funding: Einstein Kickbox - Young Scientists

Periprosthetic joint infection (PJI) is a severe complication occurring in approximately 1-5% of all patients after total joint replacement. Despite successful revision surgery, risk of aseptic loosening is significantly elevated due to the impaired capability of bone regeneration. Myeloid derived suppressor cells (MDSCs) are a heterogeneous group of immunosuppressive cells that are activated during inflammation. In PJI, MDSCs were reported to represent a large proportion of all leukocytes. Recently, their ability to differentiate into osteoclasts has been observed. Thus, we speculate MDSCs to be a viable target to prevent osteolysis and promote bone regeneration in patients suffering from PJI.

Programmed cell death 1 (PD-1) receptor, an immune regulatory receptor currently targeted in various cancer treatments, has been suggested to be linked to osteoclast differentiation. Targeting PD-1 may be beneficial for bone regeneration and an effective and safe novel therapeutic approach in patients with PJI to improve implant survival.

In our project, we aim to investigate the role of PD-1 in septic activation of MDSCs in patients with PJI. Our results may improve the understanding of the pathological mechanisms in PJI and provide a novel therapeutic approach to improve implant survival.

Team: Yi Ren, Denise Jahn, Arne Kienzle, Weijie Du

Funding: Einstein Kickbox - Young Scientists

Endothelial cells (ECs) are exposed to fluid-sheer stress (FSS) originating from blood flow, which is translated and crosstalks with a variety of signal pathways, i.e. the BMP pathway. FSS-dependent modulation of the BMP signalling pathway is highly cell type, concentration and force dependent. The key quiescence factors BMP9/10 show strong potency in the vasculature, due to the high abundance of their high-affinity receptor ALK1 on ECs. Mutations in ALK1 or in its co-receptor Endoglin alter the vascular BMP signalling response and are associated with vascular pathologies e.g. Hereditary Haemorrhagic Telangiectasia (HHT).

The visualization of BMP receptors bears difficulties due to the lack of specific antibodies. We therefore developed a Halo- and SNAP-labelled BMP receptor library, which in combination with fluorescently labelled BMP9-SiR-d12 ligand allowed for visualisation of ligand-bound receptor complexes.

Using a microfluidic flow set-up, we now aim to visualize BMP9/10 binding to ECs under fluid flow. In a later stage of the project this knowledge will be transferred to in vivo experiments. Ventricular injections of labelled BMP9/10‑SiR-d12 into developing zebrafish embryos carrying an endothelial specific reporter will allow for visualization of vascular bed-specific BMP9/10-SiR-d12 binding towards ECs.

We further want to assess dependence of ligands, co-receptors or FSS on BMP9/10 signalling in rare-signalling receptor complexes and therefore intend to develop a cell-impermeable dual-dye FRET sensor.

Team: Wiktor Burdzinski, Olya Oppenheim, Jerome Jatzlau

Funding: Einstein Kickbox - Young Scientists

Regeneration of the intestinal epithelium and its modulation by signalling molecules of other cell types plays a critical role in many diseases. An often neglected and to date scarcely investigated cell type in this context are adipocytes. Besides storing lipids, adipose tissue produces various humoral factors, so called adipokines, that are known to critically affect immune function. We want to investigate the effects of adipokines on epithelial regeneration using primary epithelial organoids. With this in vitro system, stem cells of the intestinal epithelium are grown into 3-dimensional self-organizing mini-organs that model not only basic tissue architecture but also regeneration after injury. Modifications of the culture medium and basement membrane matrix further allow for controlling cell identity and signalling activity. Following induction of a regenerative phenotype through specific culture conditions, the cells are treated with different adipokines to analyse their effects on proliferation, functionality, gene expression, protein localization, and barrier integrity. Besides the basic 3D culture model, we are using additional sophisticated in vitro systems, such as transwell filter systems or custom-made flow chambers, to investigate functional aspects and behaviour under flow. Analysis methods include real-time quantitative PCR, immunofluorescence microscopy, metabolic analysis via Seahorse, and transepithelial electrical resistance measurements.

Team: Lorenz Gerbeth, Max Brunkhorst, Jörn Felix Ziegler, Marcus Lindner

Funding: Einstein Kickbox - Young Scientists

Liver transplantation still presents the only curative treatment for end-stage liver disease, but viable donor organs are very limited. Normothermic ex vivo liver perfusion (NEVLP) has been developed as an alternative to static cold storage. NEVLP aims at maintaining the liver metabolism by perfusing the graft with oxygenated medium at 37°C and reducing ischemia time. Gene modification during NEVLP may be a possibility to tackle organ failure through immunogenic injury but may also allow for future therapies. Aim of the proposed project would be to develop an effective method to influence the gene expression of liver grafts via transfection with coding RNA sequences during small animal NEVLP. A plasmid coding for luciferase mRNA as well as a shRNA sequence against the CIITA gene expression is used for the downregulation of the expression of major histocompatibility antigens in rats, allowing for detection by recipient immune system after transplantation, thus reducing the need for immunosuppressive therapy after transplantation.

Team: Joseph Gaßner, Nathanael Raschzok, Manfred Gossen, Igor Sauer, Julian Michelotto

Funding: Einstein Kickbox - Advanced Scientists

Mesenchymal stem cell (MSC) therapy was proposed as a treatment for alloreactive conditions such as transplant rejection or GvHD due to their immunomodulatory effects observed in controlled 2D culture conditions. However, in a clinical setting MSCs are delivered into a harsh and poorly understood in vivo environment. Thus, when evaluating the therapeutic potential of a cell product, the predictive value of effects observed in standard 2D culture is low:

As the microenvironment (be it a tissue or an engineered delivery carrier) is known to heavily influence MSC biology, we propose that isolated 2D culture should no longer be the state-of-the-art testing platform to predict therapeutic potential in vivo. Instead we aim to evaluate MSC-mediated immunomodulatory effects in an integrated 3D co-culture model, where a tissue organoid is infiltrated by alloreactive immune cells and treated with MSCs at the same time.

For our first model, we will look at patient-derived whole bone marrow aspirate. Novel engineering challenges lie in designing a biomaterial that allows for immune cell infiltration, as well as in developing a biofabrication technique that will bring together all 3 components in a meaningful 3D architecture. Readouts will include immune profiling of different in-house MSC products, flow cytometry and cytokine analysis of immune cells and histological analysis of co-culture constructs.

Team: Norman Drzeniek, Stefania Martini

Funding: Einstein Kickbox - Young Scientists

As a standard of care, titanium miniplates are used for osteosynthesis in midfacial and mandible fractures. Titanium implants can be either permanently left in situ or removed once the bone healing is achieved. In the first case, negative long-term side effects such as particle debris leading to a foreign-body-reaction, possible stress-shielding effects, interference with skeletal growth as well as diagnostic imaging artifacts may occur. On the other hand, plate removal exposes the patient to all risks linked to a second surgery.

Therefore, biodegradable implants gained increasing interest to overcome the limitations of traditional biomaterials for fracture fixation. As a promising product biodegradable magnesium implants are characterized by Young’s modulus similar to cortical bone, which we hypothesize can also guarantee enough stability at the fracture site. A known complication associated with magnesium degradation is the release of hydrogen ions, which negatively impacts the bone healing process. A possible solution is given by slow-degrading magnesium alloy WE43 and new coating techniques, as Plasma Electrolytic Oxidation (PEO).

In this study, we aim to test the novel WE43-based magnesium implants with PEO-surface modifications in a sheep mandible fracture model ex vivo in order to compare their mechanical performance to conventional miniplates.

We hypothesize that magnesium miniplates are suitable for load sharing indications if the mechanical properties are respected.

Team: Claudius Steffen, Heilwig Fischer, Vincenzo Orassi, Dag Wulsten, Oskar Schmidt-Bleek

Funding: Einstein Kickbox - Young Scientists

Successful wound repair and tissue regeneration rely on controlled re-vascularization of the trauma region by angiogenic sprouting. Maturation of newly formed blood vessels notably depends on BMP9/10/Alk1 signaling, for which deficiency can result in the rare disease Hereditary Hemorrhagic Telangiectasias. BMP9/10 signal via Alk1 in endothelial cells (ECs) and prompts SMAD1/5/9 signaling, thereby promoting vascular quiescence and curbing post-injury angiogenesis which, if left unchecked, can impair scarring.

However, recent work reported on pro-angiogenic effects of BMP9, highlighting BMP9 functions as context-dependent and cell-type specific; but the mechanisms modulating the bipartite role of BMP9/alk1 signaling as pro- or anti-angiogenic are still unclear.

To study the processes regulating BMP9 dual roles, we will use fibrin-embedded endothelial spheroids as 3D models of angiogenesis, using diverse endothelial cell types and ligand concentrations. Super resolution confocal imaging and immunofluorescence will help assess sprouts morphology. Moreover, we will use 3D traction force microscopy to evaluate the crosstalk between endothelial BMP9/Alk1 signaling and mechanobiology, and 3D biomaterials to investigate the influence of BMP9/Alk1 signaling on stromal cell-induced ECM remodeling during angiogenesis.

A better mechanistic understanding of BMP9/Alk1-signaling roles during angiogenesis and vascular homeostasis could help develop therapeutics to better control the angiogenic response after injury, and thus improve scarring and tissue repair.

Team: Mounir Benamar, Erik Brauer

Funding: Einstein Kickbox - Young Scientists

Heart failure (HF) is responsible for substantial morbidity and mortality and is increasing in prevalence. Up to one half of HF patients have preserved ejection fraction (HFpEF). Although there has been remarkable progress in the treatment of HF with reduced ejection fraction (HFrEF), translation of those therapies to HFpEF has been disappointing, indicating the need to further understand the emergence of HFpEF. HFpEF and HFrEF differ in their underlying pathogenesis. In contrast to HFrEF, which is driven by an initial cardiac/cardiomyocyte damage, a low-grade systemic inflammation induced by comorbidities underlies HFpEF-specific cardiac remodeling and dysfunction.

Serum S100A8/A9 alarmin levels are increased in HFpEF patients as well as in patients with systemic sclerosis, which are characterized by skin fibrosis and cardiac diastolic dysfunction. These findings support the rationale that a systemic inflammatory response can provoke fibroblast activation in the heart and in the skin. They further raise the hypothesis whether skin fibroblasts from HFpEF patients could be used as readouts for the activated state of fibroblasts in the heart and could hereby potentially serve as a prognostic tool and a tool to test anti-fibrotic therapies. Here, we investigate whether skin fibroblasts from HFpEF patients can be used to mirror cardiac fibroblast activation (upon a pro-inflammatory stimulus), when they are grown under stiffness conditions mimicking the cardiac environment.

Team: Isabell Matz, Erik Brauer

Funding: Einstein Kickbox - Young Scientists

T lymphocytes are immune cells that defend the body from pathogens and cancer. They can be genetically modified to express designed receptors such as Chimeric Antigen Receptors (CARs), enabling them to recognize antigens of choice. Such CAR T cells have been employed in the treatment of several oncological disorders, the best clinical example being CD19-specific CAR T cells used to treat patients suffering from B cell leukaemia and lymphomas.

We recently improved a method that allows for efficient manufacturing of T cell receptor (TCR) replaced CAR T cells.  We employ CRISPR-Cas9 to introduce a CAR transgene into the TCR alpha chain (TRAC) gene locus. To this end, sgRNA/Cas9 complexes and DNA molecules carrying a CAR-transgene are co-transfected into T cells. By exploitation of Homology-directed DNA Repair, the CAR-transgene can be introduced under the control of the TRAC promoter, which furthermore prevents formation of functional TCRs.

This method mainly produces TCR-replaced CAR T cells carrying the CAR as their only specificity-determining receptor, and TCR^-/CAR^- double negative T cells, incapable of any antigen-specific action. While these cells would be fit for third-party use, a small fraction of unedited or CAR⁺/TCR⁺ double positive T cells remains, which, if transfused in an allogeneic setting, might cause Graft-vs-Host disease. This project aims to eradicate all remaining TCR⁺ contaminants in order to generate off-the-shelf CAR T cell products.

Team: Jonas Kath, Stefania Martini, Magdi Elsallab, Weijie Du

Funding: Einstein Kickbox - Young Scientists

Ketone bodies and their derivates in Type 1 Diabetes (T1D): a secondary clinical manifestation or a perpetrator of the disease?

Type 1 Diabetes (T1D) is an autoimmune disease characterized by the destruction of b-pancreatic cells by infiltrating CD8⁺ T cells into the pancreas. Under homeostatic conditions, b-pancreatic cells produce the necessary amount of insulin to promote GLUT(1-4) trafficking to the plasma membrane enabling the cells to uptake glucose and use it as a source of energy. However, during the onset of T1D, the autoimmune response that takes place in the pancreas results in decreased insulin production. Suboptimal insulin levels are not enough to keep the glucose uptake required to fulfill the metabolic demands of the organism. As a result, the liver starts oxidizing fatty acids (FAO) in order to provide alternative metabolites for the cells. The metabolic intermediates released to the bloodstream by the increased rates of FAO augment the concentration of ketone bodies (KBs) and their derivates compared to homeostatic conditions. In this study, we want to address whether the high concentrations of KBs and derivates are harmless or if it is enhancing the T1D autoimmune process. For that purpose, we will examine patient cohorts with different KBs plasma levels. We will ex vivo assess different compartments of the immune response that have been shown to be regulated by KBs in in vitro experiments.

Team: Adrian Madrigal, Valerie Plajer, Mikie Phan

Funding: Einstein Kickbox - Young Scientists

Most malignant hematological diseases are treated with hematopoietic stem cell transplantations (HSCT). On the other hand, the allogeneic transplantation is based on transplants from non-related donors whereby the chance of rejection is high when the matching of antigens is not absolute. Because of decreased development of severe side effects syngeneic stem cell transplantation is better tolerated but on the other hand less effective. The reason is the anti-tumor immune effect of allogeneic HSCT. After HSCT patients suffer from delayed immune reconstitution as well as a higher fracture rate. On the one hand, the syngeneic transplantation uses transplants from either twins or previously harvested and stimulated cells from the patient itself. This transplantation has less risk of rejections or reactions of the immune system.

The molecular basis of changes in properties and delay of reconstruction of the bone structure are sparsely examined in patients as well as experimental setups.

With the planned model we will simulate the process of stem cell transplantation in a 3D in vitro model by using human cells and investigate its effects on the bone marrow and immune cells of the model. Our long-term perspective is to develop possibilities to reduce severe side effects on the bone in HSCT patients. With a successful establishment of the 3D model, we will be able to study changes in the course of HSCTs in vitro and predict possible effects when applied to patients.

Team: Radost Anika Saß, Melanie-Jasmin Ort, Lisa-Marie Burkhardt

Funding: Einstein Kickbox - Young Scientists

Osteoarthritis (OA) is the leading cause of joint pain and age-related disability worldwide, due to the increase of life expectancy and an active, aging population. Unlike rheumatoid arthritis, therapies used in OA are still limited to pain control. Therefore, novel targets for the development of disease-modifying drugs are required.

In recent years, fibroblast-like synoviocytes (FLS), which maintain the structural and dynamic integrity of joints physiologically, were identified as key drivers of cartilage degradation. FLS can be divided into two major populations. The destructive phenotype which is restricted to the synovial lining layer stimulates osteoclastogenesis, thereby promoting bone erosion. On the other hand, FLS of the sublining layer are classified as invasive effector cells driving synovitis. Recent research has revealed that glucose metabolism shapes the FLS phenotype, promoting disease progression. Among them, the proliferative and metabolically active FLS are marked by an increased glycolysis compared to the non-invasive, quiescent FLS. Inhibition of glycolytic enzymes already attenuated the severity of cartilage damage in vivo. Our data suggest that pyruvate dehydrogenase kinase (PDK) 3 may play a crucial role in the pathogenesis.

Therefore, we hypothesize that shifting the metabolism of FLS from glycolysis to mitochondrial respiration via PDK3 that regulates the mitochondrial flux might be a novel target for the development of disease-modifying drugs.

Team: Alexandra Damerau, Nayar Alejandro Durán Hernández, Tazio Maleitzke

Funding: Einstein Kickbox - Young Scientists

If the bone fails to restore a bony continuity over more than six months after a fracture or bony reconstruction, we speak of a pseudarthrosis. For the reconstruction of critical size defects in long bones and the mandible, autologous osteomyocutanous transplants are used. Bony healing between transplant and original bone often fails. In mandible reconstructions, metal removal and following dental rehabilitation is delayed or impeded if a non-union situation occurs. There are many possible reasons for non-unions, including insufficient callus formation and a lack of vascularization.

For non-unions, the revision and the treatment with autologous cancellous bone are considered the gold standard. In larger defects, an autologous bone block is needed to fill the defect, involving the possible creation of a donor site morbidity.

Magnesium holds promising attributes for the use as a resorbable implant material. We hypothesize that, by supporting angiogenesis and its osteostimulative properties, magnesium implants are suitable for treating non-union situations.

In this project, we want to create a mouse model for treating non-union situations to evaluate magnesium implants' influence on existing pseudarthroses. This study aims to find a new treatment option for situations where increased bone formation is needed, for example, in patients suffering from non-unions after fractures and for oncological patients who have lost parts of their bone due to cancer treatment.

Team: Heilwig Fischer, Katharina Schmidt-Bleek, Carsten Rendenbach, Agnes Ellinghaus, Sabine Stumpp

Funding: Einstein Kickbox - Advanced Scientists

Gene editing, the targeted modification of DNA, allows the controlled repair of mutations to fix genetic diseases. The most important safety concern for the translation of gene-edited cell products is the risk of off-target events. Off-target editing could introduce mutations or translocations with oncogenic potential. Standard assays for the unbiased identification of potential offtargets are currently performed in surrogate cell lines or using in vitro digested genomic DNA. Here, we propose a new simplified approach that allows the cost-efficient off-target identification directly from the DNA of the gene-edited cells. To this end, we plan to adapt existing workflows of targeted enrichment sequencing for the quick and sensitive identification of structural variants and off-target integration of DNA donor templates during gene editing procedures.

Team: Dimitrios Wagner, Björn Fischer-Zirnsak, Dominik Seelow

Funding: Einstein Kickbox - Advanced Scientists

The heterogeneity of lung cancer is a major cause of its high mortality and personalized treatment strategies are urgently needed to overcome this fatal disease. Patient-derived lung cancer organoids (PDLCOs) are capable of modeling individual tumor biology and could serve as platforms to investigate the effect of therapies. However, so far, the low efficacy and reproducibility in generating PDLCOs hinders their use in preclinical research and diagnostics. Additionally, the current tumor organoids lack immune components, which are critical for the evaluation of anticancer therapies. In our project, we strive to establish a robust protocol for the generation of PDLCOs from surgically resected tissue to create a biobank for the in vitro evaluation of personalized antitumor therapies and immunotherapies in particular. We will evaluate different culture protocols to unravel niche factor dependencies of tumor stem cells, which are suggested to be essential for proliferation. Stringent testing of PDLCOs and the primary tissue they derived from will be performed to ensure their in vivo comparability. Furthermore, the capacities of including tumor microenvironment components into the respective cultures and their immunogenicity will be evaluated. Ultimately, the generated organoids could be installed as prediction models of therapeutic efficacy and could be valuable tools in personalized medicine.

Team: Lukas Ehlen, Regina Stark, Birgit Sawitzki, Janine Arndt

Funding: Einstein Kickbox - Advanced Scientists

In biomedical sciences, the interaction between organism and biomaterials is defined at the interface. When body and material enter in contact, they react to each other. As a result, rejection, absorption, oxidation, bonding, integration, failure or success can occur. The amount of contact between materials can be a good parameter for evaluating how stable their attachment is. Our aim is to create an image-computational tool that can calculate and display the contact surfaces between materials and tissues. In the present project, non-destructive imaging data from classical X-rays, Cone Beam Computed Tomography (CBCT), Micro-CT and Phase contrast-enhanced micro-CT (PCE-CT) will be used. We will develop an image-computational tool to assess the contact at the interface between materials from grayscale images to obtain a statistical quantification of interfacial contact. The output of the proposed approach will reveal detailed information about the superficial area of contact between the biomaterial and hard tissue. In the future, the use of our tool may help getting better insights about the correlation between interfacial contact and biomechanical properties. Thus supporting the development of new materials and design optimization.

Team: Ana Prates Soares, Andreia Sousa da Silveira, Heilwig Hinzmann

Funding: Einstein Kickbox - Young Scientists

The outset of all regenerative processes is defined by an inflammatory reaction. Inflammatory cells remove necrotic tissue, release inflammatory and proangiogenic factors and exert chemotactic effects. A tight regulation of the immune reaction is a prerequisite for an undisturbed healing. A prolonged pro-inflammatory reaction and an imbalance towards pro-inflammatory T cells impaired bone healing, while the transfer of anti-inflammatory T cells supported healing. Consequently, a bivalent influence of pro-/ anti- inflammatory T cells on fracture healing is suggested. Besides the known chemotactic and angiogenic functions, recent work describes a novel role of T cells in regulating extracellular matrix formation during bone healing. It was suggested that T cells help to organize the collagenous matrix and slow down the process of cartilage mineralization. Here we hypothesize that negative or positive effects of pro- or anti- inflammatory T cells, respectively, are at least partially related to modulations of ECM deposition. The link between T cells and ECM formation is however still poorly understood.

Hence, in this study we are planning to further dissect the influence of pro-/anti-inflammatory cytokines on extracellular matrix formation in a well-characterized in vitro 3D tissue formation model.  We aim to identify target cytokines which hinder or promote ECM formation. The expected insights are of high relevance for a better understanding of the crosstalk between the immune system and early ECM formation processes in bone healing and might contribute to develop future therapeutic concepts.

Team: Sophie Görlitz, Lisa-Marie Burkhardt, Aaron Herrera, Julia Berkmann, Christian Bucher

Funding: Einstein Kickbox - Young Scientists

T cells are a major part of our adaptive immunity, being able to eliminate infected or transformed target cells and are increasingly exploited as powerful tools in oncology. They can be redirected to recognize and eliminate cancer cells using Chimeric Antigen Receptors (CARs), fusion proteins of antibodies for antigen recognition and T cell-specific signaling domains. While already effective in patients suffering from certain blood cancers, the generation of an individual autologous CAR-T-Cell product for a single patient is a difficult and costly endeavor. Treatment delay due to production processes and high costs hinder their broad application in the clinic. This kickbox application aims to test a novel approach to create CAR-T-Cell product from healthy donors that are unable to attack the patients cells via their endogenous T cell receptors in an allogenic treatment setting. Therefore, this "next generation" of CAR-T cells may enable the generation of potent but cost-effective treatments of cancers. 

Team: Dimitrios L. Wagner, Jonas Kath, Leila Amini

Funding: Einstein Kickbox - Young Scientists

The regenerative potential of the heart is not sufficient to repair damage caused by myocardial infarction. Thus, there is an urgent need for approaches that reverse cardiac remodeling, e.g. by providing a pro-regenerative environment or generating de novo heart muscle cells. Research on multipotent cardiac progenitor cells (CPCs) has gained traction as they can differentiate into cardiomyocytes, endothelial cells, and smooth muscle cells in vivo, potentially overcoming the limitations of current cell therapy strategies. In humans, various populations of CPCs, including Sca-1⁺, have been shown to improve cardiac function in preclinical models of myocardial infarction, contributing to the regeneration of the infarcted area. However, limitations associated with these cells, particularly poor accessibility and ethical concerns, hamper clinical translatability. Human induced pluripotent stem cells (hiPSCs) represent a readily available source of autologous fetal-like CPCs. We found that Sca-1⁺ cells isolated from hiPSCs during cardiomyocyte differentiation exhibited a stable phenotype through in vitro cultivation and were similar to human fetal CPCs in terms of morphology and gene expression of cardiac markers such as GATA4 and Nkx2.5. Our aim is to further characterize Sca-1⁺ cells to gain a better understanding of different Sca-1⁺ subpopulations and to identify GMP-compliant isolation and expansion strategies using single-cell RNA sequencing and cell surface marker screening.

Team: Ana Garcia Duran, Timo Nazari-Shafti, Sebastian Neuber, Andranik Ivanov

Funding: Einstein Kickbox - Young Scientists

Obesity is a worldwide epidemic that has developed into a major global health problem affecting all ages and socioeconomic groups. This condition is often associated with an increased risk of stroke, diabetes mellitus type 2 as well as a reduced healing capacity. Interestingly, a weight reduction of only 5-10% can circumvent severe health conditions such as diabetes. It can be achieved through changes in diet, exercise or bariatric surgeries. However, adjustments in lifestyle often fail and surgeries are associated with increased risks for obese patients. Additionally, to date only a small number of drugs have been approved for clinical use. Thus, there is a demand for alternative strategies such as the development of efficient and safe anti-obesity drugs.

In this regard, a group of small peptides derived from the Calca gene may be of interest as targets for anti-obesity therapies – Procalcitonin (PCT) and alpha Calcitonin gene related peptide (αCGRP). Recently, we showed that mice lacking PCT and αCGRP kept on a high fat diet have a significantly improved health status compared to wild-type controls. Furthermore, both peptides are suggested to bind to the calcitonin receptor like receptor (CRLR), a G-protein coupled receptor and therefore an excellent drug target.

Hence, in our study we are planning to analyse the eligibility of CRLR antagonists as possible strategy for the fight against obesity, associated comorbidities and the related drop in regeneration capacity.

Team: Jessika Appelt, George Soultoukis, Denise Jahn, Paul Köhli

Funding: Einstein Kickbox - Young Scientists

Osteoarthritis (OA) is a severe disease with a very high prevalence world-wide. Only recently, the involvement of an inflammatory component in OA was recognized. Inflammatory cell infiltrates are frequently found in the synovium and the adjacent infrapatellar fat pad of the affected joints. In addition, also joint traumata are associated with a phase of synovial inflammation and it is generally accepted that patients that have experienced such a trauma have an increased likelihood to develop OA in the respective joint later in their life. Intraarticular soft tissue inflammation can be detected very early in the course of OA and is correlated with more rapid cartilage destruction. Besides macrophages and neutrophils, infiltrates of T cells are found in OA-associated joint inflammation. Mouse models have shown a substantial contribution of CD4+ and CD8+ T cells to the development of posttraumatic OA. Hence, using flow cytometry, we will study which T cell subtypes are present in the inflamed intraarticular soft tissues early after traumatic events such as meniscal tears compared with those in late-stage OA patients undergoing knee-replacement surgery. Thus, we can potentially identify specific pathogenic T cell subpopulations that are present both in acute and chronic joint inflammation and that could present a potential therapeutic target for the early treatment of posttraumatic OA.

Team: Caroline Peine, Nayar Durán, Tazio Maleitzke, Philipp Burt

Funding: Einstein Kickbox - Young Scientists

The only commercially available drug for the treatment of non-union of long bone fracture, local recombinant BMP-7, was taken off the market due to serious side effects, leaving orthopedic surgeons without any further pharmacological solutions for a challenging pathology.

Since then, IGF-1 has been investigated among other cytokines as a promising alternative for bone healing. However, the key limitation of cytokine therapy remains the short in vivo half life of just a few minutes and so far no alternative to BMP-7 made it to clinical application.

To address this issue we want to design a novel type of "in vivo drug factory" that would prolong the release of IGF-1 in vivo but could also be easily assembled during surgery and injected into the fracture gap. Our strategy involves the combination of mRNA-transfected hMSC as a transplantable source of IGF-1 with a biomaterial vehicle that would interact with cell-secreted cytokines and modulate their release kinetics.An additional engineering challenge lies in combining cell transfection with cell encapsulation and material injection into a simple procedure that respects the clinical setting of use.

Team: Norman Drzeniek, Paul Köhli

Funding: Einstein Kickbox - Young Scientists

Commensal bacteria play a hugely important role in maintaining gut health by producing essential vitamins and breaking down non-digestive carbohydrates. Previous studies have shown that the microbial communities and their metabolites provide crucial signals upon inflammatory insults that alter the immune response of the host. Of note, the microbiome itself and its metabolites can be altered in human diseases such as inflammatory bowel disease (IBD). It is thought that these metabolic changes can impact the phenotype of immune cells within the gut. Intestinal macrophages are highly sensitive immune cells reacting to environmental cues. They adjust their pro-inflammatory and anti-inflammatory properties to modulate immune responses and maintain mucosal homeostasis. Moreover, numerous studies have shown the anti-microbial impact of bacterial metabolites on macrophages and their pro-inflammatory properties in IBD patients. To date, it is unknown which bacterial species are able to drive this impact and whether this effect can be recapitulated in vivo. In our project, we aim to identify a common molecular signature that drives an anti-microbial phenotype in macrophages by combining functional bacteria killing assays with high throughput techniques for gene expression and cytokine secretion. This will allow us to rapidly screen bacteria, which have regenerative therapeutic potential based on their ability to ameliorate the pro-inflammatory macrophage phenotype found in inflammatory diseases.

Team: Elena von Coburg, Caitlin Jukes, Philipp Burt, Liviana Ricci

Funding: Einstein Kickbox - Young Scientists

The interplay of cells with their surrounding matrix is essential in homeostasis, regeneration and diseases. Apart from cell morphology being affected by mechanical cues from their microenvironment, it was found that early native extracellular matrix (ECM) development affects cell function. The interactions of structural ECM components were shown to be mechanoregulated as cells mediate the remodeling of fibronectin (Fn) matrix into a predominantly collagen matrix.

Fn is one of the most abundant ECM proteins and exhibits different conformational states depending on local strains in early ECM development which directly affects ECM formation. For example, in vitro experiments in 2D systems revealed that the tensional state of Fn regulates the nucleation of other ECM proteins such as collagen I. Here, we want to use a physiological 3D microenvironment with viscoelastic material properties to investigate early ECM formation in vitro.

ECM is a dynamic network undergoing constant modifications during regeneration. Understanding the mechanoregulation of early ECM formation by elucidating the spatiotemporal tensional states of the Fn matrix could answer why Fn is crucial for the assembly of other ECM proteins and how it is affected by tissue mechanical properties. We propose a novel strategy combining 3D alginate hydrogels with Fn visualization techniques which will be beneficial to regenerative therapies and tissue engineering and could be extrapolated to model degenerative diseases.

Team: Matthias Kollert, Georgios Kotsaris, Julia Mehl

Funding: Einstein Kickbox - Young Scientists

Suppressing unfavorable immunity towards the allograft and the complications that this immune suppression entails are main challenges in organ transplantation. Several immunosuppressive agents and protocols are currently in use and applied according to a rough risk assessment using the HLA-mismatch, the level of antibodies against alloantigens, and individual experience of the treating clinician. Thus, the initial treatment can be adjusted with the onset of complications due to too strong or too weak immunosuppression. However, complications harming the allograft are currently only detected when the graft function is already impaired, often leading to irreversible graft damage. This deteriorates the long-term graft survival. Therefore, there is an urgent clinical need for guidance of immunosuppressive therapy. The research project that we will execute with support of the Einstein Kickbox grand is based on a method established and patented in our group that uses kidney transplant cells collected and cultivated from urine of kidney transplant patients. We envision redefining our protocol in order to make it more simple and applicable in a clinical context. The Einstein Kickbox grant with the BioThinking support will enable us to generate ideas and hypothesis that facilitate the assay optimization under consideration of the clinical and diagnostic demands. It also will help to find support from funding institutions and potential collaboration partners in the industry.

Team: Constantin Thieme, Nina Babel, Toralf Roch

Funding: Einstein Kickbox - Advanced Scientists

Osteoarthritis (OA) is a chronic joint disease featured by cartilage deterioration and chronic pain. Regardless of the complexity of the initial causes, chondrocytes residing in cartilage are exposed to endogenous TLR agonists that are generated during cartilage breakdown. For instance, the 32-mer and 29-kDa fibronectin fragment, enzymatic products of matrix proteins, have been detected in the synovial fluid of OA patients and revealed an anti-anabolic, pro-catabolic and pro-inflammatory function by activating TLR2-mediated signalling pathways. Mesenchymal stem cells (MSC) hold great promise as treatment for the inflammation-accelerated degenerative disease OA, as they simultaneously carry regeneration potency and immunomodulatory capacity. On the other hand, MSCs are sensitive and respond to the inflammatory and degenerative cues, such as TLR agonists in the diseased microenvironment, to secrete inflammatory cytokine IL-6, IL-8 and G-CSF. We plan to overcome this limitation by endowing MSCs with a resistance to the local TLR2 agonists by silencing TLR2 expression. We will use menstrual blood–derived mesenchymal stem cells (MenSC) which hold, among other benefits, the advantage of non-invasive and periodical acquisition. Thus, we will transplant TLR2ko MenSCs to the knee joints of the OA-diseased guinea pigs and evaluate whether the knockout of TLR2 will endow MenSCs with an advanced therapeutic effects comparing to wild type MenSCs.

Team: Ping Shen, Max Löhning, Hans-Dieter Volk, Tobias Winkler, Tazio Maleitzke, Lisa Grunwald

Funding: Einstein Kickbox - Advanced Scientists

Tendon pathologies are very common and represent a significant burden for the patient. The pathomechanisms of chronic tendon pathologies (Tendinopathy) as well as failed tendon regeneration after acute rupture are not fully understood. Therefore, to date there exist no treatment option to support tendon healing, or to prevent the development of tendinopathy.

Excessive mechanical forces can cause acute ruptures and recurrent mechanical overload induces micro ruptures that lead to chronic conditions. Furthermore, inflammation is crucial for tendon healing and the development of tendon pathologies. In autoimmune-driven Achilles tendon enthesitis, IL23 Receptor (IL23R) expressing γδ T-cells that secrete IL17 have a driving pathogenic role. Previously, we showed that IL17/IL23R signaling is also present in non-autoimmune tendon pathologies. We hypothesize that mechanical strain together with inflammatory signals can modify the tenocyte-immune cell communication.

We will use an ex-vivo model of mechanical force-induced tenocyte-immune cell communication and gather information on factors influencing the IL17/IL23R signaling. In addition to varying mechanical strain or time, we will test different inflammatory triggers and cellular compositions on the secretion of IL6/IL23/IL17 associated cytokines and MMP classes. The obtained results will be used to develop a mechano-biochemical mathematical model, which aims on investigating the spatio-temporal regulation of IL6/IL23/IL17 signaling under mechanical and inflammatory stimulation.

Team: Franka Klatte-Schulz, Birgit Sawitzki, Sara Checa Esteban, Georg Duda, Christiane Gäbel, Serafeim Tsitsilonis

Funding: Einstein Kickbox - Advanced Scientists

Impaired bone healing occurs in up to 10 percent of fractures and leads to pain, a long-term reduction in the quality of life and high socio-economic costs. More than 50 years ago, clinicians observed the phenomenon of traumatic brain injury (TBI) accelerating fracture healing. Until now, the underlying pathophysiology remained unknown, but we previously found compelling evidence that the stimulatory effect of TBI on fracture healing is transmitted through an increased adrenergic signaling, caused by an interrupted circadian clock. Several studies have shown a circadian rhythm in bone metabolism, affecting osteoclast activity, osteocytes and osteoblast function. Furthermore, the circadian clock regulates the body physiology through the sympathetic nervous system, which has a strong impact on bone tissue. Although there are a number of studies investigating the influence of the circadian clock on bone metabolism, the impact on fracture healing is largely unknown. In our project, we would like to investigate the role of the circadian clock in bone healing. Therefore, we will use a mouse line with a disrupted circadian clock in our standard osteotomy model. In addition, we will monitor the circadian rhythm of these mice as well as the released catecholamine. The understanding of the role of the circadian clock in fracture healing holds great potential to aid in the development of new drugs to improve fracture healing.

Team: Denise Jahn, Serafeim Tsitsilonis, Achim Kramer, Johannes Keller

Funding: Einstein Kickbox - Advanced Scientists

Team: Kieu Nhi Tran Vo, Su-Jun Oh, Valeria Fernandez Vallone, Ute Scholl, Harald Stachelscheid, Andreas Kurtz

Funding: Einstein Kickbox - Young Scientists (BSRT funded)

Preclinical testing of cardiac regenerative therapies such as direct reprogramming of fibroblasts into cardiomyocytes (CM) usually starts in vitro, with factor screens in 2D-cultured healthy cells, and proceeds in vivo, with candidate evaluation in animal models of heart disease. 2D models insufficiently emulate the real situation, whereas 3D models more accurately mimic cardiac multicellularity and extracellular matrix (cECM). However, an in vitro 3D testing platform that bridges the gap between 2D models and the in vivo scenario by adequately modeling cardiac pathologies is not yet available.

Therefore, the main goal of the proposed project is to engineer for the first time μ-hearts that mimic acute myocardial infarction (AMI) and chronic heart failure (CHF) and to demonstrate their utility for therapy testing. To do so, uniformly-sized spheroid μ-hearts will be assembled from human iPSC-derived cardiac cells and cECM at disease-specific ratios and culture conditions. After viability confirmation and immunohistochemical characterization, in situ genetic CM reprogramming will be tested. Finally, contractile motion analysis and imaging mass cytometry (IMC) will be evaluated for outcome analysis.

In the future, using iPSCs from patients with genetic cardiac defects will render this innovative testing platform suitable for personalized medicine. Eventually this may help to accelerate therapy development and maximize its effect while reducing the number of animal experiments.

Team: Kristin Klose, Dipthi Bachamanda Somesh, Enrico Fritsche, Christof Stamm, Andreas Kurtz, Manfred Gossen

Funding: Einstein Kickbox - Young Scientists (BSRT funded)

Team: Jacob Spinnen, Michael Sittinger, Tilo Dehne, Anja Kühl, Henrik Mei, Ufuk Sentürk

Funding: Einstein Kickbox - Young Scientists (BSRT funded)

Ageing affects the regenerative abilities of skeletal muscle resulting in compromised healing associated with fibrosis, impairing mobility and affecting quality of life. Current research is largely focused on intrinsic muscle stem cell function, but local extrinsic changes are mostly neglected. However, skeletal muscle repair relies on a dynamic interplay between muscle satellite cells (SCs) and the extracellular matrix (ECM) microenvironment. We have preliminary evidence for a transient developmental-like ECM during early phases of muscle regeneration. We believe that this creates a biomechanical three-dimensional micro-niche conducive to SC expansion, differentiation and self-renewal during regeneration, and that its derailment in aging or disease contributes to SC malfunction. However, the mechanical properties of the pro-regenerative ECM, especially during aging, has not been analyzed and the influence of the bio-mechanical properties of the aged ECM on stem cell behavior remains unclear. By combining experimental and mathematical analysis in an interdisciplinary approach, we aim to comparatively determine and quantify the dynamic spatio-temporal structure, composition and functionality of the transitory ECM in young and old mice. Iterative mathematical modeling via integration of biological data in a stepwise fashion will in the future develop a predictive landscape to achieve a new level of understanding of ageing processes prospectively transferable to muscle-degenerative disorders.

Team: Sigmar Stricker, Arunima Murgai, Georgios Kotsaris, Max von Kleist, Vikram Sunkara

Funding: Einstein Kickbox - Advanced Scientists

In this project we aim to establish a new scientific research focus at the ECRT, mechanogenomics. Multiple redundant interactions connect the plasma membrane to the nucleus allowing for external mechanical forces to alter nuclear morphology, a process which has been shown to have influence on the positioning of chromatin within the nucleus. Chromatin that associates with the nuclear membrane, so called lamina-associated domains (LADs), contains mostly inactive genes. We therefore hypothesize that mechanical forces directly influence gene expression via re-positioning chromatin in 3D nuclear space. Alterations in the extracellular matrix (ECM) composition might therefore modulate gene transcription during disease and regeneration. Preliminary data with CRISPR/Cas9 edited BMP receptor mutant endothelial cells show striking changes in cellular mechanics, ECM and nuclear lamina composition, as well as increased F-actin bundling and tubulin acetylation, resulting in increased cellular tension and nuclear deformation. In this context, we suggest that altered BMP signaling accounts only for a subset of differential gene expression, while other genes are regulated as a consequence of nuclear adaptation to novel mechanical inputs. Here, we aim to characterize the nuclear modifications with classical biochemical techniques and combine this, together with genome-wide identification of LADs and the corresponding transcriptome. Understanding the changes in the underlying regulatory gene networks and the synergism between BMP and mechano-signaling would provide novel insights for basic science as well as opportunities to translate this knowledge towards future treatment options in the clinics.

Team: Petra Knaus, Stefan Mundlos, Michael Robson, Jerome Jatzlau

Funding: Einstein Kickbox - Advanced Scientists

The human body is constantly challenged with manifold pathogenic, chemical and physical stimuli. One of the major tasks of the immune system is to distinguish between harmless and harmful challenges in particular at barrier sites such as the skin. How do local T cells in such situations sense “danger”, localize challenges at defined tissues or cells and finally dose and center a right suitable reaction? How are regenerative processes induced, and do they differ in the context of pathogen- versus non-pathogen-type challenges? It seems to be logical that while a virus-infected cell has to be eliminated by cytotoxic effector T cells, such a reaction would be unsuitable for cells that have been activated by harmless chemical or mechanical stress. The immune system has to tightly control any inexpedient cytotoxic T cell action in particular at barrier sites. However, it is so far not known, how the activation of non-cytotoxic T cells is regulated at barrier sites.

In this Einstein Kickbox application we aim to perform multidimensional cytometric and histological analyses on circulating and tissue resident T cells to decode and delineate the human universe of T cell subsets according to novel cell surface signatures we have identified in our prework. Based on the achieved results we aim in further future activities to characterize the function of non-cytotoxic helper T cells in human tissues in particular in response to non-pathogenic stressors.

Team: Andreas Thiel, Christos Nikolaou, Lucie Loyal, Kerstin Wolk, Demetrios Christou, Roland Lauster

Funding: Einstein Kickbox - Advanced Scientists

Team: Michael Schmück-Henneresse, Sybille Landwehr-Kenzel, Dimitrios L. Wagner

Funding: Einstein Kickbox - Advanced Scientists

Team: Il-Kang Na, Olaf Penack, Sarah Mertlitz, Georg Duda, Peter Fratzl

Funding: Einstein Kickbox - Advanced Scientists

Osteoarthritis of the knee (OAK) is one of the top five most disabling conditions that affects more than one-­third of people over 65 years of age and over 100 million individuals globally. OAK is characterised by an erosion of the articular cartilage, which leads to inflammation and pain accompanied by joint stiffness and immobility. Despite its prevalence, the molecular mechanisms triggering OAK are poorly characterised and current therapies cannot halt-­ or revert OAK. Consequently, knee joint replacement is often the only rehabilitative intervention. While animal models of OAK exist, the causative mechanisms leading to cartilage erosion are poorly understood. It is often unclear which of the OA-­associated factors are causing the disease, and which are a consequence or a mere side-­effect of it. Backed by literature evidence and own preliminary results on chondrocyte dynamics, we hypothesise that a disease-­associated chondrocyte (DAC) phenotype exists in early OA, causing subsequent cartilage erosion. Thus, identifying and characterizing DACs would pave the way for personalized diagnostic and therapeutic strategies to tackle an unmet clinical need. Within this project, we will identify the transcriptome signature of DACs from a mixed sample (healthy + DAC + noise). To do this, sophisticated bioinformatics and mathematical tools will be developed and applied to new experimental data from early OA time points, ensuring that DACs are sampled. Candidate transcripts will be compared to previously reported early OA-­associated factors, which typically contain mixtures of causative and consequential factors.

Team: Vikram Sunkara, Annemarie Lang, Lisa von Kleist, Max von Kleist, Christoph Schütte

Funding: Einstein Kickbox - Advanced Scientists

Team: Birgit Sawitzki, Britt Wildemann, Franka Schulz-Klatte, Anja Kühl, Uta Syrbe, Martina Seifert

Funding: Einstein Kickbox - Advanced Scientists

The aim of the project is to set up an as possible true-to-life model to study the microenvironment of primary acute lymphoblastic leukemia (ALL) cells on cell survival and resistance development. It is planned to investigate the effect of different drugs especially on the microenvironment but also on the ALL cells. While most cancer research focuses mainly on the pharmaceutical impact on tumor cells, the approach of this project will concentrate on the bone marrow stroma. We plan to establish a co-culture of ALL cells with 3D cultured bone marrow Mesenchymal Stroma Cells (BM-MSCs), as published by Sieber et al. (2017). For that, a hydroxyapatit coated zirconium oxide ceramic is seeded with patient derived MSCs and ALL cells to mimic the in vivo conditions. The whole cultivation is performed dynamically in the "Multi-Organ-Chip" (MOC). The MOC-platform, which has been developed at the chair of Medical Biotechnology at Technical University, is a microfluidic device consisting of a circular channel system which connects wells for the culture of different small functional human organ units, called organoids. Currently used 2D stroma cultures fail in their ability to support primary ALL cell proliferation and are therefore limited to test regenerative potentials of MSC cultures. This minimized model of ALL could be used for drug testing and dosage estimation. The in vitro leukemia model would present a breakthrough for detailed studies of the human marrow and for personalized drug screenings.

Team: Kübrah Keskin, Tessa-Lara Skroblyn, Domenic Schlauch, Cornelia Eckert, Mark Rosowsky

Funding: Einstein Kickbox - Young Scientists

Team: Melanie Ort, Janosch Schoon, Christine Consentius, Alessandro Camponeschi, Georg Duda, Roland Lauster, Sven Geissler, Anastasia Rakow

Funding: Einstein Kickbox - Young Scientists

Team: Leila Amini, Dimitrios L. Wagner, Uta Rössler, Petra Reinke, Andy Röhmhild, Hans-Dieter Volk, Michael Schmück-Henneresse, Uwe Kornak

Funding: Einstein Kickbox - Young Scientists

The ability to walk independently and pain free directly affects mobility and quality of life1,2. An essential function involved in walking is foot plantarflexion, articulated by a muscle-tendon unit containing the Achilles tendon (AT). Acute injury, like Achilles tendon rupture (ATR), leads to lowered strength and long-term functional outcomes3–5 regardless of treatment6, and puts patients at a higher risk for further injury7,8. The incomplete regeneration of the AT is the main contributor to functional deficits9, yet few studies have directly investigated regenerative capacity with function.

The possibility to utilize biomarkers to predict tendon healing has been suggested but requires further investigation for validation. After ATR, late surgical intervention leads to an imbalance in tendon homeostasis that is time-dependent10, but has yet to be related with AT function.

Properties of the healed tissue are difficult to characterize in vivo. One available method measures AT length changes using ultrasonography during active muscle function11. The AT’s capacity to store and transfer forces is due to its compliance, which allows muscle efficiency12; following ATR, this capacity is diminished.

This research aims to link the AT’s intrinsic regenerative capacity at the time of surgery with resultant healed AT tissue function. Our goal is to identify markers predictive of human ATR healing outcomes by combining investigations of early healing events with late-stage tissue function.

Tearm: Alison Agres, Kirsten Legerlotz, Britt Wildemann, Sebastian Manegold

Funding: Einstein Kickbox - Young Scientists

Team: Susanne Hildebrandt, Petra Knaus, Jacob Piehler, Harald Stachelscheid

Funding: Einstein Kickbox - Young Scientists

Team: Krithika Hariharan, Su-Jun Oh, Andreas Kurtz

Funding: Einstein Kickbox - Young Scientists

Team: Erik Brauer, Sophie Schreivogel, Daniela Mau, Uwe Kornak, Ansgar Petersen

Funding: Einstein Kickbox - Young Scientists

Team: Marcus Weber, Konstantin Fackeldey

Funding: Einstein Kickbox - Young Scientists

Liver cirrhosis is one of the main indications for liver transplantation. Due to the organ shortage, this therapy option is limited to the minority of patients suffering from cirrhosis. Therefore, there is a need of alternative treatment options.The aim of our project is to establish a decellularization protocol for human cirrhotic livers slices, which preserves the natural extracellular matrix (ECM) of cirrhotic livers. These decellularized liver slices will serve as a 3 dimensional model to study cell matrix interactions. If we are able to establish a protocol which will preserve the ECM, we will conduct in vitro recellularization experiments to study how the cirrhotic ECM will change the genotype and phenotype of different cell types. With this knowledge we aim to modify specific cell types in vivo or vitro for example prior to cell transplantation. Our ambition is to steer the cell matrix interaction via these modified cells after their transplantation and thereby halt or even reverse the progress of liver cirrhosis. This approach may offer an alternative treatment option in the future.

Team: Karl Hillebrandt, Oliver Klein, Igor Sauer    

Funding: Einstein Kickbox - Young Scientists

Angiogenesis is a fundamental process in development, cancer, and regeneration. Complex signaling pathways regulate tip selection, tip migration, and stalk cell proliferation to shape the emerging vascular network. To improve our basic understanding of how signaling interactions within individual cells determine vascular network architecture, we need multi-scale computational models that integrate the cellular signaling dynamics into tissue level dynamics. In the kickbox phase, we will implement the software necessary to efficiently simulate intracellular dynamics in multi scale agent based models and generate a proof of concept study.In the follow up project, we aim to collaborate with experimental scientists to iteratively generate, test, and refine hypotheses on how intracellular signalling defines vascular network structure.

Team: Clemens Kühn, Judith Wodke, Sara Checa Esteban, Edda Klipp, Georg Duda

Funding: Einstein Kickbox - Young Scientists

After stroke, the brain can compensate for lost neuronal tissue functions by rewiring of the neuronal network. However, in adult brains, post-ischemic healing is compromised, since neuroplasticity is limited by the adult shape of ECM consisting of a very dense meshwork of ECM proteins. We have shown, that endothelial cells can steer the post ischemic ECM remodeling depending on the inflammatory Stat3 pathway (Hoffmann et al., 2015). In this project, we want to shape the inflammatory endothelial cell response after stroke using a targeted drug for endothelial Stat3 activation. This will steer the remodeling of the ECM towards a neuroplasticity permissive status. This might help to overcome the compromised healing and regeneration after stroke. We will evaluate the drug in mice using a transient filamentous occlusion of the middle cerebral artery (MCAo) as stroke model and behavioral tests to determine effects on long-term functional outcome. We will monitor regenerative effects by visualization of neuronal network changes by viral and chemical tracing techniques as well as DTI and resting state MRI sequences. ECM remodeling will be determined histologically with an emphasis on the perineuronal net and CNS Ranvier´s node ECM.    

Team: Christian Johannes Hoffmann, Christoph Harms, Gisela Lättig, Philipp Böhm-Sturm, Ulrich Dirnagl

Funding: Einstein Kickbox - Young Scientists

Our project is aimed to prototype new mathematical models which explain the forces produced by the quadricep in exercise meditated OA therapies. We wish to extend existing isometric quadricep models to general motions by enriching the equations using prosthesis data. We are looking for groups working in biomechanics and focused on quantifying qualitatively the effects of exercise mediated therapies.

Team: Vikram Sunkara, Max von Kleist, Georg Bergmann

Funding: Einstein Kickbox - Young Scientists

Team: Katharina Hörst, Sarah Hedtrich, Nan Ma, Uwe von Fritschen, Susan Gibbs

Funding: Einstein Kickbox - Young Scientists

Team: Evi Lippens, Rosa Schmuck, Amaia Cipitria, Dag Wulsten, Daniela Garske

Funding: Einstein Kickbox - Young Scientists

The objective of the project is to uncover the function of IL-4 in cartilage homeostasis with available murine and human material applying different methods. Furthermore, we will work on a concept to implement in silico methods as a strong tool to study biological processes and signalling pathways and to use the power of an interdisciplinary collaboration towards a common goal. In a following (advanced) project, we aim to clarify the role of IL-4 in the pathogenesis of OA, to provide evidence-based approaches on how to overcome these compromised conditions and to identify new therapeutic targets towards OA to challenge an unmet clinical need that has remained unresolved for far too long. 

Team: Annemarie Lang, Max Löhning, Susanna Röblitz, Katharina Schmidt-Bleek, Frank Buttgereit

Funding: Einstein Kickbox - Young Scientists

Team: Bella Roßbach, Philipp Enghard, Khadija El-Amrani

Funding: Einstein Kickbox - Young Scientists

The extracellular matrix (ECM) instructs cells to orchestrate crucial aspects of their biological behavior. As a multidisciplinary team of cell biologists, biochemists and biophysicists we set out to investigate biophysical and biochemical features of the ECM from endothelial cells with different signalling backgrounds and found striking changes in its composition and growth factor reservoir capacity. We uncovered feedback regulations affecting transcriptional responses, adaptations of the cytoskeleton and the ECM and altered mechanical properties explaining the vicious cycle in the development of vascular diseases.

The ECRT support enables us to characterize these biochemical and biophysical characteristics of the altered ECM with unprecedented resolution and to provide new mechanisms that potentially help to understand the involvement of BMPs and TGFb in successful and failing endogenous repair scenarios.

Team: Petra Knaus, Christian Hiepen, Kerstin Blank, Peter Fratzl

Funding: Einstein Kickbox - Advanced Scientists

Extracellular vesicles (EVs) enable intercellular communication by delivering different messenger molecules, like micro RNAs, receptors, enzymes, and others, from a donor cell to a recipient cell. Because of this ability, EVs from regenerative cells are considered as a potent new tool to treat diverse diseases.

The idea of our application is to generate “milieu-designed” EVs from a regenerative cell type, the so-called right atrial appendages (RAA)-derived cells. Herein, we want to make use of the fact that the microenvironment/condition applied to a cell during the EV biogenesis highly influences which molecules will be transported by their EVs. Thus, it can be hypothesized that the regenerative potential of released EVs can be targeted for the desired therapeutic purpose by considering different conditions during their biogenesis. In that context, we plan to investigate with this Kickbox Seed Grant how oxygen limitation affects EVs from RAA-derived cells in regard to their phenotype, transported molecules, and, most importantly, desired functional effect. With the help of this study, we believe to set foundation for the development of a new therapeutic option for the treatment of different types of cardiac damage.

Team: Martina Seifert, Marion Haag, Volkmar Falk, Michael Sittinger, Christof Stamm

Funding: Einstein Kickbox - Advanced Scientists

Team: Katharina Schmidt-Bleek, Sophie van Linthout, Georg Duda, Carsten Tschöpe, Christian Bucher

Funding: Einstein Kickbox - Advanced Scientists

Team: Reiner Jumpertz von Schwartzenberg, Joachiam Spranger, Hans-Dieter Volk, Julia Kind, Rainer Glauben, Stephan Schlickeiser, Desiree Kunkel

Funding: Einstein Kickbox - Advanced Scientists

Team: Nathanael Raschzok, Angelika Kusch, Duska Dragun, Igor Sauer

Funding: Einstein Kickbox - Advanced Scientists

With increasing life expectancy and concurrent high demands regarding personal mobility, the numbers of total joint arthroplasties (TJA) are rising. Accordingly, larger numbers of infections are documented. Periprosthetic Joint Infections (PJIs) account for the majority of implant failure, occurring in 2- 3% of all primary TJAs with even higher rates of 3-10% in revision surgeries. A consistent diagnostic after TJA is critical for the therapeutic success. Particularly the diagnostics of persistent low grade infections is still insufficient, calling for the development of highly sensitive diagnostic tools for early detection.

Immunologically reactive molecules are released from T-cells upon cell activation. We hypothesize that selected molecules can not only be used to assess the level of T-cell activation and the reactivity of the adaptive immune response, but also as a marker for lingering infections. Furthermore, their level may serve to clarify an underlying molecular predisposition for endogenous infection management and potentially even resume a protective role in infections.

Defining a biomarker on protein level, that enables local, minimally invasive and reliable diagnostics in the field of PJI while giving insight into the patients’ general immune status, is an ambitious, novel and innovative approach.

The project elements for the KickBoxSeed-Grant are in the focus area of “cellular mechanisms in compromised healing” while the long-term vision of the project additionally addresses the local modulation of inflammation. The concept provides an interface between cell-based immunology and pathogen-based infectiology/immunology with endoprosthetic infections as a clinically highly relevant field of application.

Team: Andrea Sass, Carsten Perka, Juri Rappsilber, Andrej Trampuz, Michael Fuchs, Simon Reinke, Anke Dienelt

Funding Scheme: Einstein Kickbox - Advanced Scientists

Team: Christof Stamm, Andreas Kurtz, Manfred Gossen, Nan Ma, Krithika Hariharan

Funding: Einstein Kickbox - Advanced Scientists

Team: Kostja Renko, Harald Stachelscheid, Özlem Vural, Roland Lauster, Manfred Gossen

Funding: Einstein Kickbox - Advanced Scientists

Team: Salim Seyfried, Juliane Münch, Motahareh Moghtadaei, Sigmar Stricker, Ulf Landmesser, Philipp Jakob, Peter Fratzl, Hans-Georg Simon

Funding: Einstein Kickbox - Advanced Scientists