Metanavigation:

Hier finden Sie den Zugang zur Notfallseite, Kontaktinformationen, Barrierefreiheits-Einstellungen, die Sprachwahl und die Suchfunktion.

Navigation öffnen

ECRT Projekte

Das ECRT will insbesondere den wissenschaftlichen Nachwuchs beim Aufbau eigener Forschungsvorhaben unterstützen. Dafür bietet das ECRT Einstein Kickboxen mit einer Startfinanzierung für erste Experimente zur Validierung der Forschungsideen sowie ECRT Research oder Consumable Grants zur Weiterentwicklung der Forschungsvorhaben.

Die Zusammenfassungen der Projekte stehen nur auf Englsich zur Verfügung.

Sie befinden sich hier:

Laufende ECRT geförderte Projekte

ECRT Career Kickoff - Förderperiode 2022-2023

Bone metabolism and vascular calcification: understanding the immunological regulatory network of the bi-directional interplay

Clinical and epidemiological studies have demonstrated increased rates of vascular calcification in patients with osteoporosis. Common risk factors for both disorders such as age, lack of physical activity, smoking, diabetes, hypertension or hormonal changes after menopause are insufficient to explain a mutual pathogenic mechanism. Broad evidence emphasises the important role of systemic inflammation in both diseases, indicating a pathologically immunological regulation. To address the question of a potential systemic link, extensive analyses of osteoporotic sera will be carried out and the immune cell status of the patients will be determined. The influence of osteoporotic serum and possible mediators on vascular calcification will be investigated using an established in vitro assay that is based on the osteogenic differentiation of human vascular smooth muscle cells.

Team: Wera Pustlauk, Sven Geißler, Nina Babel, Katja Hanack

Funding: ECRT Career Kickoff Grant - PhD

Effects of cellular senescence on mechano-sensation: implications for tissue regeneration

Cellular senescence is an irreversible stress program that is present in numerous regenerative processes. Despite the extensive research of its roles in cancer and age-related disorders, the effects of cellular senescence on tissue regeneration remains largely unexplored. Our group recently observed how senescence modulates biomechanical factors and affects extracellular matrix (ECM) tension inside a 3D macroporous collagen scaffold. While these findings provide novel insights into the consequences of senescence on the mechanical properties of cells and the ECM, the mechanosensing pathways that connect the two remains unknown. The aim of this project is to investigate changes in cellular mechanosensing in senescent human dermal fibroblasts (hdFs) and to link these findings to differences in their 3D behaviour. We compare three experimental approaches to induce senescence (DNA-damage, replicative senescence and over-expression of cell-cycle-inhibitor p16). To understand how these distinct senescent cells interact with their physical environment, we study their response to substrate stiffness using 2D hydrogels. Further, using a substrate with precise meso-scale geometries, we investigate their response to curvature to understand senescence-associated alterations of cell-organisation in 3D environments. Overall, these findings could help predict their response in physiological relevant processes and identify particular mechanical and geometrical environments favoured by these cells, which could be integrated into current strategies to promote tissue regeneration.

Team: Mina Sohrabi-Zadeh, Ansgar Petersen, Sven Geißler, Erik Brauer

Funding: ECRT Career Kickoff Grant - PhD

Immunosuppression-resistant Treg products for advanced adoptive T cell therapy 

End-stage organ failure requires solid organ transplantation (SOT), which needs permanent immunosuppression to prevent rejection. To minimize the adverse effects of immunosuppressants, adoptive regulatory T cells (Treg) are being investigated. A phase I /IIa clinical trial found that adoptive transfer of in vitro expanded Treg to SOT patients is safe and effective enabling patients to taper immunosuppression to Tacrolimus (Tac) monotherapy. However, the latter can interfere with Treg functionality. Thus, we developed Treg products, which are resistant to Tac. Treg were isolated with high purity by a flow cytometry-based sorting system. The gene FKBP12, which is required for the immunosuppressive function of Tac, was efficiently knocked out using vector-free CRISPR-Cas9 technology. A modified Treg product was characterized phenotypically and qualitatively in vitro. Phenotypic analysis of modified Treg products showed preserved FOXP3 expression entirely after expansion. Also,  expansion data indicate that modified Treg demonstrated resistance to Tac while suppressed by alternative immunosuppressants. Overall, we developed a novel approach for generating Tac-resistant Treg products to promote adoptive T cell therapy under Tac treatment in solid organ transplant recipients. Now, we need further data on functionality in vitro and in vivo systems to build the basis for a first in human clinical trial.

Team: Ghazaleh Zarrinrad, Petra Reinke, Julia Polansky-Biskup, Michael Schmück-Henneresse

Funding: ECRT Career Kickoff Grant - PhD

Revealing the molecular nuances of TRPC6 in focal segmental glomerulosclerosis in an in vitro human podocyte model

Focal segmental glomerulosclerosis (FSGS) is the leading glomerular cause of end-stage renal disease worldwide. This disease progresses from podocyte injury to complete nephron degeneration. A non-selective transient receptor potential calcium cation channel-6 (TRPC6) gene is associated with the development of FSGS. The process of podocyte damage and loss has been, however, difficult to address in the past, because of deficiencies in animal models and a paucity of suitable human in vitro models. Therefore, in this project, we aim to establish a novel human in vitro model system that shall help to unravel the mechanism behind podocyte loss and is suitable to develop therapeutic approaches. To model FSGS, a collection of human-induced pluripotent stem cells (hiPSCs) is established carrying gain or loss of function TRPC6 mutations and used for differentiation into induced podocytes (iPodocytes). In order to mimic the effects of mutation on the glomerular basement membrane, we want to use the transgenic TRPC6 iPodocytes in a microfluidic chip-based model system. The novel glomerulus platform offers a deeper understanding of the TRPC6-associated mechanism of podocyte loss by allowing the comparison between healthy and diseased iPodocytes in their morphology and behavior. We strongly believe that this humanized and personalized system can provide insights for future therapeutic interventions that can slow down or even abolish the progress of FSGS and other CKDs.

Team: Lilas Batool, Andreas Kurtz, Petra Reinke, Manfred Gossen, Krithika Hariharan

Funding: ECRT Career Kickoff Grant - PhD

Role of Human Extracellular Matrix Properties in Central Nervous System Regeneration

While the determinants of CNS regeneration are still up for investigation, some evidence points to the involvement of micro environmental factors, including biochemical ECM properties. However, most data on the effect of ECM on oligodendrocyte function and regeneration are based on animal models and the knowledge on the effect of the ECM in a human system is quite limited. This project is focused on a more holistic and human approach towards ECM-driven myelin regeneration. We have successfully established a decellularized scaffold from postmortem human brain tissue to investigate stem cell fate and myelination. With this working model, opportunities have risen to test and compare how monocyte phenotype changes and how neural stem cells (NSCs) and oligodendrocyte precursor cells (OPCs) differentiate on different brain regions, different MS lesions, and different disease entities. Hopefully, this project will allow us to link biochemical properties of diseased human brain tissue with functional myelination and CNS regeneration in an ex vivo setting.

Team: Roemel Jeusep Bueno, Sarah Staroßom, Ansgar Petersen, Harald Stachelscheid

Funding: ECRT Career Kickoff Grant - PhD

Einstein Kickbox - Förderperiode 2022

Amyloid-β plaque targeted anti-inflammatory macrophages: Resolving neuroinflammation, increasing phagocytosis and providing neuroprotective cytokines

Alzheimer’s disease (AD) is the most common neurodegenerative disease with insufficient approved treatments available. Amyloid-β aggregates are known as the pathological protein hallmark of this disease, accumulated due to inefficient phagocytosis by dystrophic microglia. Increase of pro-inflammatory cytokines concomitant with deficient levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) can lead to progressive dystrophy of neural cells and eventually result in cognitive decline at later stages of disease. Active transport through the blood brain barrier is one of the major challenges limiting the success of most therapeutic approaches. Given that the neuro-inflammatory environment in the brain of AD patients attracts peripheral blood cells to migrate across the blood brain barrier, these cells can be harnessed to actively transport the candidate therapeutic small molecules. Thus, the aim of this project is to explore the potential of transiently engineered peripheral monocytes and monocyte-derived macrophages by transfection with synthetic mRNA as a therapeutic strategy. We will investigate the transient overexpression of some candidate receptors to increase phagocytosis of amyloid-β aggregate species, while maintaining an anti-inflammatory environment. In the next step, we are keen to engineer macrophages to overexpress neuronal support factors, e.g. BDNF, NGF, both beneficial for increase in neural survival.

Team: Charlotte Dunne, Hanieh Moradian

Funding: Einstein Kickbox - Young Scientists

Codon Reassignment Technology as a Novel Tool for Stoechiometric and Directed Protein Labelling

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

Human iPSC-derived Schwann cell myelinated sensory neuron co-culture as an in vitro model of inflammatory demyelinating polyneuropathies

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

Improving non-viral knock-in in sensitive cell types using modified single stranded DNA templates

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

Influence of the peri-implant granulation tissue on bone healing

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

Regeneration of Motor Function after Stroke

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

Hydrogels for RNA delivery: exploration of the nucleic acid - polymer interface

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

 

Brain organoids as a human model system to study chemotherapy-induced CNS toxicity

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

Impaired cardiac oxalate homeostasis triggers atrial fibrillation in chronic kidney disease

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

Funding: Einstein Kickbox - Young Scientists

Novel ex vivo setup to assay the impact of NK cell mediated ADCC for tissue regeneration in cancer immunotherapy

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

Unravelling the influence of a deletion in the LAMA3 locus on osteopetrosis by generating a CRISPR/Cas engineered disease model

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

Quo vadis? Modulation of hiPSC differentiation into brain-specific endothelial cells by substrate stiffness

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

Funding: Einstein Kickbox - Advanced Scientists

ECRT Consumable Grants - Förderperiode 2022

Homeostatic proliferation of naïve CD4+ T cells, induces epigenetic changes, which contributes to immune aging

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) 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 with a greater homeostatic proliferation history 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.
Our working hypothesis is that the accumulation of homeostatic proliferation events over the life-time of a human being in CD4+ TN induces changes in the cell’s epigenome, which impact T cell function and contribute to the impaired immune responses in elderly. Indeed, our preliminary data show that accumulation of homeostatic proliferation events induces stable epigenetic changes in ex vivo derived CD4 TN of young healthy donors.With the support of the Einstein funding we want to show that these differences increase in elderly healthy donors and further characterize the potential differences in transcriptional regulation in CD31+ and CD31- TN.

Team: Dania Hamo, Mai Phan, Andreas Magg

Funding: ECRT Consumable Grant - Young Scientists

Immune modulation as a potential treatment strategy against periprosthetic joint infection

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. Programmed cell death protein-1 (PD-1), a receptor expressed on various cell types, has been recently shown to affect the bone metabolism, specifically on osteoclasts. We hypothesize that targeting PD-1 is a promising strategy to improve bone quality during PJI. Here we plan to introduce a bone explant culture mimicking in vivo environment. This model will allow us to test the overall change of the bony scaffold and the behavior of the different cells inside ex vivo under the influence of PD-1 inhibitor. Understanding the phenomenon and related mechanisms will provide us deeper knowledge on this unique therapeutic approach to improve implant survival.

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

Funding: ECRT Consumable Grant - Young Scientists

Oxalate-induced RyR dysfunction as a substrate for cardiac arrhythmias and contractile impairment

Prevalence estimates indicate that approximately 13% of the US adult population suffers from CKD. In 2019, the G-BA registered 93.089 dialysis patients in Germany. Both conditions are associated with an increased cardiovascular risk. In patients undergoing dialysis, sudden cardiac death is the most frequent cause of death (~25%). Tailored therapies related to underlying disease mechanisms may prove to be effective in reducing cardiovascular morbidity and mortality in these patients.

Reduced renal function leads to increased levels of plasma oxalate (POx). Several lines of evidence indicate that oxalate may have detrimental effects on the cardiovascular system. For example, post-hoc analysis of the German Diabetes Dialysis study unveiled a 62% higher risk for sudden cardiac death and 40% for cardiovascular events in dialysis patients of the highest Pox quartile compared to the lowest quartile. Elevated Pox concentrations are associated with cardiac fibrosis and altered hemodynamic parameters (pulse wave velocity, blood pressure). While these reports are intriguing, mechanisms of myocardial oxalate homeostasis and potential toxicity remain elusive.

Our previous experiments indicate that oxalate induces RyR dysfunction in cardiomyocytes in-vitro, which is considered key component of cardiac arrhythmogenesis and contractile function. In this proposal, we hypothesize that elevated concentrations of plasma oxalate affect cardiac contractility and susceptibility to arrhythmias through functional impairment of the cardiac ryanodine receptor (RyR) in-vivo.

Team: David Bode, Gerlineke Hawkins-van der Clingel, Cristian Sotomayor-Flores

Funding: ECRT Consumable Grant - Young Scientists

Deciphering the cancer stem cell niche – The road to standardize patient-derived lung cancer or-ganoid culture

Lung cancer is characterized by potent and individual drug resistance due to genetic and molecular diversity. Patient-derived lung cancer organoids (PDLCOs), 3D multicellular structures derived from patient tumors, could serve as preclinical biomarkers of individual responses to therapies. Lung cancer has been suggested to originate from cancer stem cells (CSCs). CSCs are stimulated by niche factors, secreted by non-epithelial cells in vivo. These niche factors need to be substituted in PDLCO cultures as organoids comprise only epithelial cells. However, heterogeneous trajectories of lung CSCs, such as gene expression, impede standardized and efficient PDLCO culture so far. We could identify putative trajectories of lung CSCs and their niche factor requirements. In our project, we plan to generate PDLCOs with four different growth factor cocktails (artificial niche). We will perform RNA sequencing of organoids and primary tumors. Comparing the transcriptomic signature of the individual CSC niche with the respective growth factor cocktail that successfully enables organoid growth (=successful mimicking of the niche) will enable us to predict the specific growth factors requirements for different tumor / CSC signatures. With our proof of concept approach, we could individualize protocols and efficiently generate PDLCOs as platforms for personalized medicine.

Team: Lukas Ehlen, Martin Szyska, Michael Schmück-Henneresse, Martí Farrera I Sal, Janine Arndt

Funding: ECRT Consumable Grant - Advanced Scientists

On the other side of the table – Defining the pathogenic and regenerative role of renal transplant tubular epithelial cells in ischemia-reperfusion-injury and alloreactivity

In our previous Einstein Kickbox project, we developed an assay using cells from the urine of kidney transplant patients to measure transplant reactive T cells. This allows the monitoring of immune reactions towards the allograft and the detection of acute rejection episodes, enabling personalized medicine. With the Einstein Kickbox consumable grant, we aim to deepen our understanding of the kidney transplant tubular epithelial cells (TEC). After explantation, the donor kidney is subject to warm and cold ischemia. The severity of the subsequent organ inflammation determines future immune reactivity and fibrosis. A clinical symptom of these processes is a delayed graft function, which is associated with a reduced long-term graft and patient survival. Thus, the goal of this project is to investigate the role of transplant TEC in pathologies and regeneration following kidney transplantation. Therefore, we will use the consumable grant to perform comprehensive analyses of the urine-derived transplant TEC. We will analyze changes in protein expression and epigenetic regulation in different patient cohorts (kidney transplant patients with and without delayed graft function or acute rejection and healthy donors) and different conditions (inflammation and hypoxia). All in all, we plan to gain knowledge about the pathogenic or regenerative capacities of kidney cells in transplantation and open new doors for future translational research projects and new therapeutic approaches.

Team: Constantin Thieme, Nina Babel, Julia Polánsky-Biskup, Toralf Roch, Luka Queitsch

Funding: ECRT Consumable Grant - Advanced Scientists

Cell-mediated depletion of residual TCR+ cells for allogeneic CAR T cell products

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 B cell derived leukaemia and lymphoma.

We recently improved a method 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 constant locus (TRAC). 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 brought under the transcriptional control of the TRAC promoter. CAR integration at this site 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 allogeneic use, there are also small fractions of unedited and CAR+/TCR+ double positive T cells. These remaining TCR+ cells could potentially induce Graft-vs-Host disease if transfused in an allogeneic setting. This project aims to eradicate all remaining TCR+ contaminants in order to generate safe off-the-shelf CAR T cell products.

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 B cell derived leukaemia and lymphoma.

We recently improved a method 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 constant locus (TRAC). 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 brought under the transcriptional control of the TRAC promoter. CAR integration at this site 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 allogeneic use, there are also small fractions of unedited and CAR+/TCR+ double positive T cells. These remaining TCR+ cells could potentially induce Graft-vs-Host disease if transfused in an allogeneic setting. This project aims to eradicate all remaining TCR+ contaminants in order to generate safe off-the-shelf CAR T cell products.

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

Funding: ECRT Consumable Grant

ECRT Research Grants - Förderperiode 2021-2023

3D tissue models for preclinical testing of augmented CAR T cells

T cells have been identified as one of the main defense lines of the immune system against tumors. The scientific break throughs of the recent years with immunologic checkpoint inhibitors and adoptive T-cell therapy are offering novel treatment options when the body defense mechanisms fail and T cells become dysfunctional. T cells expressing a chimeric antigen receptor (CAR) are one of the recent advancements and combine the specificity of an antibody to target-specific antigens on tumor cells with the ability of T cells to kill tumor cells.

CAR T cells have shown remarkable effects in the treatment of late stage lymphoma patients. Unfortunately, CAR T cell therapy for solid tumors has not been nearly as successful and further research is needed to improve the existing treatment options. However, current used test systems for CAR T cells such as conventional 2D tumor cell cultures and murine xenograft models lack important features of human tumors in vivo.

In this project we want to establish novel test systems for the development and preclinical testing of CAR T cells that closer resemble the situation in the patient. For this we will employ iPSC derived organoids that will be generated in close collaboration with the BIH Core Facility Stem Cells as well as patient derived organoids. Introduction of fluorescent reporters will allow high throughput screening of CAR-T cell induced toxicity using the confocal high content imaging platform at the BCRT. The focus will be on generating a model for lung-cancer using overexpression of the epidermal growth factor receptor (EGFR), a very common tumor associated antigen with tumorigenic potential. Once the organoid models are established, we can test the different CAR T cell products that are currently used and further developed in the institute. Next to the direct tumoricidal activity, also homing and invasion in the established tissue models as well as the capacity of CAR T cells to initiate durable responses will be studied in static and microfluidic systems.

Team: Leila Amini, Regina Stark, Harald Stachelscheid, Hans-Dieter Volk, Ugarit Daher

Funding: ECRT Research Grant

Resolving the Activin A Receptor complex and its endothelial function in Fibrodysplasia Ossificans Progressiva (FOP)

The TGF-β superfamily member Activin A induces signal transduction via two type I (ALK4/7) and two type II (ACVR2A/B) receptors, which upon ligand binding form heterotetrameric receptor complexes and induce phosphorylation of SMAD2/3. Fibrodysplasia Ossificans Progressiva (FOP) is a rare, severely disabling disease characterized by gain-of-function mutations in the Bone morphogenetic protein (BMP) type I receptor ALK2, the most prominent of which is R206H. This arginine to histidine change turns a normally silent ALK2 signaling complex  responsive to Activin A, and finally results in aberrant SMAD1/5 signaling leading to heterotopic ossification (HO). While there is no approved treatment for FOP; several treatment options targeting Activin A or ALK2 are in research and development. Previous studies propose an involvement of the endothelium to the progression of FOP, as the aberrant Activin A signaling is recapitulated in FOP ECs. However, 1) the role of ECs in the formation of HO is not yet fully understood, 2) the composition of the receptor complex is not resolved and 3) the detailed molecular and cellular mechanism of Activin A binding to FOP-ALK2 remains to be explored.  Therefor the PhD candidate will perform (a): siRNA based approaches, in combination with co-immunoprecipitation experiments to determine the composition of the receptor complexes. Further the applicant will take advantage of an existing SNAP- and HALO-tagged BMP/TGFβ type I and II receptor library, and perform Stimulated Emission Depletion (STED)-based super resolution imaging. Finally the applicant (c) will set-up a new drug screening plat form for yet unapproached targeting strategies, to functionally interfere with both WT and FOP-ALK2 in ECs treated with Activin A and other ligands of the family. This project is particularly interesting for master students with an interest and/or background in signal transduction, imaging and translational research.

Team: Jerome Jatzlau, Petra Knaus, Holger Gerhardt, Wiktor Burdzinski

Funding: ECRT Research Grant

The search for the holy stromal cell - Identification and characterization of specialized and regenerative stromal cell populations to accelerate cartilage repair

Knee osteoarthritis (KOA) is one of the most common form of joint disease among elderly patients and remains a major orthopedic challenge since mature hyaline articular cartilage has only a limited capacity for intrinsic repair. Despite its prevalence, the molecular mechanisms triggering KOA are poorly characterized and there are currently no disease modifying therapies that can halt or revert KOA. Cell therapeutic approaches using autologous chondrocytes or mesenchymal stromal cells (MSCs) have been developed and tested in the last decade. MSCs can be found in bone marrow, adipose-tissue, synovium and cartilage itself, and are capable of differentiating into osteogenic, chondrogenic and adipogenic lineages. First attempts to inject MSCs in KOA patients have provided promising results during clinical trials, showing a significant decrease in inflammation and enhanced joint function. However, in most studies, regeneration of cartilage was not observed. Our preliminary results and current literature suggest that the MSCs inhibiting inflammation and the MSCs regenerating cartilage are not one and the same.

The overall aim of this project is to delineate the MSC subpopulations in the synovial membrane and the epiphyseal bone marrow area, to conceptualize a novel mixed MSC population therapeutic strategy for KOA. The project has both a wet-lab and dry-lab outcomes. The wet-lab outcomes will entail delineating MSC subpopulations using novel scRNA-seq techniques and perform functional assays to characterize the cells for their differentiation, regeneration, and immunomodulatory capacity. In the dry-lab, mathematical models will be derived from the data to explain the macro-level dynamics; attractants and surrounding factors that are required for maintenance and re-activation of chondrogenic MSCs during ageing.

Team: Annemarie Lang, Mir-Farzin Mashreghi, Christof Schütte, Vikram Sunkara, Sebastian Kurmies

Funding: ECRT Research Grant

ECRT Research Grants - Förderperiode 2020-2022

A chemokine system to unleash T cells against solid cancer - Regenerate immune responses against tumor

The immune system harbors intrinsic capacity to fight malignancy. Tumor-specific T cells play a crucial role in combatting cancer by recognizing and killing malignant cells. To enhance tumor-specific immune responses, T cells can be redirected using genetically engineered receptors against tumor antigens. Clinical success of engineered chimeric antigen receptor (CAR)-T cell has been achieved for hematological malignancies. However, success of CAR-T cell therapy in treating solid tumors has been limited, since transferred CAR-T cells could not infiltrate and persist in the hostile tumor environment. To improve adoptive transfer of CAR-T cells against solid tumors, we initiated a study to learn from intrinsic cues that promote T cell infiltration and persistence in human solid tumors.

We joined forces with Karolinska Institutet and Umeå University (Sweden) to study solid cancer disease. Here, we unraveled chemotactic mechanisms in muscle-invasive bladder cancer (BC). BC is a solid tumor disease with poor prognosis, yet, T cell infiltration into the BC can resuscitate patients to reject the tumor. We addressed the question which signals in BC favor T cell trafficking to the tumor and intratumor expansion. In this approach, we identified a distinct Th1 chemokine (C1, see notes) as main driver of T cell infiltration at the tumor site. In-vitro, we found that protective T cell subsets expressed a C1-specific receptor variant (R1*) and functionally, R1*+ T cells enriched when targeted by the ligand C1. Strikingly, analyzing the novel R1*-C1 axis in the tumor, we could predict overall survival and successful response to therapy in BC-patients. We hypothesize that R1*-C1 is a T cell-activating pathway in cancer with therapeutic potential.

In this project, we continue this approach within our European translational group. We pursue two aims:

1) Understanding how C1 improves anti-tumor T cell function.

2) Setting up a new therapy via R1*+ CAR-T cells.

1) We want to test the use of C1 for in-vitro culture and potential clinical application. For this, we will study signaling induced by the R1*-C1 pathway by applying i) our established flow cytometry method on phosphorylation targets (e.g. STAT-proteins) ii) multiplex-mass cytometry (in-house available in the cytometry core facility) and iii) a unique DIGI-West approach (with Tübingen in the EU-network Reshape).

2) We address to select natively R1*-expressing T cells from human PBMC as a starting population for CAR-T cell production (nR1-CAR-T cells). Further, we plan to endow ex-vivo generated tumor-specific T cells with the genetically engineered receptor R1* (eR1-CAR-T cells). In addition, we intend to access HER2-specific CAR-T cell products that were previously applied against mamma carcinoma (with Baylor College of Medicine Houston, TX, USA). Here, we seek to verify if low expression of R1* in products correlates with poor response rates following adoptive therapy.

In this project, we aim to gain insights into the newly described R1*-C1 axis in human solid cancer by native pre-selection of R1*-expressing cells or R1*-engineering for CAR-T cell generation. Thereby, we employ novel therapeutic approaches to boost the efficacy of tumor-specific CAR-T cells to reach and survive in the solid cancer microenvironment.

Team: Tino Vollmer, Michael Schmück-Henneresse, Hans-Dieter Volk, Leila Amini, Jacqueline Wendering, Sarah Schulenberg

Funding: ECRT Research Grant

Building a Platform for Fast Track Development and Characterization of Engineered Antigen Receptors

Figure 1) Flow cytometric Evaluation of CAR Signaling (FLECS) platform for inexpensive, standardized in vitro assessment of CAR function. In reporter cells, T cell receptor (TCR) or chimeric antigen receptor (CAR) signaling activates transcription of a TCR-inducible gene which is fused to a GFP reporter transgene via a P2A linker. Upon translation, GFP is freed by self-cleavage of the P2A linker; b) anti-CD3 antibody activates TCR, leading to GFP expression (pos. control); c) CD19-specific CAR is activated in CD19 antigen-coated well leading to GFP expression (pos. control); d) Upon binding their specific antigen (Ag), test CAR constructs may vary in signaling strength and consecutive GFP expression; e) test CARs in the absence of their specific antigen ideally show no/little basal GFP expression. CD19-CARs in absence of their antigen may serve as neg. controls; f) Hypothetical results of flow cytometric analysis of GFP expression for positive controls, test CARs 1-4 (with differing signaling properties) and a negative control. (Fig. generated on biorender.com
Figure 2) Chimeric auto-antibody receptor design for Neuromyelitis Optica/Multiple Sclerosis: Chimeric auto-antibody receptors (CAARs) mediate recognition of pathogenic B cells by engineered CAAR-T cells through binding to surface-bound immunoglobulins (sIgG) specific for an immunogenic self-antigen. The CAAR consists of a peptide derived from the auto-antigen as its extracellular recognition domain which interacts with pathogenic auto-antibodies, a hinge domain for optimal intermembrane distance of the immunological synapse, a CD8 transmembrane (TM) domain to facilitate dimerization, a 4-1BB costimulatory domain and a CD3 zeta chain for signal transduction. (Fig. generated on biorender.com)

Redirecting T lymphocytes using chimeric antigen receptors (CARs) is a powerful tool in the emerging field of regenerative medicine. T cells reprogrammed to recognize and kill CD19 positive cancer cells dramatically improved leukemia and lymphoma treatment, whereas the transfer of CARs specific for allogeneic antigens (Ag) into regulatory T cells was shown to induce immune tolerance in transplantation models. Recent reports suggest CARs may facilitate innovative treatments for autoimmune diseases as novel receptor designs enable CAR-T mediated depletion of pathogenic auto-Ag specific B cells and prevent autoantibody (Ab) mediated diseases (1).

CARs are fusion proteins made of a target-specific extracellular domain (usually a single chain variable fragment derived from an Ab) and an intracellular signal transduction region (varying costimulatory domains and a CD3 zeta chain) which imitate T cell receptor (TCR) signaling. Optimal signaling strength of CARs is paramount for T cell activation and the clinical success, while binding affinity/avidity and minimal tonic signaling are also of importance (2). Surprisingly, careful evaluation of CAR signaling properties is rarely performed and no standardized assays have been described. Existing assays to predict in vivo CAR-T cell function are limited to tumor-specific CARs and rely on lengthy serial co-culture experiments. Thus, they are not practical for fast iterative optimization of CAR design.

We hypothesize that signaling properties of a given CAR can predict its in vivo performance. To this end, we plan to create a reporter cell line that generates an optical output in response to Ag-receptor signaling. In Jurkat cells, which are a standard model to study TCR signaling, the expression of a variety of genes is induced upon TCR ligation, some of which correlate exclusively and gradually with Ag-mediated TCR signaling strength.

To obtain a good reporter system, Jurkat cells will be genetically modified by inserting a reporter green fluorescent protein (GFP) transgene into a TCR-inducible gene via CRISPR/Cas9 (3). Ag-receptor (TCR or CAR) signaling will lead to the expression free GFP through the self-cleaving activity of a P2A linker (Fig. 1). CAR constructs to be tested can be readily transferred to the reporter cells via the delivery system of choice (retroviral, transposon, site-specific integration). After subsequent Ag stimulation, fluorescence intensity of CAR-mediated signaling will be detected by flow cytometry over multiple time points. Results from TCR/CD19 CAR signaling will serve as performance benchmarks.

We propose to name this platform FLECS as an acronym for Flow cytometric Evaluation of CAR Signaling. It will enable standardized mass-testing of new CAR constructs and ensure the selection of the most efficient CAR for any given Ag. It will be deposited in public cell banks and shared openly to advance CAR-redirected T cell therapy in regenerative medicine.

After establishing the FLECS platform, we will focus on the development of CARs for the severe, rapidly exacerbating, demyelinating, Multiple Sclerosis (MS)-like disease Neuromyelitis Optica (NMO). In NMO, auto-Abs against immunogenic self-Ag are thought to play a causative role. Similar to a recent report (1), we designed novel antigen receptors that incorporate the immunogenic epitope derived from the self-Ag to form a chimeric auto-Ab receptor (CAAR). Thus, cytotoxic T cells will be able to recognize B cells that display surface bound immunoglobulin (sIgG) specific for the auto-Ag (Fig.2). After FLECS analysis, the CAAR with best performance will be subjected to further functional analysis in vitro and in the mouse model. Eventually, targeted elimination of auto-Ag specific B cells may offer a novel therapeutic approach to prevent chronic inflammation through auto-Ab in NMO, while FLECS might facilitate the development of similar therapeutics for other autoimmune diseases.

Team: Dimitrios L. Wagner, Michael Schmück-Henneresse, Hans-Dieter Volk, Petra Reinke, Anja Hauser, Helena Radbruch, Jonas Kath

Funding: ECRT Research Grant

Cellular senescence in mechano-sensation and ECM organization

Aging is an irreversible, progressive process resulting in a decline of tissue functionality and its regenerative capacity. With an increasingly aged population in developed countries, understanding deviations in healing processes after injury in aged is key for developing novel treatment strategies. During life the body accumulates with cells which lack a proliferative capacity due to an irreversible cell cycle arrest which is termed cellular senescence. Senescent cells secrete a variety of pro-inflammatory cytokines which recruit immune cells for tissue clearance during development and to prevent cancer. Although this feature confers early-life benefits, the increasing pro-inflammatory environment contributes to late-life debility (Campisi 2013). This antagonistic function is even further emphasized by recent observations which not only show the accumulation of senescent cells within an injured tissue but that elimination of senescent cells delays wound healing in vivo (Demaria et al. 2014). While this investigation related the effect to the secretory phenotype, little is known how senescent cells directly contribute to tissue formation through ECM formation.

We investigated how the senescence program affects extracellular matrix deposition and tissue tension using a recently published in vitro wound healing model system (Brauer et al. 2019). We observed that cellular senescence strongly affected macroscopic tissue tension and fibrillar collagen secretion and assembly in an antagonistic manner. While senescence due to over-expression of cell cycle inhibitors (p16INK4/p21CIP) resulted in an increased contraction, DNA-damage-induced (Mitomycin C-treated) senescence strongly reduced tissue contraction. This indicates a direct and antagonistic role of cellular senescence in establishing tissue tension. Aside of the tensile state of the resulting ECM, cellular senescence also affected the biochemical composition via differential regulation of ECM proteins, e.g. collagen, fibronectin, decorin, and tenascin C. Furthermore, we found evidence that these macroscopic perturbations might be influenced by an altered mechanotransduction of senescent cells already on the single cell level (2D). The expression of integrins was altered correlating with an enlarged cell morphology and higher abundance of large focal adhesions particularly in cells that were driven into senescence by selective over-expression (p16/p21). How these two observations are linked remains so far unknown.

Based on these preliminary data, we propose a novel research question which we believe to be relevant and of high interest for a PhD project. Based on the understanding that senescent cells seem to strongly alter the extracellular niche we want to better understand the relevance of this finding for the in vivo tissue regeneration process. While the in vitro experiments focused on the behavior of a homogeneous population, senescent cells in vivo are found in relatively low abundance. We believe that senescent cells nevertheless impact the regeneration process and hypothesize that the ECM formed by senescent cells exhibits specific cell-instructive cues which influence cellular processes, e.g. migration, survival but also differentiation of surrounding stromal cells. A distinct matrix phenotype thereby might contribute to the impact senescent cells exert during tissue regeneration aside of their secretory phenotype. The PhD student will optimize a recently established procedure for decellularization in order to obtain cell-free ECM generated by senescent cells. The matrix will be used as a template for studying the cellular response both of fibroblasts and MSCs. Differences in ECM-dependent cellular behavior would support the hypothesis that an ECM created by senescent cells affects behavior of surrounding stromal cells. The student will further focus on the consequences of senescence-modulated cellular mechano-sensation on tissue formation. Starting from a basic description of cellular mechano-sensation, cellular organization will be studied on simplified geometries to describe differences in multi-scale cell organization (Werner et al. 2016). Finally collective cell organization, which leads to ECM formation, will be studied in more detail inside the established scaffold-based 3D tissue model to further understand the impact of senescent cells on early tissue regeneration.

Together, the results will contribute to a principle understanding of (i) how the resulting matrix properties steer the behavior of non-senescent through a distinct cell-instructive matrix code and (ii) how senescent cells sense and respond to their environment with regard of cell organization and tissue formation.

Team: Erik Brauer, Ansgar Petersen, Sven Geißler, Mina Sohrabi-Zadeh

Funding: ECRT Research Grant

Differentiation of induced pluripotent stem cells (iPSCs) into specific T cell subtypes using epigenetic editing

Immuno-suppressive CD4+ regulatory T cells (Tregs) are the main natural preventer of auto-immune diseases and chronic inflammation and are known to support tissue regeneration after injury and immuno-pathology. Therefore, Tregs are currently being extensively studied as "living drugs" in adoptive cellular therapy against pathogenic inflammation and to foster tissue regeneration.

Classical approaches of adoptive Treg therapy, which are based on the extensive in vitro expansion of pre-existing Treg populations, are currently being hampered by several obstacles:

1) the acquisition of proliferation-induced cellular senescence during in vitro expansion resulting in limited survival;

2) a functional instability of Tregs during inflammatory conditions;

3) the absence of pre-existing functional Treg populations in certain auto-immune disease patients and

4) a lack of pre-existing Treg populations displaying the required antigen-specificity or migration capacity, as the antigen-specific T cells in auto-immune patients have acquired a (pathogenic) pro-inflammatory phenotype.

Our group is trying to address these limitations from a new molecular angle: from epigenetics. We have identified several critical epigenetic control elements ('Epi-stabilizers') in T cells which are involved in maintaining a given T cell phenotype (Durek et al, Immunity 2016). The best characterized one is the so-called 'Treg-specific demethylated region – TSDR' in the FOXP3 gene, which is determining Treg function and cellular identity (Huehn et al, Nat Rev Immunol., 2009). To date, the demethylated state of the TSDR is the most reliable biomarker for human Tregs.

Previous work in the group established a CRISPR/Cas9 based system of 'epigenetic editing' allowing the targeted switching of methylation states at Epi-stabilizer elements. With this, Epi-stabilizers can be switched on (=demethylation) and off (=methylation) at will. While this system was successful to switch DNA methylation states and with that, regulate expression of the associated gene, the functional phenotype of the T cells was not completely switched probably due to the remaining pre-imprinted T cell epigenetic landscape.

This is why I now suggest to take a small detour in the functional re-programming of T cell phenotypes and include a re-programming step towards induced pluripotent stem cells (iPSCs). During this, the original T cells can be rejuvenated (addresses obstacle 1 of current Treg therapy, above), while the original TCR can be maintained (obstacle 4). This approach would allow Treg therapy even in Treg-deficient patients (obstacle 3). The resulting iPSCs can be grown in virtually unlimited numbers without senescence acquisition (obstacle 1), are easy to manipulate by epi-/genetic editing and can be stored for repetitive transfusions if needed (obstacle 1). In addition, iPSCs can also be generated from other cellular sources but still be re-differentiated into T cells. The epigenetic editing will be introduced during the T cell differentiation step from iPSC-derived hematopoietic precursor cells, which mimics the normal T cell development in the thymus (7) and thus, is a promising approach to induce stable functional T cell subsets (obstacle 2).

With this project, we expect to establish a system, with which large numbers of storable, fully functional T cell populations can be generated displaying defined advantageous characteristics (e.g. functional stabilization, re-juvenation, selected TCR-specificity). This system can be extended to induce features like a defined migration behavior (obstacle 4) or cytokine expression profile, since Epi-stabilizers in homing receptor (Szilagyi et al., Mucosal Immunol., 2017; Pink et al., J Immunol., 2016) and cytokine genes (Lee et al., Immunity, 2006) have also been identified. With this, we assume to break down several road blocks on the way towards a successful clinical application of adoptive T cell therapies.

Team: Christopher Kressler, Julia Polansky-Biskup, Harald Stachelscheid, Marcel Finke

Funding: ECRT Research Grant

Organoid disease models for spinal muscular atrophy

Team: Angélica Garciá Pérez, Mina Gouti, Markus Schülke, Lan Vi Nguyen

Funding: ECRT Research Grant

Role of human extracellular matrix properties in central nervous system regeneration

Oligodendrocytes constitute one of the four principal central nervous system (CNS) cell types - next to neurons, astrocytes and microglia. Oligodendrocytes form myelin sheaths around neuronal axons, ensuring efficient signal conduction in these axons. In Multiple Sclerosis (MS), a chronic immune-mediated demyelinating disease of unknown etiology, loss of myelin appears during both an initial relapsing-remitting phase and a subsequent secondary-progressive phase. The loss of myelin is associated with clinical disability, including pain, paralysis, vision loss and cognitive incline. Conversely, the generation of new myelinating oligodendrocytes and the repair of myelin, called oligodendrogenesis and remyelination respectively, are prerequisites for functional recovery. The pathological hallmark of MS is the presence of focal demyelinated lesions with partial axonal preservation and reactive astrogliosis. Focal demyelinated lesions can be partly or completely repaired by spontaneous remyelination. However, these regenerative processes are efficient only in a small subset of MS patients. Thus, for the development of highly effective remyelinating therapies it is particularly important to identify the factors that are suppressive or permissive of myelin regeneration.

While the determinants of lesion progression versus lesion repair in MS are still completely unknown, evidence points to the involvement of microenvironmental factors, including biomechanical and compositional extracellular matrix (ECM) properties. While past experimental approaches were often based on animal models, and have lead to the identification of a a few of individual ECM components as regulators of myelin repair. However, a fast growing body of conflicting data on the functional role of such singular components points to the necessity of a more holistic and human approach towards ECM-driven myelin regeneration:

In this project, we aim at identifying the correlative and causal relations between EMC mechanical as well as structural properties, ECM composition and the regenerative potential of human demyelinating and remyelinting MS lesions. Neuropathologically characterized human MS lesion tissue will be divided and subjected to 1) testing of biomechanical properties using microindentation together with structural characterization using label-free two-photon autofluorescence and second harmonic imaging and 2) decellularization of the ECM. The latter will allow us to study the lesion’s ECM as a whole without interference of cellular components. Isolated ECM will be again characterized for its biomechanical and structural properties and used as matrix for human myelinating stem cell cultures for the study of ECMmediated myelination efficiency. Stem cell fate and functional myelination studies will be performed in a microscopy based setting. These experiments will, for the first time, allow us to connect the biomechanical and structural properties of individual MS lesions and lesion-derived ECM with the efficiency of functional myelination in an ex vivo setting.

While we have broad insight into the promyelinating and myelin-supressive signaling effects of singular brain matrix components, nothing is known about the effect of the lesion ECM as a whole. Thus, in a next step, we aim at identifying the lesion type-specific ECM-mediated signaling pattern. Experimentally, we will look at phosphorylation patterns of multiple pro-oligodendrogenic and anti-myelinating signaling pathways on a single cell basis using high-contenct microscopy and advanced image analysis. Immunohistochemical and proteomic analysis of the individual decellularized lesion ECM will further enable us to characterize the different ECM components that underlie the individual biomechanical and regenerative properties. Lastly, through in vitro-mimicry studies, we aim at identifying which mechanical and biochemical properties may be used to functionally overcome brain ECM-associate inhibition of remyelination.

Taken together, through the integration of knowledge and methods from the fields of biomechanical engineering, biochemistry and regenerative cell biology, this project aims at characterizing key regulating brain ECM properties and identifying functional cellular regenerative mechanisms.

Team: Sarah-Christin Staroßom, Ansgar Petersen, Harald Stachelscheid, Erik Brauer, Juliana Campo Garcia, Roemel Jeusep Bueno

Funding: ECRT Research Garnt

ECRT Research Grants - Förderpeiode 06/2019-06/2022

Expansion of SpCas9-specific regulatory T cells as an approach to prevent hazardous inflammatory damage to CRISPR/Cas9-edited tissues

The field of gene therapy has been galvanized by the discovery of the highly efficient sitespecific nuclease system CRISPR/Cas9 from bacteria. Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues. Most applications aim to express the Cas9 nuclease in or deliver the protein directly into the target cell. Hence, intracellular protein degradation processes lead to presentation of Cas9 fragments on the cellular surface of gene-edited cells that can be recognized by T cells. Recently, we found a ubiquitous memory/effector T cell response directed toward the most popular Cas9 homolog from S. pyogenes (SpCas9) within healthy human subjects (Nature Medicine 2019). Intriguingly, SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9-directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with the magnitude of the respective effector T cell (TEFF) response indicating that SpCas9-reactive TREG cells may control respective TEFF responses. Consequently, SpCas9-specific TREG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response in vivo, especially in patients with low SpCas9-specific TREG/TEFF ratio. Therefore, we aim to test the enrichment and in vitro expansion of SpCas9-specific TREG ultimately aiming at their use in an adoptive immunotherapeutic approach.

Team: Michael Schmück-Henneresse, Sybille Landwehr-Kenzel, Petra Reinke, Hans-Dieter Volk, Dimitrios L. Wagner, Lena Peter

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant

Immunosuppressant-resistant anti-viral T cells for advanced adoptive T cell therapy

Due to the immunosuppressive therapy required for prevention of organ rejection after transplantation, some patients suffer from severe complications due to normally harmless chronic viral infections. Mostly potent anti-viral drugs can manage complications, however many of these bare toxicities, some patients are irresponsive or resistances can be developed. Thus, specific regeneration of the endogenous anti-viral immune response, without risking organ rejection by alloreactive T cells, by the means of adoptive anti-viral T cell therapy is an attractive alternative treatment. In some cases, our conventional approach of T cell therapy can only control the virus temporally, probably due to malfunctions of transferred T cells caused by the immunosuppressive therapy. Hence, we plan to generate T cells which are resistant to immunosuppressive treatment with widely used Tacrolimus for adoptive transfer. Tacrolimusresistant T cells shall be generated by knock out (k.o.) of the adapter protein FKBP12, which is required for the immunosuppressive function of Tacrolimus. The k.o. shall be achieved by a vector-free transfer of nucleoprotein complexes of the nuclease CRISPR associated protein 9 (Cas9) with a site-specific guide RNA by electroporation into virus-specific T cells. We plan to confirm specificity of the k.o. by sequencing of possible off target areas. Ultimately, we want to integrate resistance introduction into our GMP-conform protocol to facilitate clinical translation.

Team: Leila Amini, Dimitrios L. Wagner, Uta Rössler, Michael Schmück-Henneresse, Petra Reinke, Uwe Kornak, Daniel Kaiser, Andy Römhild, Ghazaleh Zarrinrad

Funding: Einstein Kickbox - Young Scientists & ECRT Research Grant

Serial high-resolution 3D-immunofluorescence bone imaging in patients undergoing hematopoietic stem cell transplantation

Little is known about the micro- and macro-structural changes in bone and their consequences on immune reconstitution in patients after hematopoietic stem cell transplantation (HSCT). We will study patients undergoing autologous (lymphoma/myeloma) and allogeneic (AML) HSCT. Both patient cohorts are treated with high-dose chemotherapy and are challenged with steroids, either before autologous HSCT as part of the standard treatment (lymphoma/ myeloma) or after allogeneic HSCT (AML) in case of graft-versus-host disease (GVHD). We have recently established high-resolution 3D-immunofluorescence bone imaging in experimental animal models. However, animal models of human tumors have important limitations and may not adequately reflect the clinical situation. Having immediate access to biopsies of HSCT patients, we are planning to apply the new imaging technique to bone biopsies from patients undergoing HSCT and to compare them to normal bone biopsies (e.g. from patients with malignant lymphoma at initial diagnosis and without BM manifestation). We will specifically investigate the bone micro-structure as well as the bone marrow niche for the physiological hematopoiesis and immune cells. In parallel, we will collect clinical data, such as cytopenia, chimerism, persistence of malignant cells, GVHD and infections. We will correlate the results of high-resolution 3D-immunofluorescence bone imaging with clinical characteristics, in order to identify clinically relevant structural changes in the bones.

Team: Il-Kang Na, Olaf Penack, Katarina Riesner, Sarah Mertlitz, Georg Duda, Katharina Schmidt-Bleek, Radost Anika Saß

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant

Tendon healing, scar formation or chronic inflammation – a matter of miscommunication between tenocytes and immune cells?

Tendon disorders can have diverse etiologies such as acute or chronic mechanical overload or enthesial autoimmunity & inflammation. Interestingly, although the causes are different the clinical outcome shows overlapping features with tendon degeneration and fibrosis. For autoimmune-mediated tendon disorders such as spondyloarthritis (SpA) the contribution of immune cell infiltration and activation is well accepted. For tendinopathies, due to chronic mechanical overload and tendon remodelling, infiltration of immune cells has been observed. Although the exact and especially individual pathology and the role of immune cells is far from being fully understood. With overlapping outcomes, it seems obvious that miscommunications between tenocytes and immune cells are shared pathomechanisms of these disorders. With the kick-box seed money we will start a comparative in depth analysis of immune cell composition and distribution applying multiplex immune histology. This will be continued within the three-year PhD project. Furthermore, we will dissect tenocyte & immune cell communications operational during healing upon acute injury as well as failed tendon regeneration in case of tendinopathy and enthesitis applying single cell RNA-seq. In future, elucidated signaling pathways and potential triggers driving tendon healing or degeneration will be investigated in tenocyte-immune cell co-cultures incorporating mechanical stimulation with the goal to identify novel therapeutic candidates.

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

Funding: Einstein Kickbox - Advanced Scientists & ECRT Research Grant