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ECRT Projekte

Das ECRT will insbesondere den wissenschaftlichen Nachwuchs beim Aufbau eigener Forschungsvorhaben unterstützen. Dafür bietet das 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 Englisch zur Verfügung.

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Laufende ECRT geförderte Projekte

Einstein Kickbox - Förderperiode 2023

Biomechanical potential of advanced osteotomy using CARLO® laser-cutting device for mandibular reconstruction

Extensive plating designs can lead to bone resorption in mandibular reconstruction with fibula free flap1–3. Therefore, self-stabilization between the bones could enhance bone healing due to the potential of plating material reduction. This self-stabilization could be achieved using the tongue and groove method. However, the current manual bone-cutting device widely used in the maxillofacial field, PIEZOSURGERY®, is limited to straight osteotomy lines. A more precise cutting and exact osteotomy customization can be achieved using the new Cold Ablation Robot‐guided Laser Osteotome (CARLO®). CARLO® includes a laser head and a robotic arm to perform precise osteotomies in mandibular reconstruction, but with unlimited custom geometries and low cutting temperatures, to preserve bone tissue structure and prevent device-related injuries. Finite element models have been previously established on the reconstructed mandible4.

The aim of this study is to investigate the biomechanical conditions induced with a tongue and groove approach in mandibular reconstruction. Using a previously established finite element model4 of the fibula-reconstructed mandible, the biomechanical conditions within the healing regions of a reconstructed mandible will be evaluated for different tongue-groove osteotomies feasible with PIEZOSURGERY® and CARLO®. Thereafter, the potential to reduce the plating material in the tongue-groove approach will be investigated.

References

1.    Rendenbach C, et al. International Journal of Oral and Maxillofacial Surgery. 2019;48:1156-1162

2.    Knitschke M, et al. Cancers. 2022;14

3.    Knitschke M, et al. Current oncology (Toronto, Ont.). 2022;29:3375-3392

4.    Ruf P, et al. Frontiers in bioengineering and biotechnology. 2022;10

Team: Philipp Ruf

Funding: Einstein Kickbox - Young Scientists

Defective mucosal barrier integrity links periodontitis to rheumatoid arthritis

Periodontal disease (PD) is the most common chronic inflammatory disease of the oral cavity, affecting up to 47% of the population worldwide. PD leads to the destruction of the tooth supporting tissue and has systemic implications coupled with an increased risk of rheumatoid arthritis (RA). Both PD and RA display systemic markers of inflammation and chronic inflammatory pathways. Moreover, RA patients with untreated PD show increased disease activity and seem more likely to develop a treat-refractory RA. However, the mechanisms by which PD affects the pathogenesis of RA and vice versa remain elusive.

Recent data show that gingival fibroblasts (GFs) are essential for the maintenance and repair of periodontal tissue and play an active role in host defense mechanisms. On the other hand, innate lymphoid cells (ILCs) are involved in tissue repair and immune homeostasis at barrier surfaces and are critical players in the first line of immune host defense against pathogens. We hypothesize that ILCs and GFs are key players in oral inflammatory diseases such as PD, and their dysfunction may lead to disruption of oral mucosal integrity contributing to the pathogenesis of PD and RA, respectively.

Therefore, this project aims to shed light on the key immune mechanisms underlying PD by focusing on ILCs - GFs interactions and their putative role in immune activation and disease progression, leading to barrier dysfunction.

Team: Alexandra Damerau, Aysegül Adam

Funding: Einstein Kickbox - Young Scientists

Evaluation of electrical current effect on implant-associated biofilm infections alone and in combination with antibiotics

Biofilms are involved in about two-third of all human infections causing challenging-to-treat infections since biofilms are considerably less susceptible to antimicrobials than planktonic counterparts. Novel and innovative therapeutic approaches are needed to improve the outcome of implant-associated infections, if possible, without implant removal (current common cure of infection). 

Application of electricity to the conductive implant is an attractive option, as electrolytic procedure has shown a strong anti-biofilm effect. Electricity could be applied during the surgery to avoid early infection and contamination of newly implanted foreign bodies. Moreover, implantable devices might be used to generate effective electric fields, which would improve the perioperative administration of antimicrobials to eliminate growing bacterial biofilms and prevent device-related infections. It is hoped that the “bioelectric effect” be effective in the control of biofilm infections on indwelling medical devices. The main goal of this study is to evaluate the efficacy of electricity against multidrug-resistant bacterial biofilms alone and in combination with different antibiotics. 

We aim to develop an electricity-based approach that, when combined with antibiotics, can significantly outperform antibiotic therapy against biofilms and lead to a superior treatment outcome in difficult-to-treat implant-associated infections. This could streamline surgical procedures, minimize the number of revisions, and shorten the time patients spend in the hospital, lowering hospitalization costs.  

Team: Rima Pirlar, Maximilian Fenko

Funding: Einstein Kickbox - Young Scientists

Improving production of single-stranded DNA templates for homology-directed integration using phagemid vectors

Non-viral gene editing allows the insertion of transgenes in defined locations of the genome, which can be used to engineer potent cellular therapies. Currently, the most commonly used template format to induce homology-directed repair (HDR) after nuclease-induced DNA breaks is double-stranded DNA (dsDNA). However, electroporation of high doses of dsDNA is toxic to many cell types. To address this issue, we developed a platform for reprogramming of T cells with chimeric antigen receptors using the CRISPR-Cas12a system and modified ssDNA templates. Comparing different non-viral DNA templates, we found that modified ssDNA consistently outperformed other conditions in both knock-in efficiency and cell viability, regardless of the targeted locus. However, there are challenges that come with the production of linear ssDNA, such as yield and size limit. As an alternative, we propose exploring circular ssDNA (cssDNA) produced from phagemids as a template for HDR-mediated integration of DNA. Our study involves designing phagemids encoding therapeutically relevant genes for generation of cssDNA and assessing efficacy and toxicity of these constructs in different cell types, including primary T cells and induced pluripotent stem cells, utilizing the CRISPR-Cas12a system. The scalability and cost-effectiveness of producing phage-derived cssDNA would streamline the generation of high yields and large HDR templates, presenting a valuable asset to advance ssDNA-based gene editing.

Team: Ana Nitulescu, Viktor Glaser, Rima Pirlar

Funding: Einstein Kickbox - Young Scientists

Investigating the Influence of Receptor Affinity on T Cell Activation in Response to Varying Antigen Density

Genetic engineering has revolutionized T cell therapy with designed antigen receptors, as seen in CAR T cell success. However, the impact of receptor affinity on CAR T cell activation against varying antigen levels is poorly understood. This study addresses this gap by assessing engineered T cells' performance with different affinities for a solid tumor antigen. Various receptor architectures, including classical CARs and novel designs like STAR and eTruC, will be compared. Synthetic receptor architectures will be generated by in-frame fusion of exogenously delivered binder-encoding transgenes into endogenous TCR-complex genes. Serial co-cultures, live imaging, and cytokine analysis will reveal immediate and long-term cytotoxicity, proliferation, and phenotype changes. This research dissects the intricate link between receptor affinity, expression, and target density in engineered T cells, promising insights for solid tumor immunotherapies.

Team: Jonas Kath, Lena Andersch, Vanessa Drosdek

Funding: Einstein Kickbox - Young Scientists

Be-aware of the metabolism - Characterizing metabolic changes in anti-viral T cells with different specificities for successful adoptive T cell therapy in transplantation

Solid organ transplantation (SOT) is the ultimate savior for end-stage organ failure. However, viral complications are frequent, due to decreased T cell surveillance under immunosuppression. Adoptive anti-viral T-cell therapy represents a promising treatment option. While confirming safety and efficacy of preclinical virus-specific T-cell products (TCPs) during manufacturing, the functional metabolic state of TCPs was not considered in depth.  

In our project, we would like to emphasize the importance of metabolic investigation of anti-viral TCPs for adoptive T cell therapy in the transplant setting. Inclusion of metabolic characterization as read-out parameter in manufacturing may lead to enhanced product safety, efficacy and long-term survival in the patient. To investigate this, we plan to analyze the metabolic patterns of four different anti-viral TCPs using the Seahorse XF Mito and Glycolysis test to gain insights into the mitochondrial respiration as well as glucose metabolism. Anti-viral TCPs with the specificities cytomegalovirus virus (CMV), influenza-A-virus (IAV), Epstein-Barr-virus (EBV) and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will undergo Seahorse XF analysis yielding first proof-of-concept data.

We will be able to gain novel insights into an up to now scarcely studied field and envision to integrate the evaluation of metabolic features in future preclinical testing and translation towards the clinic.

Team: Lisa Burkhardt, Michael Ristow, Michael Schmück-Henneresse, Leila Amini, Caroline Frädrich, Kim Zarse

Funding: Einstein Kickbox - Advanced Scientists

Development of a biocompatible, stable coating to improve thromboresistance of recellularized tissue engineered vascularized organs

Type 1 diabetes mellitus results in the destruction of insulin-secreting β-cells. Islet transplantation is a minimally invasive alternative to subcutaneous insulin and pancreas transplantation, but intraportal infusion has several limitations. To address these challenges, our group has developed a functional endocrine "neo-pancreas", which involves repurposing the decellularized rat liver by recellularizing the vascular structures with endothelial cells, and the parenchymal compartment with islets of Langerhans. To ensure thromboresistance of the vascular tree and to avoid organ failure due to thrombosis upon implantation the time between ex vivoendothelialization and completion of in vivo reendothelialization must be bridged. The goal of this project is to develop a functional coating for the lining of the vascular tree in the endocrine neo-pancreas and evaluate its performance. The project consists of two work packages: 1) establishment of the optimal coating(s) in a decellularized rat abdominal aorta chip model in vitro and 2) coating the endocrine neo-pancreas and testing its functionality in an ex vivo whole blood perfusion system. This project holds importance as it seeks to generate findings applicable not only to tissue-engineered whole organs but also to tissue-engineered vascular grafts. By addressing these critical areas, the project has the potential to advance multiple fields within tissue engineering and contribute to improved biomedical solutions.

Team: Eriselda Keshi, Igor Sauer, Marie Weinhart, Karl Herbert Hillebrandt

Funding: Einstein Kickbox - Advanced Scientists

Immunomodulatory features of tubular epithelial cells after kidney transplantation

Kidney transplantation is an effective treatment for end-stage renal disease, but the short- and long-term success of the transplantation depends on the recipient's immune response to the transplanted kidney. Tubular epithelial cells play a key role in this immune response to the transplanted kidney. Ischemia and reperfusion injury following hypoxia during the transplantation procedure, as well as early immune responses and infections after transplantation cause an inflammatory milieu in the transplanted kidney. This shifts the tubular epithelial cells into a pro-inflammatory and immunogenic state, therefore itself promoting alloimmune responses. Thus, we aim to characterize the expression of inflammatory and anti-inflammatory, co-stimulating and co-inhibiting molecules by kidney tubular epithelial cells in vitro in response to inflammatory stimuli and hypoxia. Therefore, we will cultivate tubular epithelial cells from the urine of healthy probands and kidney transplant patients and stimulate them with various pro-inflammatory cytokines and under hypoxic conditions followed by flow cytometric analysis of changes in surface protein expression and secreted molecules with the potential to modulate immune responses. The results of this study may contribute to deepen our understanding of the role of tubular epithelial cells in alloimmunity and to the development of new strategies for preventing immune rejection and improving the success of kidney transplantation.

Team: Constantin Thieme, Hanieh Moradian, Nina Babel, Manfred Gossen, Julian Autrata

Funding: Einstein Kickbox - Advanced Scientists

Somatic Mutations as a Novel Cause of Autoinflammation

In our project we aim to identify novel causes of autoinflammatory diseases. We will analyze blood samples from patients with symptoms of autoinflammation (fever spells, arthritis, rash, chondritis) and perform whole exome sequencing to identify somatic mutations of leukocytes in the peripheral blood. Using bioinformatic tools, we will identify the mutations that are most likely to be associated with the identified phenotype and validate them using myeloid cell lines.

Team: Lennard Ostendorf, Robin Kempkens, Dimitrios Laurin Wagner, Dominik Seelow, Frederik Damm

Funding: Einstein Kickbox - Advanced Scientists

T cell-derived hiPSCs - towards an unlimited source for therapeutic regulatory T cells

Team: Christopher Kreßler, Marcel Finke, Julia Polansky, Harald Stachelscheid

Funding: Einstein Kickbox - Advanced Scientists

Regenerative effects of CD31+ cells isolated from peripheral blood on osteoarthritic chondrocytes

Osteoarthritis (OA) is the most common degenerative joint disease worldwide. Despite the increasing pathophysiological understanding of this disease, current therapy options are still limited. Recently CD31+ cells isolated from peripheral blood cells (CD31+ (PBC)) have been demonstrated to contribute to bone regeneration through a combination of immunomodulation and anti-inflammation both in vitro and in a rat model of impaired bone healing. However, to date, studies exploring the role of CD31+ (PBC) cells may play in the development of OA is still missing.

Thus, the aim of this project is to investigate the anti-inflammatory and immunomodulatory effects of CD31+ (PBC) cells on OA. To isolate CD31+ (PBC) cells and primary osteoarthritic articular chondrocytes (ACs), peripheral blood and osteoarthritic knee cartilage are collected from the same patients with end-stage knee OA undergoing knee arthroplasty. CD31+ (PBC) cells are characterized using flow cytometry, and the cell composition is analyzed. CD31+ (PBC) cells and ACs at passage 0 from a single patient are cocultured in monolayer and in pellets. Besides, the monolayer and pellet culture of ACs are also used as controls. Samples are collected at different time points for further analyses.

Our study will provide a new perspective to understand the regenerative effects of CD31+ (PBC) cells, easily obtained from OA patients, on cartilage and chondrocytes, which may be beneficial for therapeutic intervention in OA patients.

Team: Sijia Zhou, Luis Lauterbach, Tazio Maleitzke, Yi Ren

Funding: Einstein Kickbox - Young Scientists

From rapid diagnostics of rare monogenic diseases to therapeutic strategies – SGMS1-deficiency as a proof of concept

By the implementation of “next generation sequencing” (NGS) in the clinical routine, the diagnostic yield of rare genetic diseases has drastically improved. After the progress in the diagnosis of rare diseases, the development of personalized therapies for affected patients will be the next important step.

Via Trio Exome sequencing we identified compound heterozygous missense variants in the gene SGMS1 in a patient with molecularly unsolved neurodevelopmental disorder. SGMS1 encodes the enzyme spingomyelinsynthase 1, which synthesizes sphingomyelin from ceramide within the sphingolipid metabolism. So far, pathogenic variants in the SGMS1 gene have not been published as causative for a Mendelian disease. However, deficiencies in the other enzymes of the same biochemical pathway are well-studied, such as Niemann-Pick disease, Morbus Fabry or Morbus Gaucher. For these sphingolipidoses, different therapeutic strategies (e.g. enzyme replacement, gene therapy) have already been approved.

With the support of the Einstein funding, we aim to characterize the cellular phenotype in patient derived fibroblasts. Furthermore, we plan to generate knock-in HeLa cell lines via base editing to validate SGMS1 as the disease-causing gene and to establish a cellular disease model. This will be the basis to test different therapeutic strategies in the future.

Team: Johannes Kopp, Hristiana Lyubenova, Viktor Glaser, Felix Boschann, Dimitrios Laurin Wagner, Björn Fischer-Zirnsak

Funding: Einstein Kickbox - Advanced Scientists

Human cell lineage tracing in atherosclerosis using mtscATACseq

Cardiovascular disease is the leading cause of death world-wide and account for approximately 32 % of all global deaths. Of those, 85 % are due to heart attack and stroke with atherosclerosis as the underlying cause. In atherosclerosis, lipids accumulate at inflammatory sites in the vessel walls, followed by invasion of immune cells and other cell types, which leads to the buildup of a so-called plaque. Depending on their stability, these plaques may rupture and obstruct vessels, leading to myocardial infarction, stroke, or thrombosis. It has been shown that different cellular trans-differentiation processes are essential drivers of plaque formation. However, these assumptions are mainly based on indirect approaches (lineage tracing via pseudotime-analysis, lineage-tracking in mice). Thus, the need for a translational approach analysing cellular plasticity (i.e. trans-differentiation processes) and clonality in human plaque material is imminent. We now aim to use mitochondrial single-cell ATAC sequencing, a new sequencing method, which allows to directly infer clonal relationships of cell populations by analysing naturally occurring mutations in mitochondrial DNA. We want to apply this to analyse plaque material and corresponding blood samples from carotid endarterectomy operations at the Charité Berlin, comparing patients who are asymptomatic (i.e., those who did not suffer from heart attack or stroke) against those who are symptomatic. We aim to characterize the cellular composition of plaques and immune cell populations in the circulation and infer how cells are clonally related. We will use gene loci accessibility, motif enrichment and transcription factor footprinting analysis to resolve which genes and transcription factors may drive cellular plasticity in human atherosclerotic plaques.
 

Team: Paul-Lennard Mendez, Jan Frese, Leif Ludwig, Petra Knaus

Funding: Einstein Kickbox - Advanced Scientists

Investigation of the CIDEB genetic variants and their role in preventing PNPLA3 I148M-triggered non-alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is the leading cause of end-stage liver disease in developed countries, and its prevalence is projected to increase over the next decade. NAFLD is characterized by hepatic steatosis, with heritability playing a significant role in disease occurrence and progression. The PNPLA3-I148M variant has been described as one of the strongest genetic risk factors for NAFLD. We and others found that this variant alters lipid metabolism, leading to a significant increase in lipid accumulation in hepatocytes. Newly discovered rare genetic mutations in a cysteine-type endopeptidase, have been proposed to exert a protective role for NAFLD, particularly for PNPLA3 I148M carriers. However, the mechanisms have not yet been investigated. This project aims to elucidate the expression patterns of the endopeptidase in different disease stages of NAFLD. The protein’s function on lipid droplet formation and lipid metabolism will be investigated using live cell imaging and metabolomic analysis. Overall, this project will just be the first step in unraveling the protective mechanism against NAFLD associated with mutations in the cysteine-type endopeptidase. 

Team: Nils Haep, Igor Sauer, Nikolaus Berndt, Anja Selke-Reutzel

Funding: Einstein Kickbox - Advanced Scientists

Understanding the chemistry-dependent immune signature of synthetic mRNAs in engineered cells: a deep dive into translatome by ribosome profiling

Clinical use of synthetic messenger RNA as a therapeutic entity was broadly accepted upon successful performance of mRNA vaccines against SARS-Cov-2 virus. This momentum, however, was delayed for two decades due to the potential immunogenicity of exogenous mRNA. Depending on the intended therapeutic application, however, the immunostimulatory effect can be beneficial, e.g. as adjuvant in cancer vaccines, or problematic in case of protein replacement therapies. Incorporation of chemically modified nucleotides, optimization of nucleotides sequence, extra chromatography-based purification of transcripts from dsRNA by-products are main approaches, which were reported to successfully mitigate the immunogenicity of synthetic mRNA. In this project, we aim to identify molecular mechanisms behind immune stimulation cause by mRNA depending on corresponding chemical modification scheme by studying cells translatome using ribosome profiling assay (Ribo-seq). Compared to conventional RNA-seq methods, Ribo-seq measures mRNAs which are actively participating in translation. Thus it provides information about dynamic process of gene expression instead of cells steady-state transcriptome. Moreover, by quantifying the correlation between immune-response to expression for individual chemistries, we can unravel (perhaps misstated) adverse influence of immune-response on transgene expression. The result of this pilot study can pave the way toward rational selection nucleotide modification of IVT-mRNA, best fitting the particular demands defined by clinical application.

Team: Hanieh Moradian, Manfred Gossen, Hans-Dieter Volk, Toralf Roch

Funding: Einstein Kickbox - Advanced Scientists

ECRT Consumable Grants - Förderperiode 2023

Regeneration of Motor Function after Stroke via JAK/STAT3 Pathway

In spite of state-of-the-art medical care provided in contemporary hospitals, motor recovery of stroke patients is often incomplete. To this end, basic research in animal models has shown that substantial degrees of spontaneous recovery after cortical stroke rely on the rewiring of spinal circuits, which attract new synaptic innervation from multiple supraspinal brain areas including serotonergic nuclei in the brainstem. Our Kickbox project within the Einstein Center for Regenerative Therapies aimed to further stimulate the rewiring of corticospinal serotonergic neurons in mice by amplifying their post-stroke growth capacity through the use of contemporary viral tracing tools. Here, we further seek to selectively activate the JAK/STAT3 signalling pathway, a major biochemical pathway involved in cellular processes such as proliferation, differentiation, migration and growth. In parallel, we want to document the effects of enhanced corticospinal sprouting on motor performance of the mice through extensive recovery testing across multiple movement domains. In case we are able to demonstrate a positive effect of the upregulation of targeted fibers on motor recovery, we would achieve far-reaching results that could one day become essential to improving the motor ability and quality of life of stroke patients.

Team: Matej Skrobot, Rafael De Sa, Remmora Gomaid

Funding: ECRT Consumabel Grant - Young Scientists

ECRT Career Kickoff - Förderperiode 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

Characterization of a novel human long FOXP3 mRNA isoform

The transcription factor FOXP3 is the master regulator for immuno-suppressive regulatory T cells (TREG), a population of critical importance to maintain a well-balanced immune system. Recently independent groups have reported transcriptional activity upstream of the annotated transcriptional start site of the FOXP3 gene in naturally-occurring TREG, both in mice and human. However, there is little agreement in terms of structure and function of the novel transcript.

Taking advantage of direct RNA long-read sequencing (Nanopore technology), we addressed what polyAdenylated transcripts originated from the FOXP3 locus in in vitro-expanded human blood-derived TREG. Among other unreported transcripts, we identified a novel FOXP3 mRNA isoform with an extended 5`-UTR ('longFOXP3'). We believe that longFOXP3 might be functional, either a) giving rise to a FOXP3 proteoform with an N-terminus extension or b) translating into peptides coded by open reading frames found in the extended 5`-UTR, or c) translating into the canonical FOXP3 protein only under specific conditions, or d)  by a constitutive low-level translation, representing a long-undecoded transcript isoform.

Taken together our data reveals the existence of a novel human long FOXP3 mRNA isoform raising the possibility of further layers of regulation within the complex gene expression circuitry that operates during the development and function of human nTREG.

Team: Marcos Cases, Julia Polansky

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

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

Targeting the CXCR3-chemokine system to unleash T cells against solid cancer

T cell therapy for solid tumors is one of the most promising live drugs in precision medicine. However, the
main obstacle is the selective delivery into the tumor microenvironment and the targeted efficacy. Thus,
T cells must be equipped with the ability to migrate and act specifically in malignant tissues. Early differentiated
memory T cells constitute ideal candidates as they exhibit high proliferative and migratory capacities. The
increased migration potential is attributable to the abundant expression of CXCR3, a receptor critical for
infection-induced migration. Since this chemokine system has the dual ability to guide both cell migration and
activation depending on the engaging ligand, we intend to investigate the underlying signaling pathways and
regulatory mechanisms to ultimately harness its therapeutic potential. To this end, we aim at a comprehensive
characterization of the CXCR3 chemokine system and examine signaling patterns upon stimulation with the
CXCR3 ligands (CXCL9/10/11) in primary human T-cells compared to CXCR3-deficient T cells. Our data thus far
suggest that CXCR3 is involved not only in cell migration but also in T cell survival by triggering JAK-STAT
signaling and colocalizing with the T cell receptor after activation. To ultimately impact CXCR3 variant
expression in therapeutic T cells, we plan to discover, initiate, and test regulatory factors. In the upcoming
year, we aim to evaluate their ability to improve anti-tumor potency of current approaches with preclinical
models developed in our group. We are given the chance to characterize clinical T cell products targeting brain
tumors and thus to correlate our measures on the CXCR3 chemokine system to patient’s therapy outcome.
These final explorations will contribute substantively to the understanding of the CXCR3 system to harness its
potential for adoptive T-cell therapies, and effectively leverage it in the context of solid tumors.

Team: Sarah Schulenberg, Michael Schmück-Henneresse

Funding: ECRT Career Kickoff - PhD

ECRT Career Kickoff - Förderperiode 2022-2023

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

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