ECRT Projects

Extensive plating designs can lead to bone resorption in mandibular reconstruction with fibula free flap^1–3. 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

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

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

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

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

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

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

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

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

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

Funding: Einstein Kickbox - Advanced Scientists

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

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

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

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

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

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