Research

Osteoarthritis (OA) represents a considerable societal and economic burden in today’s society. Despite the tremendous developments in the field of articular cartilage tissue engineering (AC TE) in the recent decade, none of the TE-based approaches has been able to regenerate the cartilage to levels of native tissue. The established paradigm of AC TE involves employment of undifferentiated MSCs in combination with 3D scaffolds/hydrogels and appropriate growth factors to induce chondrogenic differentiation of cells and deposition of ECM components like collagen and glycosaminoglycans. Once successful tissue has been formed in vitro, engineered cartilage grafts can be studied in vivo in large animal models to assess safety and efficacy of such grafts. Unfortunately, a considerable amount of grafts fails in vivo, which indicates the overall unsuitability and immaturity of the engineered tissues to function in the mechanically demanding environment of the joint in vivo. More importantly, there is no incentive to publish or submit for publication unsuccessful studies, which indicates that the number of failed studies employing large animal models could be considerably higher. Therefore, there is a need for novel strategies to screen and identify in vitro engineered cartilage grafts that have higher chances of success in vivo. In addition to increasing the success rate of such studies, this approach would have a great potential to reduce the number of animals utilized in such studies. In this context, there is evidence suggesting that chondrogenically differentiating MSCs respond anabolically to mechanical stress at later stages of differentiation by producing ECM components like glycosaminoglycans. Interestingly, the differentiation of MSCs is also associated with metabolic changes, where glycolysis is reduced and oxidative phosphorylation is enhanced as maturation progresses. The goal of this project is to develop a platform that could be used to assess such metabolic changes by sampling metabolites in- and outside the developing cartilage grafts to make statements concerning the maturity. By establishing such platform an additional readout would be available, in addition to commonly used biochemical and histological techniques within AC TE, that would facilitate a more informed decision making prior to an in vivo transition of a potential cartilage graft.

Development of a process to produce recombinant Influenza Neuraminidase (rNA) antigen in the baculovirus system, and especially downstream processing/purification will be performed in collaboration between the Icahn School of Medicine at Mount Sinai and the University of Natural Resources and Life Sciences to optimize an affinity purification-based downstream process for production of his-tagged rNA which can be successfully implemented at the CMO Expression Systems to produce enough rNA for a Collaborative Influenza Vaccine Innovation Centers (CIVICs) phase I clinical trial. Furthermore, to develop a high-yielding tag-less purification process that would allow us to get sufficient protein yields for post-phase I clinical development and lastly, to perform testing of alternative rNA expression constructs and expression systems. Doing this work will enable us to test rNA vaccines in clinical trials and may also provide a commercial path forward for rNA protein-based vaccine development in general.

Theoretical framework One of the first specific defense mechanisms against invading pathogens and self-antigens is the complement system, activated by immunoglobulins (Igs). Igs bind specifically to the antigen on the pathogen and thereby enable the docking of the C1q-complement initiation complex. Two factors influencing the complement activation were so far not investigated in detail. First, the format of the antigen, described by the chemical nature, the molecular size and the mode of presentation (as soluble substance or embedded in vesicles for mimicking the cell surface). Second, the huge difference in complement activation resulting from the degree of oligomerization of the IgMs. Objectives The overall goal of the pent/hexIgM project is to elucidate the activation sites of C1q and the IgM-Fc after binding of pentameric and hexameric IgMs to different antigen formats. Approach/methods We will produce recombinant IgMs and the C1q protein in mammalian cells and generate mutants thereof by yeast surface display. Next, we will confirm the biological activities of generated proteins in vitro by immunochemical and biophysical analyses as well as functionality tests. Most important, we will elucidate in influence of the antigen format and the degree of oligomerization of the IgMs (pentameric versus hexameric IgMs) on the activation of the complement system. Level of originality Although IgMs in combination with complement proteins have an important function in the human body, these proteins are not yet widely used in therapy and diagnosis. The results of the pent/hexIgM project will contribute to understanding the complex mechanisms underlying the activation of the complement cascade and will provide data of particular importance for developing new diagnostics and efficient treatment methods for various infectious, inflammatory, chronical and cancerous diseases.