No drug can be effective without an appropriate delivery system; this is why the development of formulations for effective and safe drug delivery currently raises major interests, in all clinical applications. While the use of therapeutic proteins has substantially increased in the last four decades, there is an entire class of proteins that has not yet been explored as drugs: the transmembrane receptors. In fact, the delivery of receptors for clinical applications remains very challenging and has only been achieved via genetic engineering of cells, referred as gene therapy. To date, most gene therapies remain extremely expensive and therefore, poorly accessible to patients. DRESSCODE proposes to innovate cutting-edge protein engineering technologies to deliver and enhance receptors at the cell membrane, using recombinant proteins only. By doing so, we aim to create a paradigm shift from genetic engineering to protein engineering of the cell membrane, with the ambition of developing cost-effective protein-based therapies. In DRESSCODE, we will first engineer independently the extracellular, intracellular, and transmembrane parts of a receptor, before combining our technologies to reconstruct functional receptors with enhanced bioactivity. Particularly, we will generate fusion proteins that 1) target the cell surface with super-avidity, to enhance receptor sensing; 2) penetrate the cell membrane and target its inner side, to enhance receptor signaling; and 3) insert across the cell membrane to reconstitute the receptor transmembrane domain using pH-low inserting peptides. DRESSCODE will demonstrate Proof-of-Technologies in two high-impact clinical applications, focusing on the engineering of VEGFR-2 for therapeutic angiogenesis and of the CAR for T cell-based cancer immunotherapy. Its success will lead to the development of novel therapeutic proteins and inspire the future use of receptors as drugs, while providing valuable tools for biological research.

Despite intensive study in the past on the problem of how information is processed in the brain to enable individual organisms to adapt to their continuously changing environment, little progress has been made on how new similar but discrete memory traces emerge in neuronal networks during learning. Current theories suggest that experience-dependent modifications in excitation-inhibition balance enable a selected group of neurons to form a new cell association during learning which represent the new memory trace. It was further proposed that particularly GABAergic inhibitory interneurons (INs) have a large impact on population activity in neuronal networks by means of their inhibitory output synapses. However, how cell associations emerge in space and time and how INs may contribute to this process is still largely unknown. This complex topic was so far difficult to address due to technical constraints. IN-Fo-Trace-DG aims to address this fundamental question in the dentate gyrus (DG), a brain structure essential for the acquisition of similar but discrete new memories. Based on our detailed knowledge on DG’s cellular elements, their interconnectivity and our recently established molecular interference tools, we will first, visualize the spatial and temporal activity patterns of cell populations during spatial learning in a virtual-reality using 2-Photon imaging. Second, we will determine the role of IN recruitment and plasticity in assembly formation by optogenetic and molecular interference. Third, we will analyze changes in excitatory and inhibitory signals in granule cells (GCs), the principal cells in this brain area, and INs during learning using whole-cell recordings in vivo. Finally, we will examine whether adult-born GCs contribute differently to learning-associated population activity compared to mature ones in the adult DG. This innovative multi-disciplinary approach will provide new insights on the mechanisms of new memory formation in cortical networks.

Protein N-termini represent a wealth of biological information, such as their generation by limited proteolysis, which sculpts proteome functionality by yielding protein-sized cleavage products with novel functionality. N-terminomics is a branch of functional proteomics and enables the analysis of thousands of protein N-termini in an unbiased manner. By targeting native, proteolytically generated or acetylated protein N-termini as well as uncovering novel translation initiation sites, N-terminomics bears relevance to proteolysis and proteogenomics research. Unlike proteomic assays to study modifications such as phosphorylation, N-terminomics has remained inaccessible to non-expert laboratories due to a lack of ready-to-use kit formats or commercial services. Based on a recently published N-terminomic workflow by my laboratory, the present project aims to fill this niche by laying the foundation to represent N-terminomics (sample preparation up to mass spectrometry analysis) in a kit-like format together with online data analysis. Development steps include inclusion of multiple internal standards for improved sample-to-sample comparability, sole usage of cost-effective, disposable “small-scale” equipment in sample preparation, minimization of required input material, adaption to different input material including cryopreserved and formalin-fixed, paraffin-embedded samples, as well as web-based mass spectrometry data analysis and annotation. This endeavor is based on multiple collaborative projects of the applicant with pharmaceutical companies and backed by interest of proteomic supply companies. In a small forward-looking activity, the project will explore the application N-terminomics in molecular diagnostics of bladder cancer; based on the rationale local infiltration by solid tumors is based on differential proteolysis. This activity serves an initial step to add N-terminomics to the toolbox of molecular pathology.