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Advancing novel immunotherapy strategies requires refined tools in preclinical research to thoroughly assess drug targets, biodistribution, safety, and efficacy. Light sheet fluorescence microscopy (LSFM) offers unprecedented fast volumetric ex vivo imaging of large tissue samples in high resolution. Yet, to date laborious and unstandardized tissue processing procedures have limited throughput and broader applications in immunological research. Therefore, we developed a simple and harmonized protocol for processing, clearing and imaging of all mouse organs and even entire mouse bodies. Applying this Rapid Optical Clearing Kit for Enhanced Tissue Scanning (ROCKETS) in combination with LSFM allowed us to comprehensively study the in vivo biodistribution of an antibody targeting Epithelial Cell Adhesion Molecule (EpCAM) in 3D. Quantitative high-resolution scans of whole organs did not only reveal known EpCAM expression patterns but, importantly, uncovered several new EpCAM-binding sites. We identified gustatory papillae of the tongue, choroid plexi in the brain and duodenal papillae as previously unanticipated locations of high EpCAM expression. Subsequently, we confirmed high EpCAM expression also in human tongue and duodenal specimens. Choroid plexi and duodenal papillae may be considered as particularly sensitive sites due to their importance for liquor production or as critical junctions draining bile and digestive pancreatic enzymes into the small bowel, respectively. These newly gained insights appear highly relevant for clinical translation of EpCAM-addressing immunotherapies. Thus, ROCKETS in combination with LSFM may help to set new standards for preclinical evaluation of immunotherapeutic strategies. In conclusion, we propose ROCKETS as an ideal platform for a broader application of LSFM in immunological research optimally suited for quantitative co-localization studies of immunotherapeutic drugs and defined cell populations in the microanatomical context of organs or even whole mice.

SUMMARYThe generation of organs from stem cells by blastocyst complementation is a promising approach to cover the clinical need for transplants. In order to generate rejection-free organs, complementation of both parenchymal and vascular cells must be achieved, as endothelial cells play a key role in graft rejection. Here we used a lineage-specific cell ablation system to produce mouse embryos unable to form both the cardiac and vascular systems. By mouse intraspecies blastocyst complementation we rescued heart and vascular development separately and in combination, obtaining complemented hearts with cardiomyocytes and endothelial cells of exogenous origin. Complemented chimeras were viable and reached adult stage, showing normal cardiac function and no signs of histopathological defects in the heart. Furthermore, we implemented the cell ablation system for rat-to-mouse blastocyst complementation, obtaining xenogeneic hearts whose cardiomyocytes were completely of rat origin. These results represent an advance in the experimentation towards the in vivo generation of transplantable organs.

At the fundamental level, messenger RNA (mRNA)-based therapeutics involves the delivery of in vitro-transcribed (IVT) mRNA into the cytoplasm of a target cell, where it is translated into the desired protein. IVT mRNA presents various advantages compared to DNA and recombinant protein-based approaches that make it ideal for a broad range of therapeutic applications. IVT mRNA, which is translated in the cytoplasm after transfection into cells, can encode virtually any target protein. Notably, it does not enter the nucleus, which avoids its integration into the genome and the risk of insertional mutagenesis. The large-scale production of IVT mRNA is less complex than production of recombinant proteins, and Good Manufacturing Practice-compliant mRNA production is easily scalable, ideally poising mRNA for not only off-the-shelf, but more personalized treatment approaches. IVT mRNA's safety profile, pharmacokinetics, and pharmacodynamics, including its inherent immunostimulatory capacity, can be optimized for different therapeutic applications by harnessing a wide array of optimized sequence elements, chemical modifications, purification techniques, and delivery methods. The value of IVT mRNA was recently proved during the COVID-19 pandemic when mRNA-based vaccines outperformed the efficacy of established technologies, and millions of doses were rapidly deployed. In this review, we will discuss chemical modifications of IVT mRNA and highlight numerous preclinical and clinical applications including vaccines for cancer and infectious diseases, cancer immunotherapy, protein replacement, gene editing, and cell reprogramming.