Feedback connections between tissue stiffness and cellular contractile forces can instruct cell identity and activity via a process referred to as mechanosensing. Specific phosphoproteome changes during mechanosensing are poorly characterized. In this work, we chart the global phosphoproteome dynamics of primary human lung fibroblasts sensing the stiffness of injury relevant fibronectin coated Poly(dimethylsiloxane) substrates. We discovered a key signaling threshold at a Young’s modulus of eight kPa stiffness, above which cells activated a large number of pathways including RhoA, CK2A1, PKA, AMPK, AKT1, and Hippo-YAP1/TAZ mediated signaling. Time-resolved phosphoproteomics of cell spreading on stiff substrates revealed the temporal dynamics of these stiffness-sensitive signaling pathways. ECM substrate stiffness above eight kPA induced fibroblast contractility, cytoskeletal rearrangements, ECM secretion, and a fibroblast to myofibroblast transition. Our data indicate that phosphorylation of the transcriptional regulator NFATC4 at S213/S217 enhances myofibroblast activity, which is the key hallmark of fibrotic diseases. NFATC4 knock down cells display reduced stiffness induced collagen secretion, cell contractility, nuclear deformation and invasion, suggesting NFATC4 as a novel target for antifibrotic therapy. Synopsis How tissue stiffness regulates identity and activity of tissue fibroblasts is unclear. Mass spectrometry based analysis of tissue stiffness dependent phosphoproteome changes reveals how primary lung fibroblasts sense the mechanical properties of their environment and identifies NFATC4 as a novel regulator of the stiffness dependent transition of fibroblasts to ECM secreting myofibroblasts. Mass spectrometry analysis reveals the signaling landscape of fibroblast mechanosensing Time-resolved phosphoproteomic analysis of cell spreading on fibronectin NFATC4 regulates myofibroblast collagen secretion, cell contractility and invasion

While all the siRNA drugs on the market target the liver, the lungs offer a variety of currently undruggable targets which could potentially be treated with RNA therapeutics. Hence, local, pulmonary delivery of RNA nanoparticles could finally enable delivery beyond the liver. The administration of RNA drugs via dry powder inhalers offers many advantages related to physical, chemical and microbial stability of RNA and nanosuspensions. The present study was therefore designed to test the feasibility of engineering spray dried lipid nanoparticle (LNP) powders. Spray drying was performed using 5% lactose solution (m/V), and the targets were set to obtain nanoparticle sizes after redispersion of spray-dried powders around 150 nm, a residual moisture level below 5%, and RNA loss below 15% at maintained RNA bioactivity. The LNPs consisted of an ionizable cationic lipid which is a sulfur-containing analog of DLin-MC3-DMA, a helper lipid, cholesterol, and PEG-DMG encapsulating siRNA. Prior to the spray drying, the latter process was simulated with a novel dual emission fluorescence spectroscopy method to preselect the highest possible drying temperature and excipient solution maintaining LNP integrity and stability. Through characterization of physicochemical and aerodynamic properties of the spray dried powders, administration criteria for delivery to the lower respiratory tract were fulfilled. Spray dried LNPs penetrated the lung mucus layer and maintained bioactivity for >90% protein downregulation with a confirmed safety profile in a lung adenocarcinoma cell line. Additionally, the spray dried LNPs successfully achieved up to 50% gene silencing of the house keeping gene GAPDH in ex vivo human precision-cut lung slices at without increasing cytokine levels. This study verifies the successful spray drying procedure of LNP-siRNA systems maintaining their integrity and mediating strong gene silencing efficiency on mRNA and protein levels both in vitro and ex vivo. The successful spray drying procedure of LNP-siRNA formulations in 5% lactose solution creates a novel siRNA-based therapy option to target respiratory diseases such as lung cancer, asthma, COPD, cystic fibrosis and viral infections.

SARS-CoV-2 has been the cause of a global pandemic since 2019 and remains a medical urgency. siRNA-based therapies are a promising strategy to fight viral infections. By targeting a specific region of the viral genome, siRNAs can efficiently downregulate viral replication and suppress viral infection. However, to achieve the desired therapeutic activity, siRNA requires a suitable delivery system. The VIPER (virus-inspired polymer for endosomal release) block copolymer has been reported as promising delivery system for both plasmid DNA and siRNA in the past years. It is composed of a hydrophilic block for condensation of nucleic acids as well as a hydrophobic, pH-sensitive block that, at acidic pH, exposes the membrane lytic peptide melittin, which enhances endosomal escape. In this study, we aimed at developing a formulation for pulmonary administration of siRNA to suppress SARS-CoV-2 replication in lung epithelial cells. After characterizing siRNA/VIPER polyplexes, the activity and safety profile were confirmed in a lung epithelial cell line. To further investigate the activity of the polyplexes in a more sophisticated cell culture system, an air-liquid interface (ALI) culture was established. siRNA/VIPER polyplexes reached the cell monolayer and penetrated through the mucus layer secreted by the cells. Additionally, the activity against wild-type SARS-CoV-2 in the ALI model was confirmed by qRT-PCR. To investigate translatability of our findings, the activity against SARS-CoV-2 was tested ex vivo in human lung explants. Here, siRNA/VIPER polyplexes efficiently inhibited SARS-CoV-2 replication. Finally, we verified the delivery of siRNA/VIPER polyplexes to lung epithelial cells in vivo, which represent the main cellular target of viral infection in the lung. In conclusion, siRNA/VIPER polyplexes efficiently delivered siRNA to lung epithelial cells and mediated robust downregulation of viral replication both in vitro and ex vivo without toxic or immunogenic side effects in vivo, demonstrating the potential of local siRNA delivery as a promising antiviral therapy in the lung.