Project Summary. Delivery of recombinant proteins into the cytosol would provide access to therapeutic targets that are not accessible within the extracellular environment, with faster pharmacokinetics than what are afforded by gene delivery approaches requiring transcription and translation. Vehicles that can shuttle active proteins across the cell membrane and into the cytosol are needed to circumvent the limited passive internalization of proteins due to their large size, charge, and hydrophilicity. An ideal vehicle would mediate rapid and efficient delivery of any protein cargo into the cytosol, regardless of protein physical properties, be fabricated under mild conditions that maintain protein activity, protect the protein from degradation during transport, and not induce adverse side-effects, such as cell death or anti-protein immunogenicity. Toward this end, the proposed research program will develop an innovative new vehicle for cytosolic protein delivery based on pairs of oppositely- charged synthetic peptides, CATCH(+) and CATCH(-), that co-assemble into b-sheet nanofibers in water. Recombinant fusion of either CATCH peptide onto the terminus of a protein provides a soluble precursor (i.e., a “CATCH-Protein”) that is incorporated into the nanofibers that form in the presence of a complementary CATCH peptide partner. Our unpublished data demonstrate that polysorbate excipients (e.g. Tween-20 and -80) drive the rapid formation of nanoparticles from dilute (i.e., µM) mixtures of a complementary CATCH(+) peptide and CATCH-Protein pair. These CATCH-Protein particles traffic into the cytosol, where the protein then exhibits biochemical activity. CATCH-Protein particles are not cytotoxic and do not induce antibodies against the protein in mice. Collectively, these observations suggest that CATCH-Protein particles are an ideal candidate vehicle for cytosolic protein delivery. Informed by these observations, we hypothesize that: (1) CATCH-Protein particles are internalized by endocytosis, where cytosolic delivery is enabled by protonation of the anionic CATCH-Protein during endosome acidification, which yields cationic nanostructures that induce endosomal vesicle rupture; and (2) CATCH-Protein particles can be employed to generate tolerogenic dendritic cells ex vivo via cytosolic delivery of the immunosuppressive enzyme indoleamine-2,3-dioxygenase. To test these hypotheses, Specific Aim 1 will characterize cytosolic protein delivery via CATCH-Protein particles using in vitro cell models and established spectroscopy, flow cytometry, and microscopy methods, alongside established endocytosis inhibitors. Specific Aim 2 will evaluate cytosolic delivery of CATCH-IDO for cell-mediated immunomodulation using the OTI and OTII antigen-specific immune response models. Success of this program will provide quantitative and mechanistic understanding of CATCH-Protein particle internalization that will be important for future translational efforts, while also e...