SUPRAMOLECULAR PEPTIDE CO-ASSEMBLIES FOR CYTOSOLIC PROTEIN DELIVERY

项目摘要。将重组蛋白递送到胞质溶胶中将提供到达治疗靶点的途径 在细胞外环境中不可及，药代动力学比提供的更快， 通过需要转录和翻译的基因传递方法。可以穿梭活性蛋白的载体 需要穿过细胞膜进入胞质溶胶，以避免 蛋白质由于它们的大尺寸、电荷和亲水性。一个理想的车辆将调解快速和有效的 将任何蛋白质货物递送到胞质溶胶中，无论蛋白质的物理性质如何，都可以在温和的环境下制造。 维持蛋白质活性、保护蛋白质在运输过程中免于降解且不诱导 不良副作用，如细胞死亡或抗蛋白免疫原性。为此，建议的研究 该计划将开发一种创新的新工具，用于细胞溶质蛋白质的传递， 带电荷的合成肽CATCH（+）和CATCH（-），其在水中共组装成b-片层纳米纤维。 任一CATCH肽到蛋白质末端的重组融合提供了可溶性前体（即，一 所述纳米纤维在互补CATCH-蛋白（“CATCH-蛋白”）的存在下形成 肽伴侣。我们未发表的数据表明，聚山梨酯辅料（如吐温-20和吐温-80）可驱动 从稀释的（即，μM）互补CATCH（+）肽和 CATCH-蛋白质对。这些CATCH-蛋白质颗粒进入胞质溶胶，在那里蛋白质然后表现出 生化活性CATCH-蛋白颗粒无细胞毒性，不会诱导针对蛋白的抗体 对小鼠总的来说，这些观察结果表明CATCH蛋白颗粒是理想的候选载体 用于细胞溶质蛋白质递送。根据这些观察，我们假设：（1）CATCH蛋白颗粒 通过内吞作用内化，其中通过阴离子CATCH蛋白的质子化使胞质递送成为可能 在内体酸化期间，其产生诱导内体囊泡破裂的阳离子纳米结构;以及 (2)CATCH-蛋白颗粒可用于通过胞质递送离体产生致耐受性树突细胞 免疫抑制酶吲哚胺-2，3-双加氧酶。为了验证这些假设，具体目标1将 使用体外细胞模型表征通过CATCH-蛋白颗粒的胞质蛋白递送，并建立了 光谱学、流式细胞术和显微镜方法，以及已建立的内吞作用抑制剂。具体 目的2将使用OTI和OTI评价CATCH-IDO的细胞溶质递送用于细胞介导的免疫调节， OTII抗原特异性免疫应答模型。该计划的成功将提供定量和 CATCH-蛋白质颗粒内化的机制理解，这将是重要的未来翻译 这些努力，同时也建立了CATCH-蛋白颗粒作为免疫工程的一种有前途的策略。

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 Our unpublished data demonstrate that polysorbate excipients (e.g. Tween-20 and -80) drive the rapid formation of nanoparticles from dilute (i.e., µM) mixtures of a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 establishing CATCH-Protein particles as a promising strategy for immune engineering.