SUPRAMOLECULAR PEPTIDE CO-ASSEMBLIES FOR CYTOSOLIC PROTEIN DELIVERY

用于胞浆蛋白递送的超分子肽共组装体

基本信息

批准号：
10704128
负责人：
Gregory Hudalla
金额：
$ 21.94万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10704128
关键词：
Adjuvant Adopted Allogenic Amyloid beta-Protein Antibodies Antigens Biochemical Cell Death Cell Death Induction Cell Therapy Cell membrane Cell model Cells Cellular immunotherapy Charge Chemicals Confocal Microscopy Cytosol Data Dendritic Cells Development Drug Kinetics Drug Targeting Dyes Endocytosis Endosomes Engineering Environment Enzymes Evaluation Excipients Exhibits Fibroblasts Flow Cytometry Fluorescence Future Gene Delivery Genetic Transcription Green Fluorescent Proteins Hydrogels Immune Immune response Immunity In Vitro Inflammation Luciferases Macrophage Measurable Measures Mediating Methods Microscopy Modeling Mus Nanostructures Peptides Polysorbates Proteins Recombinant Fusion Proteins Recombinant Proteins Recombinants Reporting Research Rupture Signal Pathway Spectrum Analysis Techniques Testing Translations Tryptophan 2,3 Dioxygenase Tweens Vesicle Water Work abeta oligomer beta pleated sheet biomaterial compatibility cytotoxicity delivery vehicle extracellular hydrophilicity immunoengineering immunogenicity immunoregulation inhibitor innovation insight meter nanofiber nanoparticle nanoscale particle physical property programs protein degradation protein distribution protonation response side effect subcutaneous success synthetic peptide therapeutic protein therapeutic target translational therapeutics uptake

项目摘要

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.

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。在细胞外环境中无法获得的药物，其药代动力学比可承受的药物更快通过需要转录和翻译的基因传递方法来运输活性蛋白质。需要穿过细胞膜并进入细胞质，以避免有限的被动内化蛋白质由于其大尺寸、电荷和亲水性，理想的载体将快速有效地介导。将任何蛋白质货物输送到细胞质中，无论蛋白质物理性质如何，都可以在温和的条件下制造维持蛋白质活性、保护蛋白质在运输过程中不被降解而不是诱导的条件不良副作用，例如细胞死亡或抗蛋白免疫原性，为此，提出了研究。该计划将开发一种基于相反对的胞质蛋白递送的创新载体带电荷的合成肽 CATCH(+) 和 CATCH(-) 在水中共同组装成 b-片纳米纤维。将任一 CATCH 肽重组融合到蛋白质末端可提供可溶性前体（即， “CATCH-蛋白质”）被纳入在互补 CATCH 存在下形成的纳米纤维中我们未发表的数据表明，聚山梨醇酯赋形剂（例如 Tween-20 和 -80）具有驱动作用。由互补的 CATCH(+) 肽和的稀释（即 µM）混合物快速形成纳米颗粒这些 CATCH-蛋白质颗粒进入胞质溶胶，然后蛋白质在胞质溶胶中展示。 CATCH-蛋白质颗粒没有细胞毒性，不会诱导针对该蛋白质的抗体。总的来说，这些观察结果表明 CATCH-蛋白质颗粒是理想的候选载体。根据这些观察结果，我们发现：(1) CATCH 蛋白颗粒。通过内吞作用内化，其中通过阴离子 CATCH 蛋白的质子化实现胞质递送在内体酸化过程中，产生诱导内体囊泡破裂的阳离子纳米结构； (2) CATCH-Protein 颗粒可用于通过胞质递送离体产生耐受性树突状细胞免疫抑制酶吲哚胺-2,3-双加氧酶的作用为了检验这些假设，具体目标 1 将进行。使用体外细胞模型表征通过 CATCH-Protein 颗粒的胞质蛋白递送，并建立光谱学、流式细胞术和显微镜方法，以及已建立的特异性内吞抑制剂。目标 2 将使用 OTI 评估用于细胞介导免疫调节的 CATCH-IDO 胞质递送该计划的成功将提供定量和 OTII 抗原特异性免疫反应模型。 CATCH-蛋白质颗粒内化的机制理解对于未来的转化非常重要努力，同时还建立了 CATCH-Protein 颗粒作为免疫工程的一种有前景的策略。