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：
10430322
负责人：
Gregory Hudalla
金额：
$ 18.13万
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/10430322
关键词：
Adjuvant Adopted Allogenic Amyloid beta-Protein Antibodies Antigens Biochemical Cations Cell Death Cell Therapy Cell membrane Cell model Cells Cellular immunotherapy Charge Chemicals Confocal Microscopy Cytosol Data Dendritic Cells Development Dioxygenases 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 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 Therapeutic Translations Tryptophan 2,3 Dioxygenase Tweens Vesicle Water Work abeta oligomer base beta pleated sheet biomaterial compatibility cytotoxicity delivery vehicle extracellular hydrophilicity immunoengineering immunogenicity immunoregulation indoleamine inhibitor innovation insight macrophage nanofiber nanoparticle nanoscale particle physical property programs protein degradation protein distribution protonation response side effect subcutaneous success synthetic peptide therapeutic protein therapeutic target 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。将重组蛋白递送到细胞质中将提供治疗靶标在细胞外环境中无法获得的，具有比提供的更快的药代动力学通过需要转录和翻译的基因输送方法。可以穿梭活跃蛋白的车辆在整个细胞膜和细胞质中都需要绕过有限的被动内在化蛋白质由于其大小，电荷和亲水性而引起的蛋白质。理想的车辆会介导快速而高效将任何蛋白质货物递送到细胞质中，无论蛋白质物理特性如何维持蛋白质活性，保护蛋白质在运输过程中免受降解的状况，不影响不良副作用，例如细胞死亡或抗蛋白质免疫原性。为此，拟议的研究计划将根据相对的成对开发一种创新的新车来用于胞质蛋白的递送带电的合成肽，捕获（+）和捕获（ - ），将其共组装成水中的B片纳米纤维。脱皮到蛋白质末端的两种捕获的重组融合提供了固体前体（即 “捕获蛋白”）将完整的捕获物纳入形成的纳米纤维中肽伴侣。我们未发表的数据表明，多吸附赋形剂（例如Tween -20和-80）驱动器从稀释（即µm）混合物（+）胡椒和捕获蛋白对。这些捕获蛋白颗粒流入细胞质，然后在其中蛋白质表现出来生化活性。捕获蛋白颗粒不是细胞毒性的，也不诱导针对蛋白质的抗体在老鼠中。总的来说，这些观察结果表明捕获蛋白颗粒是理想的候选车辆用于胞质蛋白递送。通过这些观察结果，我们假设：（1）捕获蛋白颗粒通过内吞作用内在化，在阴离子捕获蛋白的质子化中启用了胞质递送在内体酸化过程中，产生阳离子纳米结构，诱导内体囊泡破裂；和（2）可以使用捕获蛋白颗粒通过胞质递送来生成耐受性树突状细胞免疫抑制酶吲哚胺-2,3-二加氧酶。为了检验这些假设，特定的目标1将使用体外细胞模型通过捕获蛋白颗粒来表征胞质蛋白递送并确定光谱，流式细胞仪和显微镜方法以及已建立的内吞作用抑制剂。具体的 AIM 2将评估使用OTI和OTI和 OTII抗原特异性免疫响应模型。该计划的成功将提供定量和对捕获蛋白粒子内在化的机械理解，这对于将来的翻译很重要努力，同时还建立捕获蛋白颗粒作为免疫工程的承诺策略。