Development of Validation of Phage-Displayed Random Peptide Libraries Technologies for Rapid Isolation and Characterization of Extracellular Vesicles from Patients with Brain Tumors

噬菌体展示随机肽文库技术的验证开发，用于快速分离和表征脑肿瘤患者的细胞外囊泡

• 批准号: 10245053
• 负责人: Michael W. Graner
• 金额: $ 63.61万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-18 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10245053
• 关键词: Affinity; Affinity Chromatography; Antibodies; Bacteriophages; Binding; Biological; Biological Markers; Blood; Brain; Brain Neoplasms; Carbohydrates; Cell Culture Techniques; Cell Line; Cells; Central Nervous System Diseases; Central Nervous System Neoplasms; Cerebrospinal Fluid; Culture Media; Data; Density Gradient Centrifugation; Development; Disease; Environment; Extracellular Matrix; Flow Cytometry; Gender; Human; Immunoassay; Immunofluorescence Microscopy; Immunoglobulin G; Label; Link; Lipids; Liver; Lung; Magnetism; Mass Spectrum Analysis; Methodology; Methods; Modeling; Modification; Molecular; Molecular Conformation; Molecular Sieve Chromatography; Multiple Sclerosis; Nature; Neuraxis; Nucleic Acids; Nucleic acid sequencing; Pathologic; Pathology; Patients; Peptide Library; Peptide Phage Display Library; Peptides; Phage Display; Phase; Plasma; Polystyrenes; Precipitation; Preparation; Proteins; Proteomics; Publications; Random Peptide Libraries; Research; Slice; Specificity; Specimen; Spinal Cord; Spleen; Subarachnoid Hemorrhage; Surface; Techniques; Technology; Tissues; Transmission Electron Microscopy; Traumatic Brain Injury; Tumor Cell Line; Tumor-Derived; Ultracentrifugation; Validation; Western Blotting; analytical method; base; cell type; cross reactivity; extracellular vesicles; improved; multiple sclerosis patient; nanoparticle; protein aminoacid sequence; scale up; screening; tissue culture; transcriptomics

Rapid isolation/characterization of CNS-origin EVs from biofluids via phage-display peptide libraries Cells of the CNS shed extracellular vesicles (EVs) into their external environment, especially during pathologic states. Such EVs are considered high-value biomarker reservoirs due to their cell-of-origin specific protein/nucleic acid/metabolite content. Our publications and preliminary data demonstrated that 1) we can isolate high quality EVs from CNS and tumor cell lines, CSF, and plasma of patients with brain tumors' 2) we can isolate high affinity phage peptides specific to IgG antibodies from patients with multiple sclerosis (MS); 3) we identified specific phage peptides for EVs derived from a brain tumor cell line. We hypothesize that application of phage-display random peptide libraries will identify EVs of CNS origin. High-affinity phage peptides can be used for rapid isolation and characterization of EVs from biofluids of patients with CNS diseases. We propose to develop phage peptide technologies for enrichment, characterization diseases. display isolation, and of EVs derived from different CNS cell types from blood and/or CSF of patients with CNS. The unbiased nature of phage display and its ability to detect non-protein moieties makes phage a unique and powerful technique to differentially probe EV surfaces. R21 Phase Aim 1 will screen phage-display random peptide libraries with EVs from CNS cell lines, brain tumor cell lines, and human brain slice cultures to identify high-affinity peptides recognizing CNS EVs. R21 Phase Aim 2 will utilize phage peptides that recognize CNS-specific EVs to isolate such EVs from relevant biofluids (blood/plasma, cerebrospinal fluid) from patients with CNS diseases. We will achieve 2 milestones for R21 phase. Milestone #1: Development of a robust phage peptide technologies for rapid identification of purification of EVs derived from CNS cell lines and tissue cultures. Milestone #2: Demonstrate applicability and specificity of peptide affinity matrices for EVs from biofluids of patients with CNS pathologies. R33 Phase Aim 1 will validate the phage and peptides selected by EVs from CNS cells/cultures do indeed recognize cells and EVs of central nervous system origin. In Aim 2 of R33, we will produce improved peptide affinity-based methods for large-scale isolation of CNS EVs. And Aim 3 of R33 phase is to determine the biotargets bound by the CNS EV-specific phage peptides. We will achieve 3 milestones in R33 phase. Milestone #1: Demonstrate that phage and phage peptides are specific for CNS entities. Milestone #2: Generate and demonstrate improved isolation materials and early-stage scale-up models for scale-up of CNS EV isolation from biofluids. Milestone #3: Identify phage peptide-reactive molecular species from CNS EVs for validation and biologic activity purposes

通过噬菌体展示肽库从生物体液中快速分离/表征 CNS 来源的 EV CNS 细胞将细胞外囊泡 (EV) 释放到外部环境中，尤其是在病理状态下。由于其细胞源特异性蛋白质/核酸/代谢物含量，此类 EV 被认为是高价值的生物标志物库。我们的出版物和初步数据表明：1) 我们可以从脑肿瘤患者的中枢神经系统和肿瘤细胞系、脑脊液和血浆中分离出高质量的 EV 2) 我们可以从多发性硬化症患者中分离出针对 IgG 抗体的高亲和力噬菌体肽（多发性硬化症）; 3) 我们鉴定出来自脑肿瘤细胞系的 EV 的特定噬菌体肽。我们假设噬菌体展示随机肽库的应用将识别 CNS 来源的 EV。高亲和力噬菌体肽可用于从 CNS 疾病患者的生物体液中快速分离和表征 EV。我们建议开发噬菌体肽技术来富集、表征疾病。显示从 CNS 患者的血液和/或 CSF 中分离出的不同 CNS 细胞类型的 EV。噬菌体展示的公正性及其检测非蛋白质部分的能力使噬菌体成为一种独特且强大的技术来差异探测 EV 表面。 R21 阶段目标 1 将使用来自 CNS 细胞系、脑肿瘤细胞系和人脑切片培养物的 EV 筛选噬菌体展示随机肽库，以识别识别 CNS EV 的高亲和力肽。 R21 阶段目标 2 将利用识别 CNS 特异性 EV 的噬菌体肽，从 CNS 疾病患者的相关生物流体（血液/血浆、脑脊液）中分离出此类 EV。我们将在 R21 阶段实现 2 个里程碑。里程碑#1：开发强大的噬菌体肽技术，用于快速鉴定来自 CNS 细胞系和组织培养物的 EV 的纯化。里程碑#2：证明肽亲和矩阵对于来自中枢神经系统病变患者生物体液的 EV 的适用性和特异性。 R33 阶段目标 1 将验证 EV 从 CNS 细胞/培养物中选择的噬菌体和肽确实能够识别中枢神经系统来源的细胞和 EV。在 R33 的目标 2 中，我们将开发改进的基于肽亲和力的方法，用于大规模分离 CNS EV。 R33阶段的目标3是确定CNS EV特异性噬菌体肽结合的生物靶标。我们将在 R33 阶段实现 3 个里程碑。里程碑#1：证明噬菌体和噬菌体肽对 CNS 实体具有特异性。里程碑#2：生成并展示改进的隔离材料和早期放大模型，用于放大从生物流体中分离 CNS EV 的规模。里程碑#3：识别 CNS EV 中的噬菌体肽反应分子种类，用于验证和生物活性目的