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: 10019698
• 负责人: Michael W. Graner
• 金额: $ 63.22万
• 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/10019698
• 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; 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 - 播种肽库细胞从生物流体中快速分离/表征CNS-Origin EV，将CNS的肽培养基库细胞置于其外部环境中，尤其是在病理状态下。由于其细胞细胞特异性蛋白/核酸/代谢产物含量，因此将这种电动汽车视为高价值生物标志物储层。我们的出版物和初步数据表明，1）我们可以将脑肿瘤患者的中枢神经系统和肿瘤细胞系，CSF和血浆分离出高质量的EV'2）我们可以分离出具有多发性硬化症患者IgG抗体特异的高亲和力噬菌体肽（MS）； 3）我们确定了源自脑肿瘤细胞系的电动汽车的特定噬菌体肽。我们假设应用噬菌体播放随机肽库将识别中枢神经系统的电动汽车。高亲和力的噬菌体肽可用于从中枢神经系统疾病患者的生物流体中快速分离和表征EV。我们建议开发噬菌体肽技术以富集，表征疾病。显示隔离，以及来自CNS患者血液和/或CSF的不同CNS细胞类型的EV。噬菌体显示的无偏性及其检测非蛋白质部分的能力使噬菌体成为差异探测EV表面的独特而有力的技术。 R21阶段AIM 1将筛选噬菌体斑点随机肽库，其中CNS细胞系，脑肿瘤细胞系和人脑切片培养物，以识别识别CNS EVS的高亲和力肽。 R21阶段目标2将利用噬菌体肽识别CNS特异性电动汽车来分离与CNS疾病患者的相关生物流体（血/血浆，脑脊液，脑脊液）的相关生物流体（血液/血浆，脑脊液）。我们将在R21阶段获得2个里程碑。里程碑＃1：开发可靠的噬菌体肽技术，以快速鉴定CNS细胞系和组织培养的EV的纯化。里程碑＃2：证明了来自中枢神经系统病理患者的生物流体的电动汽车的肽亲和力基质的适用性和特异性。 R33期AIM 1将验证来自中枢神经系统细胞/培养的电动汽车选择的噬菌体和肽确实确实识别中枢神经系统起源的细胞和电动汽车。在R33的AIM 2中，我们将生成改进的基于肽亲和的方法，用于大规模隔离CNS EV。 R33相的AIM 3是确定由CNS EV特异性噬菌体肽结合的生物目标。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是CNS实体的特异性。里程碑＃2：生成并展示了改进的隔离材料和早期阶段尺度模型，用于从生物流体中进行CNS EV的扩展。里程碑＃3：从中枢神经系统EVS确定噬菌体肽反应性分子物种以验证和生物活性目的