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

利用噬菌体展示肽库从生物体液中快速分离/鉴定源自中枢神经系统的细胞外囊泡（ev），尤其是在病理状态下。这些电动汽车被认为是高价值的生物标志物储存库，因为它们的细胞起源特异性蛋白质/核酸/代谢物含量。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（MS）患者中分离出特异性IgG抗体的高亲和力噬菌体肽；3)我们鉴定了来自脑肿瘤细胞系的ev的特异性噬菌体肽。我们假设应用噬菌体展示随机肽文库可以识别源自中枢神经系统的ev。高亲和力噬菌体肽可用于从中枢神经系统疾病患者的体液中快速分离和鉴定ev。我们建议开发噬菌体肽技术用于富集、表征疾病。显示分离，以及来自CNS患者血液和/或脑脊液中不同CNS细胞类型的ev。噬菌体展示的无偏性及其检测非蛋白片段的能力使噬菌体成为差分探测EV表面的一种独特而强大的技术。R21 Phase Aim 1将筛选来自中枢神经系统细胞系、脑肿瘤细胞系和人脑切片培养的ev噬菌体展示随机肽库，以鉴定识别中枢神经系统ev的高亲和力肽。R21 Phase Aim 2将利用识别中枢神经系统特异性EVs的噬菌体肽从中枢神经系统疾病患者的相关生物体液（血液/血浆、脑脊液）中分离出这种EVs。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，用于快速鉴定来自中枢神经系统细胞系和组织培养物的ev纯化。里程碑2：证明肽亲和基质对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 Phase Aim 1将验证从CNS细胞/培养物中选择的噬菌体和肽确实能识别源自中枢神经系统的细胞和ev。在R33的目标2中，我们将建立改进的基于肽亲和力的CNS ev大规模分离方法。R33期的目的3是确定与CNS ev特异性噬菌体肽结合的生物靶点。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中枢神经系统实体具有特异性。里程碑2：生成并演示改进的分离材料和早期放大模型，用于从生物流体中分离CNS EV。里程碑#3：从CNS ev中鉴定噬菌体肽反应分子物种，用于验证和生物活性目的

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