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

通过噬菌体展示肽库从生物体液中快速分离/鉴定CNS来源的EV CNS的细胞将细胞外小泡(EV)释放到其外部环境中，特别是在病理状态下。这类EV因其细胞来源的特定蛋白质/核酸/代谢物含量而被认为是高价值的生物标记物储藏库。我们的出版物和初步数据表明，1)我们可以从中枢神经系统和脑肿瘤患者的肿瘤细胞系、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)患者中分离到针对抗体的高亲和力噬菌体多肽；3)我们鉴定了来自脑肿瘤细胞系的EV的特异性噬菌体多肽。我们推测，应用噬菌体展示随机多肽文库可以识别来源于中枢神经系统的EV。高亲和力噬菌体多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肠道病毒。我们建议开发噬菌体多肽技术来丰富和鉴定疾病。显示分离，并从CNS患者的血液和/或脑脊液中分离出来自不同CNS细胞类型的EV。噬菌体展示的不偏不倚的性质和它检测非蛋白质部分的能力使噬菌体成为一种独特而强大的差异探测EV表面的技术。R21阶段目标1将用来自CNS细胞系、脑肿瘤细胞系和人脑切片培养的EV筛选噬菌体展示随机肽库，以识别识别CNS EV的高亲和力多肽。R21阶段目标2将利用识别CNS特异性EV的噬菌体多肽从CNS疾病患者的相关生物液(血液/血浆、脑脊液)中分离此类EV。我们将实现R21阶段的两个里程碑。里程碑#1：开发一种强大的噬菌体多肽技术，用于快速鉴定来自中枢神经系统细胞系和组织培养的EV的纯化。里程碑2：展示从中枢神经系统病理患者的生物液中提取的EV的多肽亲和矩阵的适用性和特异性。R33阶段目标1将验证EV从中枢神经系统细胞/培养中选择的噬菌体和多肽是否确实识别起源于中枢神经系统的细胞和EV。在R33的目标2中，我们将产生改进的基于多肽亲和力的方法来大规模分离CNS EV。R33阶段的目的3是确定与CNS EV特异性噬菌体多肽结合的生物靶点。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多肽是中枢神经系统实体所特有的。里程碑2：生成并演示改进的隔离材料和早期放大模型，用于放大从生物体液中分离CNS EV。里程碑3：从CNS EV中识别噬菌体多肽活性分子物种，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