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流体中快速分离/表征CNS来源的EV CNS细胞将细胞外囊泡（EV）脱落到其外部环境中，特别是在病理状态期间。此类EV被认为是高价值的生物标志物储库，这是由于它们的起源细胞特异性蛋白质/核酸/代谢物含量。我们的出版物和初步数据表明：1）我们可以从患有脑肿瘤的患者的CNS和肿瘤细胞系、CSF和血浆中分离高质量的EV; 2）我们可以从患有多发性硬化症（MS）的患者中分离特异于IgG抗体的高亲和力噬菌体肽; 3）我们鉴定了针对源自脑肿瘤细胞系的EV的特异性噬菌体肽。我们假设噬菌体展示随机肽库的应用将确定中枢神经系统起源的EV。高亲和力噬菌体肽可用于从CNS疾病患者的生物流体中快速分离和表征EV。我们建议发展噬菌体肽技术用于富集、表征疾病。显示分离，以及来自CNS患者的血液和/或CSF的不同CNS细胞类型的EV。噬菌体展示的无偏性质及其检测非蛋白质部分的能力使得噬菌体成为差异探测EV表面的独特且强大的技术。R21 Phase Aim 1将用来自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文库，以鉴定识别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液体（血液/血浆，脑脊液）中分离此类EV。我们将在R21阶段实现两个里程碑。里程碑1：开发用于快速鉴定纯化源自CNS细胞系和组织培养物的EV的稳健噬菌体肽技术。里程碑#2：证明肽亲和基质对来自CNS病变患者生物液体的EV的适用性和特异性。R33阶段目标1将验证由来自CNS细胞/培养物的EV选择的噬菌体和肽确实识别中枢神经系统来源的细胞和EV。在R33的目标2中，我们将产生用于大规模分离CNS EV的改进的基于肽亲和力的方法。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阶段实现三个里程碑。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，用于从生物流体中放大CNS EV隔离。里程碑#3：从CNS EV中鉴定噬菌体肽反应性分子种类，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