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
Extracellular vesicles (EVs) are considered high-value disease biomarkers. We propose to develop phage peptide technologies for enrichment, types, detect blood and/or CSF of patient?s non-protein moieties makes isolation, and characterization of EVs derived from different CNS cell with CNS diseases. The unbiased nature of phage display and its ability to phage display a unique and powerful technique to differentially probe EV surface