Orter to elucidate the spatiotemporal property and mechanism(s) of cancer EVs in the course of disease progression. Funding: Ministry of Science and Technologies (MOST) grants104-2320-B-00705-MY2 (C.P.L.), 106320-B00704-MY3 (C.P.L.), and Academia Sinica Innovative Supplies and Evaluation Technologies Exploration (i-MATE) System AS-iMATE-1073 (C.P.L.)ISEV2019 B7-H4 Proteins Storage & Stability ABSTRACT BOOKSymposium Session 23: EV Engineering II Chairs: Cherie Blenkiron; Thomas Kislinger Location: Level 3, Hall B 13:004:OS23.exoTOPE: loading bioactive molecules into exosomes employing a shortpeptide fusion Russell McConnell, Madeleine Youniss, Ke Xu, Kevin Dooley, Bryan Choi, Rane Harrison, Sonya Haupt, Damian Houde, Nuruddeen Lewis, Shelly Martin, Chang Ling Sia and Sriram Sathyanarayanan Codiak BioSciences, Cambridge, USAIntroduction: Exosomes represent a promising therapeutic platform for the selective delivery of diverse classes of payloads; even so, loading exosomes with non-native cargo molecules has historically been a important barrier to unlocking this potential. We reasoned that it would be doable to load therapeutically relevant proteins into exosomes by identifying and L-Selectin/CD62L Proteins manufacturer coopting peptide sequences that natively enrich proteins in exosomes. Procedures: Differential and density gradient ultracentrifugation had been employed to purify exosomes from cell culture supernatant. LC-MS/MS was made use of to identify proteins present in purified exosomes, the amino acid sequences of hugely abundant proteins had been analysed for typical sequence functions, and plasmids encoding candidate peptide sequences fused to cargo proteins were expressed in stably selected cells. The enrichment of fusion proteins in purified exosomes was assessed using biochemical, flow cytometric and functional analyses. Results: Among one of the most abundant native exosomal proteins identified by LC/MS-MS were three members on the MARCKS family members. All three MARCKS family members have been found to strongly localize to purified exosomes when overexpressed as GFP fusions. Applying truncated and point mutant versions of sequences derived from these proteins, we identified a seven amino acid consensus peptide sequence that is definitely in a position to load non-native cargo proteins into the exosome lumen at really high levels, comprising up to ten with the total exosomal protein. Sequences containing this seven amino acid “exoTOPE” tag had been employed to load exosomes with cytosolic cargos such as fluorescent proteins, RNA-binding proteins and mRNA, Cas9, antigenic peptides and proteins, and the variety two transmembrane protein CD40 ligand (CD40L). Exosomes carrying exoTOPE-CD40L activated antigen presenting cells inPBMC assays with similar EC50 values as cost-free recombinant CD40L. Summary/Conclusion: We’ve got identified and refined a brief peptide, exoTOPE, that can be employed to load exosomes with diverse classes of cargos, which includes proteins and nucleic acids. The tiny size of this peptide tag makes this system readily adaptable to a wide number of applications and represents a considerable advance in our capability to engineer exosomes with biologically active cargos. Funding: Funded by Codiak Biosciences.OS23.Retrograde dicer-independent AGO-loading of extracellular single stranded miRNA in recipient human cells Bartika Ghoshala and Suvendra N. BhattacharyyabaCSIR_Indian Institute of Chemical Biology, Kolkata, India, Kolkata, India; Molecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Kolkata, IndiabIntroduction: microRNAs are tiny regulator of gene expression that.
