Orter to elucidate the spatiotemporal house and mechanism(s) of cancer EVs for the duration of illness progression. Funding: Ministry of Science and AMPK Activator Biological Activity Technology (MOST) grants104-2320-B-00705-MY2 (C.P.L.), 106320-B00704-MY3 (C.P.L.), and Academia Sinica Innovative Materials and Analysis Technology Exploration (i-MATE) Program AS-iMATE-1073 (C.P.L.)ISEV2019 ABSTRACT BOOKSymposium Session 23: EV Engineering II Chairs: Cherie Blenkiron; Thomas Kislinger Place: Level 3, Hall B 13:004:OS23.exoTOPE: loading bioactive molecules into exosomes working with 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; having said that, loading exosomes with non-native cargo molecules has historically been a important barrier to unlocking this potential. We reasoned that it would be achievable to load therapeutically relevant proteins into exosomes by identifying and coopting peptide sequences that natively enrich proteins in exosomes. Techniques: Differential and density gradient ultracentrifugation have been made use of to purify exosomes from cell culture supernatant. LC-MS/MS was applied to determine proteins present in purified exosomes, the amino acid sequences of highly abundant proteins were analysed for widespread sequence features, and plasmids encoding candidate peptide sequences fused to cargo proteins were expressed in stably chosen cells. The enrichment of fusion proteins in purified exosomes was assessed working with biochemical, flow cytometric and functional analyses. Results: Among the most abundant native exosomal proteins identified by LC/MS-MS have been three members with the MARCKS household. All three MARCKS members of the family were found to strongly localize to purified exosomes when overexpressed as GFP fusions. Using truncated and point mutant versions of sequences derived from these proteins, we identified a seven amino acid consensus peptide sequence that is Trypanosoma MedChemExpress certainly able to load non-native cargo proteins into the exosome lumen at very higher levels, comprising as much as ten in the total exosomal protein. Sequences containing this seven amino acid “exoTOPE” tag have been applied to load exosomes with cytosolic cargos for example fluorescent proteins, RNA-binding proteins and mRNA, Cas9, antigenic peptides and proteins, plus the type 2 transmembrane protein CD40 ligand (CD40L). Exosomes carrying exoTOPE-CD40L activated antigen presenting cells inPBMC assays with related EC50 values as cost-free recombinant CD40L. Summary/Conclusion: We’ve got identified and refined a short peptide, exoTOPE, that can be applied to load exosomes with diverse classes of cargos, like proteins and nucleic acids. The tiny size of this peptide tag tends to make this technique readily adaptable to a wide variety of applications and represents a important advance in our ability 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.