Mon. May 20th, 2024

Orter to elucidate the spatiotemporal property and mechanism(s) of cancer EVs through illness progression. Funding: Ministry of Science and Technology (MOST) grants104-2320-B-00705-MY2 (C.P.L.), 106320-B00704-MY3 (C.P.L.), and Academia Sinica Revolutionary Components and Analysis Technologies 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 three, Hall B 13:004:OS23.exoTOPE: loading bioactive molecules into exosomes making use of 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; nevertheless, loading exosomes with non-native cargo molecules has historically been a significant barrier to unlocking this potential. We reasoned that it could be feasible to load therapeutically relevant proteins into exosomes by identifying and coopting peptide sequences that natively enrich proteins in exosomes. Procedures: Differential and density gradient MT1 manufacturer ultracentrifugation were utilised to purify exosomes from cell culture supernatant. LC-MS/MS was applied to recognize proteins present in purified exosomes, the amino acid sequences of hugely abundant proteins have been analysed for typical sequence characteristics, and plasmids encoding candidate peptide sequences fused to cargo proteins have been expressed in stably chosen cells. The enrichment of fusion proteins in purified exosomes was PDE1 drug assessed using biochemical, flow cytometric and functional analyses. Benefits: Amongst by far the most abundant native exosomal proteins identified by LC/MS-MS were 3 members of your MARCKS household. All 3 MARCKS family members had been discovered to strongly localize to purified exosomes when overexpressed as GFP fusions. Making use of truncated and point mutant versions of sequences derived from these proteins, we identified a seven amino acid consensus peptide sequence which is able to load non-native cargo proteins into the exosome lumen at very higher levels, comprising up to 10 from the total exosomal protein. Sequences containing this seven amino acid “exoTOPE” tag had been employed to load exosomes with cytosolic cargos like fluorescent proteins, RNA-binding proteins and mRNA, Cas9, antigenic peptides and proteins, along with the type two transmembrane protein CD40 ligand (CD40L). Exosomes carrying exoTOPE-CD40L activated antigen presenting cells inPBMC assays with equivalent EC50 values as totally free recombinant CD40L. Summary/Conclusion: We have identified and refined a quick peptide, exoTOPE, that can be employed to load exosomes with diverse classes of cargos, which includes proteins and nucleic acids. The smaller size of this peptide tag makes this method 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.