Native and engineered human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to blood stem cells

Native and engineered extracellular vesicles (EVs) generated from human megakaryocytes (huMkEVs) or from the human megakaryocytic cell line CHRF (CHEVs) interact with tropism delivering their cargo to both human and murine hematopoietic stem and progenitor cells (HSPCs). 24 hours after intravenous infusion of huMkMPs into NOD-scid IL2R{gamma}null (NSG) mice, they induced a nearly 50% increase in murine platelet counts relative to saline control, thus demonstrating the potential of these EVs, which can be stored frozen, for treating thrombocytopenias. PKH26-labeled huMkMPs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs. Using engineered huMkEVs or CHEVs, their receptor-mediated tropism for HSPCs was explored to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to murine HSPCs both in vitro and in vivo. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases. Native and engineered human megakaryocytic extracellular vesicles for targeted non-viral cargo delivery to blood stem cells (Table of Contents): O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/536479v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@664768org.highwire.dtl.DTLVardef@d9fe13org.highwire.dtl.DTLVardef@1b795eforg.highwire.dtl.DTLVardef@1d48e2d_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical Overview: Native and engineered human megakaryocytic extracellular vesicles (huMkEVs) for provide targeted non-viral cargo delivery to blood stem cells. We demonstrate that huMkEVs as a transformational cargo-delivery system to blood stem cells (hematopoietic stem and progenitor cells, HSPCs) in NOD-scid IL2R{gamma}null (NSG) mice. Intravenous delivery of native huMkEVs enhances de novo platelet biogenesis by inducing megakaryocytic differentiation of murine HSPCs, thus demonstrating the desirable strong tropism of huMkEVs for murine HSPCs. Based on this tropism, we demonstrate that engineered huMkEVs can deliver functional plasmid-DNA cargo specifically to HSPCs.