Native and engineered extracellular vesicles 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). To develop non-viral delivery vectors to HSPCs based on MkEVs, we first confirmed, using NOD-scid IL2Rγnull (NSG™) mice, the targeting potential of the large EVs, enriched in microparticles (huMkMPs), chosen for their large cargo capacity. 24 h post intravenous infusion into NSG mice, huMkEVs induced a nearly 50% increase in murine platelet counts. PKH26-labeled huMkEVs or CHEVs localized to the HSPC-rich bone marrow preferentially interacting with murine HSPCs, thus confirming their receptor-mediated tropism for NSG HSPCs, and their potential to treat thromobocytopenias. We explored this tropism to functionally deliver synthetic cargo, notably plasmid DNA coding for a fluorescent reporter, to NSG HSPCs both in vitro and in vivo. We loaded huMkEVs with plasmid DNA either through electroporation or by generating hybrid particles with preloaded liposomes. Both methods facilitated successful functional targeted delivery of pDNA, as tissue weight-normalized fluorescence intensity of the expressed fluorescent reporter was significantly higher in bone marrow than other tissues. Furthermore, the fraction of fluorescent CD117+ HSPCs was nearly 19-fold higher than other cell types within the bone marrow 72-h following administration of the hybrid particles, further supporting that HSPC tropism is retained when using hybrid particles. These data demonstrate the potential of these EVs as a non-viral, HSPC-specific cargo vehicle for gene therapy applications to treat hematological diseases.

Chinese hamster ovary (CHO) cells release and exchange large quantities of extracellular vesicles (EVs). EVs are highly enriched in microRNAs (miRs, or miRNAs), which are responsible for most of their biological effects. We have recently shown that the miR content of CHO EVs varies significantly under culture stress conditions. Here, we provide a novel stoichiometric ("per-EV") quantification of miR and protein levels in large CHO EVs produced under ammonia, lactate, osmotic, and age-related stress. Each stress resulted in distinct EV miR levels, with selective miR loading by parent cells. Our data provide a proof of concept for the use of CHO EV cargo as a diagnostic tool for identifying culture stress. We also tested the impact of three select miRs (let-7a, miR-21, and miR-92a) on CHO cell growth and viability. Let-7a-abundant in CHO EVs from stressed cultures-reduced CHO cell viability, while miR-92a-abundant in CHO EVs from unstressed cultures-promoted cell survival. Overexpression of miR-21 had a slight detrimental impact on CHO cell growth and viability during late exponential-phase culture, an unexpected result based on the reported antiapoptotic role of miR-21 in other mammalian cell lines. These findings provide novel relationships between CHO EV cargo and cell phenotype, suggesting that CHO EVs may exert both pro- and antiapoptotic effects on target cells, depending on the conditions under which they were produced.

Gene therapy lacks tools to deliver in a safe and tissue-targeted manner. Limitations include toxicity, off-target effects due to targeting, and inability to repeat dose. We have developed a novel megakaryocyte-derived extracellular vesicle (MkEV)-based non-viral gene therapy delivery platform that preferentially targets bone marrow in vivo. Here we present work showing that STRM.BIO MkEVs: 1) pass through the liver and spleen to preferentially target bone marrow in mice and non-human primates (NHPs); 2) can selectively deliver pDNA cargo to long term HSCs to drive reporter protein expression exclusively in bone marrow following intravenous delivery in mice; and 3) present a platform to develop and deliver targeted gene therapies in vivo that are safe for repeat dosing. Our vision is to open the door to the future of medicine for patients living with rare diseases worldwide and bring gene therapy to life. Primary human CD34+ hematopoietic stem and progenitor cells (hHSPCs) were differentiated into megakaryocytes in vitro. MkEVs were harvested and loaded with pDNA via electroporation, followed by DNase treatment to remove free cargo. For in vivo biodistribution and cargo-mediated protein expression studies, STRM.BIO MkEVs were exogenously labeled with fluorescent dye (PKH26, Cell Tracker Deep Red, and/or DiD), loaded with cargo, and IV-injected into mouse tail veins (wild type or NSG) or NHPs (cynomolgus monkeys). Tissues were collected 16-48h after IV injection, with MkEV tissue biodistribution quantified by dye fluorescence (Mean Fluorescence Intensity (MFI), plate reader) normalized to tissue weight or genomic DNA input, and pDNA cargo quantified by qPCR. Additionally, quantification of MkEVs+ cells was performed by flow cytometry analysis of murine bone marrow derived sub-populations (lineage negative, c-Kit +, Sca-1 + (LSK); lineage positive (Lin +); and, long-term HSCs (LT-HSCs; Lin -/CD150 +/CD201 +)) cells. Protein expression from MkEV-loaded pDNA cargo was similarly determined by plate reader fluorescence data and confocal microscopy. In mouse biodistribution studies, DiD-labeled MkEVs were predominantly detected in bone marrow, followed by liver and spleen 16h-post tail vein injection (plate reader analyses: Bone Marrow - 1.1e 4 ± 0.2 SD; Liver - 9.6e 3 ± 1.0 SD; Spleen - 1.8e 4 ± 0.2 SD. MFI/g tissue; n=3mice/group). Flow cytometry analyses show MkEVs preferentially targeted the hematopoietic compartment (of the MkEV+ cells, 94 ± 1% CD45+ vs 6 ± 1% CD45-, respectively), specifically, the HSPCs (68%, LSK) and LT-HSCs (100%) 48h post intravenous delivery via tail vein injection. Pharmacokinetic studies show that while MkEVs can circulate through highly vascularized tissues like the liver and spleen, they accumulate in the bone marrow. Strikingly, miRFP encoded by the pDNA was exclusively detected in bone marrow (p<0.0001 vs. all other tissues; Fig. 1). In NHPs, dye-labeled MkEVSs were loaded with a MkEV-driven eGFP reporter plasmid. Studies showed preferential biodistribution to bone marrow, and preferential qPCR amplification of the pDNA cargo in bone marrow (both MFI/ ACTB and GFP/ ACTB were significantly higher vs. all other tissues: range p<0.05 through p<0.0001), Fig. 2. A tolerability and repeat dosing study was performed in NHPs. Monkeys were injected with 4 weekly doses of STRM.BIO MkEVs and serial bloodwork was obtained 6h post dose for cytokine evaluation and 5 days post dose for end organ damage assessment (n=4). The study showed no evidence of kidney, hematologic, or liver damage as evidenced by normal creatinine and electrolytes, no significant changes in complete blood counts, coagulation parameters, or liver function tests. There was no evidence of significant cytokine release or inflammatory changes on tissue histology. These data confirm that MkEVs are non-toxic and the platform is amenable to repeat dosing. Taken together, these data establish an advanced gene therapy platform that can target bone marrow specifically following in vivo administration in both mice and NHPs, potentially eliminating the need for current ex vivo approaches to treat rare blood disorders, and providing the critical ability to repeat dose as required in clinic. STRM.BIO is leveraging our platform to develop gene therapies for rare blood diseases in people.

Extracellular vesicles (EVs) are cornerstones of intercellular communication with exciting fundamental, clinical, and more broadly biotechnological applications. However, variability in EV composition, which results from the culture conditions used to generate the EVs, poses significant fundamental and applied challenges and a hurdle for scalable bioprocessing. Thus, an understanding of the relationship between EV production (and for clinical applications, manufacturing) and EV composition is increasingly recognized as important and necessary. While chemical stimulation and culture conditions such as cell density are known to influence EV biology, the impact of biomechanical forces on the generation, properties, and biological activity of EVs remains poorly understood. Given the omnipresence of these forces in EV preparation and in biomanufacturing, expanding the understanding of their impact on EV composition-and thus, activity-is vital. Although several publications have examined EV preparation and bioprocessing and briefly discussed biomechanical stresses as variables of interest, this review represents the first comprehensive evaluation of the impact of such stresses on EV production, composition and biological activity. We review how EV biogenesis, cargo, efficacy, and uptake are uniquely affected by various types, magnitudes, and durations of biomechanical forces, identifying trends that emerge both generically and for individual cell types. We also describe implications for scalable bioprocessing, evaluating processes inherent in common EV production and isolation methods, and propose a path forward for rigorous EV quality control.

Megakaryocytic extracellular vesicles (MkEVs) promote the growth and megakaryopoiesis of hematopoietic stem and progenitor cells (HSPCs) largely through endogenous miR-486-5p and miR-22-3p cargo. Here, we examine the impact of biomechanical force and culture age/differentiation on the formation, properties, and biological efficacy of MkEVs. We applied biomechanical force to Mks using two methods: shake flask cultures and a syringe pump system. Force increased MkEV production in a magnitude-dependent manner, with similar trends emerging regardless of whether flow cytometry or nanoparticle tracking analysis was used for MkEV counting. Both methods produced MkEVs that were relatively depleted of miR-486-5p and miR-22-3p cargo. However, while the shake flask-derived MkEVs were correspondingly less effective in promoting megakaryocytic differentiation of HSPCs, the syringe pump-derived MkEVs were more effective in doing so, suggesting the presence of unique, unidentified miRNA cargo components. Higher numbers of MkEVs were also produced by "older" Mk cultures, though miRNA cargo levels and MkEV bioactivity were unaffected by culture age. A reduction in MkEV production by Mks derived from late-differentiating HSPCs was also noted. Taken together, our results demonstrate that biomechanical force has an underappreciated and deeply influential role in MkEV biology, though that role may vary significantly depending on the nature of the force. Given the ubiquity of biomechanical force in vivo and in biomanufacturing, this phenomenon must be grappled with before MkEVs can attain clinical relevance.

Megakaryocytes release submicron size microparticles (MkMPs) in circulation. We have shown that MkMPs target CD34+ hematopoietic stem/progenitor cells (HSPCs) to induce megakaryocytic differentiation, and that small RNAs in MkMPs play an important role in the development of this phenotype. Here, using single-molecule real-time (SMRT) RNA sequencing (RNAseq), we identify the synergetic effect of two microRNAs (miRs), miR-486-5p and miR-22-3p (highly enriched in MkMPs), in driving the Mk differentiation of HSPCs in the absence of thrombopoietin (TPO). Separately, our data suggest that the MkMP-induced Mk differentiation of HSPCs is enabled through JNK and PI3K/Akt/mTOR signaling. The interaction between the two signaling pathways is likely mediated by a direct target of miR-486-5p and a negative regulator of PI3K/Akt signaling, the phosphatase and tensin homologue (PTEN) protein. Our data provide a possible mechanistic explanation of the biological effect of MkMPs in inducing megakaryocytic differentiation of HSPCs, a phenotype of potential physiological significance in stress megakaryopoiesis.