CN117015552A

CN117015552A - Compositions and methods relating to megakaryocyte-derived extracellular vesicles for the treatment of myeloproliferative neoplasms

Info

Publication number: CN117015552A
Application number: CN202180078649.3A
Authority: CN
Inventors: J·索恩
Original assignee: Strom Biology
Current assignee: Strom Biology
Priority date: 2020-10-23
Filing date: 2021-10-23
Publication date: 2023-11-07

Abstract

Disclosed herein are compositions and methods relating to the use of megakaryocyte-derived extracellular vesicles, including megakaryocyte-derived extracellular vesicles derived from human pluripotent stem cells, for the treatment of myeloproliferative diseases or disorders such as MPN.

Description

Compositions and methods relating to megakaryocyte-derived extracellular vesicles for the treatment of myeloproliferative neoplasms

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional patent applications filed on day 23 of 10 in 2020, 63/104,769 filed on day 4 in 2021, 63/173,735, and 63/209,084 filed on day 10 in 2021, which are all incorporated herein by reference in their entireties.

Sequence listing

The present application contains a sequence listing that is submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy was created at 2021, 10, 23, under the name "STRM-004PC_126555-5004_SL_ST25.Txt", and was 1,108 bytes in size.

Technical Field

The present disclosure relates to compositions and methods relating to megakaryocyte-derived extracellular vesicles derived from human pluripotent stem cells for use in the treatment of myeloproliferative diseases or disorders such as, for example, myeloproliferative neoplasms (MPNs).

Background

The direct administration of therapeutic agents to patients without the use of delivery vehicles has several drawbacks, including rapid clearance, poor bioavailability, low delivery to target cells or tissues, non-specific cytotoxicity, and consequent systemic side effects. Treatment with nano-delivery vehicles can have several advantages, including reduced renal clearance, improved site-specific delivery, simultaneous delivery of multiple therapeutic agents, prevention of enzymatic degradation, immune escape, sequential multi-stage release, stimulus response activation, and therapeutic diagnostic capabilities, among others. However, most of these features have not been used in the clinic, in part because of the complex and expensive manufacturing required to perform multiple functions. The largest class of clinically approved nanoparticles is the liposome, which consists of a simple lipid bilayer surrounding an aqueous compartment. However, in addition to target non-specific and inefficient unloading of therapeutic agents, liposomes can also elicit adverse effects in patients, including immune responses and cytotoxicity, as liposomes are foreign synthetic entities and have limited cell or tissue targeting mechanisms. Adenovirus, retrovirus, AAV and lentiviral vectors are currently the most popular gene therapy viral vectors; however, these methods have targeting, scalability of manufacture, immunogenicity and safety issues.

Hematological malignancies are forms of cancer that begin with hematopoietic tissue (such as bone marrow) cells or immune system cells. Examples of hematological cancers are acute and chronic leukemias, lymphomas, multiple myelomas, and myelodysplastic syndromes.

Myeloproliferative neoplasms or MPNs are hematological neoplasms caused by neoplastic hematopoietic bone marrow progenitor cells in the bone marrow (such as erythrocytes, platelets, and granulocyte precursor cells). Proliferation of tumor progenitor cells results in overproduction of any combination of leukocytes, erythrocytes and/or platelets, depending on the disease. These overproduced cells may also be abnormal, leading to additional clinical complications. Treatment of MPN is lacking and is focused mainly on symptoms rather than healing.

Thus, there is a need for delivery vehicles that can be cost-effectively mass-produced and eliminate or reduce adverse effects when administered to patients, and that can also provide new therapies for MPN that target tumor progenitor cells that lead to a malignant phenotype of the disease, particularly in individuals that are resistant to, or experience adverse events due to the adoption of, first-line therapies that are typically prescribed for such disorders, for example.

Disclosure of Invention

Disclosed herein are compositions and methods relating to megakaryocyte-derived extracellular vesicles that are useful in the treatment of myeloproliferative diseases or disorders, such as myeloproliferative neoplasms (MPNs). In particular, the megakaryocyte-derived extracellular vesicles of the invention exhibit unique biomarker profiles and/or size profiles, which make them well-suited for therapeutic delivery for the treatment of myeloproliferative diseases or disorders, such as myeloproliferative neoplasms (MPNs). In various embodiments, the compositions and methods disclosed herein are useful for drug delivery and treatment of myeloproliferative diseases or disorders, such as myeloproliferative neoplasms (MPNs). The compositions and methods disclosed herein are useful for drug delivery and treatment of myeloproliferative diseases or disorders, such as, for example, myeloproliferative neoplasms (MPNs), or diseases or disorders related to myeloproliferative diseases or disorders, such as, for example, myeloproliferative neoplasms (MPNs). The methods disclosed herein may be in vivo or ex vivo and may be used, for example, in gene replacement therapy and gene editing.

In another aspect, the invention relates to a method of gene editing a cell. In some embodiments, the method comprises (a) contacting the cell with a composition comprising a plurality of substantially purified megakaryocyte-derived extracellular vesicles disclosed herein comprising a lipid bilayer membrane surrounding a lumen, wherein: the megakaryocyte-derived extracellular vesicle lumen comprises cargo comprising an agent suitable for gene editing of a cell and/or cargo comprising an agent suitable for gene editing of a cell associates with a surface of a megakaryocyte-derived extracellular vesicle; and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein, and (b) subjecting the cell to gene editing to alter mutations in the JAK2 gene therein.

In another aspect, the invention relates to a method of treating a myeloproliferative disease or disorder, comprising: (a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles disclosed herein; (b) Incubating the plurality of substantially purified megakaryocyte-derived extracellular vesicles with a therapeutic agent to cause the therapeutic agent to fill the lumen of and/or associate with the surface of the megakaryocyte-derived extracellular vesicles and produce a deliverable therapeutic agent, wherein the therapeutic agent is capable of treating a myeloproliferative disease or disorder; and (c) administering the deliverable therapeutic agent to the patient or contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient, wherein megakaryocyte-derived extracellular vesicles are substantially purified and comprise lipid bilayer membranes surrounding a lumen, the megakaryocyte-derived extracellular vesicle lumen comprising the therapeutic agent and/or the therapeutic agent being associated with a surface of the megakaryocyte-derived extracellular vesicles; and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein. In another aspect, the invention relates to a pharmaceutical composition comprising a composition comprising megakaryocyte-derived extracellular vesicles as disclosed herein and a pharmaceutically acceptable excipient or carrier. In some embodiments, megakaryocyte-derived extracellular vesicles comprise cargo comprising one or more agents, e.g., one or more therapeutic agents, useful for treating myeloproliferative diseases or disorders, e.g., myeloproliferative neoplasms (MPNs).

In another aspect, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) Obtaining megakaryocyte-derived extracellular vesicles of the compositions disclosed herein; (b) Incubating megakaryocyte-derived extracellular vesicles with a therapeutic agent to cause the therapeutic agent to fill the lumen of and/or associate with the surface of the megakaryocyte-derived extracellular vesicles and produce a deliverable therapeutic agent; and (c) administering the deliverable therapeutic agent to the patient or contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In some embodiments, the myeloproliferative disease or disorder is MPN.

Drawings

FIG. 1A is a schematic diagram showing the differentiation steps of megakaryocyte-derived extracellular vesicles ("MKEV" or "MV"), wherein the duration of each stage, harvest time and associated yield are shown. FIG. 1B is a graph of experimental data showing the increase in yield of megakaryocyte-derived extracellular vesicles over time during Megakaryocyte (MK) differentiation in vitro. For reference, the last point, from top to bottom, is MkEV, live cells, and live MK. FIG. 1C is experimental data showing the phenotype of MKEV in culture. Upper graph: representative histograms of cell surface marker expression. The following figures: representative microscopy images of megakaryocytes (left) and harvested mkevs (right).

Figures 2A-2F show experimental data showing MkEV biomarker expression. Surface marker expression of mkevs of the disclosure was compared to Platelet Free Plasma (PFP) mkevs and platelet derived EVs (PLT EVs). Fig. 2A-2B are representative diagrams showing flow cytometry gating strategies. Fig. 2C is a representative graph showing the marker profiles of the cd4+mkev, cd4+pfp MKEV, and cd4+plt EV of the present disclosure. The MkEV of the present disclosure has a different surface marker phenotype compared to naturally occurring MKEVs and platelet-derived EVs. Differential expression of surface markers co-expressed on cd4+ STRM MkEV (black bars) compared to cd4+ naturally occurring Platelet Free Plasma (PFP) MkEV (hashed bars) and cd4+ platelet derived EV (dotted bars). For MKEV and PFP MKEV of the present disclosure, bars represent the mean percentage ± standard deviation, n=2 biological replicates. Fold change is relative to PFP EV. Fig. 2D is a representative graph showing fold-change in marker expression between mkevs and PFP mkevs of the disclosure. For CD32a, GPVI and CD18, fold changes were calculated by changing the values from 0 to 0.01. Fig. 2E is a representative graph showing fold change in marker expression between MkEV and PLT EV of the present disclosure. For CD32a, the fold change was calculated by changing the value from 0 to 0.01. The data show that the mkevs of the present disclosure exhibit different surface marker expression compared to PFP mkevs and PLT EVs, and establish a marker profile of the mkevs of the present invention relative to PFP mkevs and PLT EVs. Fig. 2F is a representative graph showing minimal DRAQ5 positive events indicating no cell contamination.

Fig. 3A-3B are electron microscopic images, including size and morphology, showing MkEV characterization. Fig. 3A is a frozen EM image of MkEV of the present disclosure with immunogold labeling of CD 41. Fig. 3B is a frozen EM image of MkEV of the present disclosure with an immunogold-labeled phosphatidylserine. Measurement of MkEV in frozen EM images showed that MkEV size ranged between 100-500nm with an average diameter of about 250nm. Fig. 3C is an image of MkEV isolated from PFP plasma co-stained with CD41 (large dots) and PS (small dots).

Fig. 4A shows the size distribution (nm) of the cd41+ mkevs of the present disclosure as compared to the cd41+ PFP mkevs and platelet EVs. Flow cytometry analysis using fluorescent cd41+ antibody markers. Fig. 4B is a graph showing the size distribution of the cd4+mkevs of the present disclosure compared to the cd4+pfp (native MkEV and platelet cd4+ev). Fig. 4C is a graph showing the percentage of size distribution (nm) of EV. Fig. 4D to 4E are frozen EM images of PFP MkEV. Fig. 4F to 4K are frozen EM images of mkevs of the present disclosure. Cd41+ immunogold markers were used, shown as black dots.

Fig. 5A-5B are graphs of experimental data showing that size exclusion filtration effectively removes aggregates from unfiltered products. Fig. 5A shows an unfiltered MkEV product. FIG. 5B shows a 650-nm filtered MKEV product. The large aggregate material (observed by EM in frozen MkEV samples) has been shown to have been successfully removed by post-harvest filtration using a 650nm size exclusion filter. The images were from flow cytometry experiments.

Fig. 6A-6H are graphs of experimental data showing EV characterization. EV is collected from medium containing mature cultured MK 24 hours after megakaryocyte isolation and purification. Isolated human platelets were stimulated with thrombin (0.1U/mL) and collagen (1. Mu.g/mL) (conventional platelet agonists) or LPS (5. Mu.g/mL). EV number/platelets and size were measured by nanoparticle tracking analysis (fig. 6A, 6B, 6E and 6F), CD41 receptor positivity and amount were measured by electron microscopy (fig. 6C, 6D, 6G and 6H).

Fig. 7A-7B show the minimum batch-to-batch variability of MkEV yields as shown by the average MkEVS/mL (fig. 7A, left) and the total MkEV yield (fig. 7A, right). Furthermore, mkEV surface marker expression was similar between batches (fig. 7B)

Figures 8A-8B show successful editing of JAK2-V617F mutations in vitro. Gene editing constructs were designed and methods of correcting JAK 2V 617F mutations by homologous recombination were developed (FIG. 8A). As shown in FIG. 8A, HEL cells, a leukemia cell line harboring the JAK2-V617F mutation, were electroporated with GFP-labeled Ribonucleoprotein (RNP) and a single stranded oligonucleotide template for homologous recombination. After 24 hours gfp+ cells were isolated, qPCR of genomic DNA confirmed successful editing of JAK2-V617F mutation (fig. 8B).

Figures 9A-9C show correction of JAK2-V617F mutation in edited cells as shown by Next Generation Sequencing (NGS) of control and edited HEL cells. NGS showed a correction efficiency of 41% for edited cells (fig. 9A), where the base ratios for uncorrected and RNP transfected HEL cells and the insertion and deletion ratios at the V617F point mutation locus in uncorrected and RNP transfected HEL cells are shown in fig. 9B and 9C, respectively.

FIGS. 10A-10B show successful correction of JAK2-V617F mutations by homologous recombination across multiple time points. GFP-tagged RNPs targeting JAK2 mutations were electroporated into HEL cells. Gfp+ cells were divided 24 and 48 hours after RNP delivery, and qPCR of genomic DNA was performed on Wild Type (WT) (fig. 10B) and mutant JAK2 (fig. 10A). Cells from 24 hours and 48 hours post electroporation showed the presence of WT JAK2 due to gene editing (fig. 10B).

FIGS. 11A to 11B show successful correction of JAK2-V617F mutation by homologous recombination using a pDNA encoded RNP. pDNA encoding RNP targeting JAK2 mutation was electroporated into HEL cells and qPCR was performed on genomic DNA for WT (FIG. 11A) and mutant JAK2 (FIG. 11B). Cells 24 hours and 48 hours post electroporation showed the presence of WT JAK2 due to gene editing (fig. 11A).

Fig. 12A-12B illustrate the loading of nucleic acid cargo into mkevs by electroporation. For these experiments, pDNA encoding the MPN gene editor was electroporated into MkEV using 4D-Nucleofector (Lonza Wakersville, inc.). pDNA was extracted after dnase 1 treatment to remove non-internalized DNA and quantified by qPCR. Controls included mkevs+pdna without electroporation ± dnase treatment (fig. 12A). As demonstrated by the bars representing the mean fold change compared to the control samples treated with dnase, pDNA was successfully internalized into MkEV by electroporation (fig. 12B).

Figure 13 demonstrates Cas9 protein loading into MkEV by electroporation. MkEV was electroporated with Cas9, treated with proteinase K to remove any uninhibited cargo, and then western blotted to quantify Cas9. Controls included MkEV plus Cas9 without electroporation ± proteinase K. Cas9 is present in electroporated mkevs but not in control non-electroporated mkevs, and subsequent proteinase K digestion indicates protection of protein cargo by mkevs after electroporation.

Fig. 14A-14C show that cargo-loaded mkevs transfer Cas9 to Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro. Primary bone marrow-derived lineage depleted cells isolated from mice bearing JAK2-V617F mutations were co-cultured in vitro with MkEV bearing GFP-tagged Cas9 RNP (ribonucleoprotein) for 4 hours. The doses were 80, 155 and 465 MKEV/cell. Controls included cells co-cultured with unloaded mkevs and cells co-cultured with RNP alone, treated in parallel with mkevs. The percentage of gfp+ cells was quantified by flow cytometry (fig. 14A). The percentage of gfp+ cells increased with increasing MkEV dose (fig. 14B). The median fluorescence intensity is shown in fig. 14C.

Fig. 15A-15C show that association and/or uptake of Hematopoietic Stem and Progenitor Cells (HSPCs) to cargo-loaded mkevs increases after longer in vitro co-culture times. Primary bone marrow-derived lineage depleted cells isolated from mice bearing JAK2-V617F mutations were co-cultured in vitro with MkEV bearing GFP-tagged Cas9 RNP (ribonucleoprotein) for 14 hours. The doses were 80, 155 and 465 MKEV/cell. Controls included cells co-cultured with unloaded mkevs and cells co-cultured with RNP alone, treated in parallel with mkevs. Gfp+ cells were quantified by flow cytometry (fig. 15A). The percentage of gfp+ cells increased with increasing MkEV dose, and the median average fluorescence intensity increased with increasing dose and time of co-culture (fig. 15B and 15C).

Figures 16A-16C show significant association and/or uptake of primary mouse bone marrow lineage depleted cell-loaded cargo MkEV after 18 hours of co-culture. Primary bone marrow-derived lineage depleted cells isolated from mice bearing JAK2-V617F mutations were co-cultured in vitro with MkEV bearing GFP-tagged Cas9 RNP (ribonucleoprotein) for 18 hours. The doses were 80, 155 and 465 MKEV/cell. Controls included cells alone and cells co-cultured with RNP alone, in parallel with MkEV. Gfp+ cells were quantified by flow cytometry (fig. 16A). The percentage of gfp+ cells increased with increasing MkEV dose, with 17% of cells positive for MkEV association at 450 MkEV/cell dose (fig. 16A and 16B).

Fig. 17A-17C show that MkEV preferentially targets primary HSPCs. Lineage depleted cells co-cultured for 18 hours with RNP-loaded MKEVs were stained for HSPC specific markers (c-Kit and Sca-1) to determine the phenotype of cells targeted by the MKEV. Flow analysis of lineage negative/c-kit+/Sca-1+ (LSK) cells (a primary HSPC population) was performed on GFP+ and GFP-cell fractions (FIG. 17A). MkEV: an increase in the cell ratio was accompanied by an increase in the proportion of LSK cells in the gfp+ fraction (fig. 17A). The 600 MkEV/cell dose was sufficient to target most LSK cells (fig. 17A, 17B), data indicating that MkEV selectively targeted the first tested HSPC compartment in vitro. Confocal microscopy of gfp+ cells after co-culture at 600 MkEV/cell confirmed MkEV-mediated delivery of RNP complexes to target cells (fig. 17C). Imaging revealed internalization of GFP signal in target cells (fig. 17C).

FIGS. 18A-18B show co-culture of cargo-loaded MKEV with primary murine lineage negative/c-kit+/Sca-1+ (LSK) cells derived from JAK2-V617F mutant homozygous mice. Mkevs incubated with GFP-tagged cas9 RNP with or without electroporation were co-cultured with LSK cells at doses of 670 and 2000 MkEV/cell and analyzed for 14 hours. When incubated with MKEV-RNP, MKEV promoted LSK association and RNP uptake, as evidenced by increased GFP+ cells (FIG. 18A). There was a dose response with increasing percent gfp+ cells with increasing MkEV to cell ratio (figure 18B).

FIGS. 19A-19B show co-culture of cargo-loaded MKEV using primary murine lineage negative/c-kit+/Sca-1+ (LSK) cells derived from JAK2-V617F mutant heterozygous mice and 890 and 2570 MKEV/cell dose. As seen for homozygous LSK cells, there was MkEV-mediated cargo uptake in LSK cells, as determined by flow cytometry, with increasing MkEV/cell dose, the number of gfp+ cells increased in a dose-dependent manner (fig. 19A and 19B).

Detailed Description

The present invention is based in part on the discovery of compositions and methods that can be used to treat myeloproliferative diseases or disorders, such as, for example, myeloproliferative neoplasms (MPNs). In some embodiments, the compositions comprise substantially purified megakaryocyte-derived extracellular vesicles characterized by a particular set of physical properties, such as biomarker composition (e.g., presence, absence, or amount of biomarkers) and size, and can carry cargo in a lumen for delivery of an agent, such as a therapeutic agent for treating a myeloproliferative disease or disorder, such as, for example, myeloproliferative neoplasms (MPNs). In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are distinct from naturally occurring products that are collected from whole blood (platelet-free plasma) or derived from activated platelets (platelet EVs). Thus, in some aspects, the present invention provides compositions and methods for treating myeloproliferative diseases or disorders, such as, for example, myeloproliferative neoplasms (MPNs), using megakaryocyte-derived extracellular vesicles that are continually produced, have desirable properties, and carry specific cargo-making their therapeutic use for treating myeloproliferative diseases or disorders more likely to be successful.

Methods of treatment using megakaryocyte-derived extracellular vesicles

Myeloproliferative diseases

Myeloproliferative neoplasms (MPNs) are a class of hematological malignancies caused by hematopoietic progenitors, including Chronic Myelogenous Leukemia (CML), polycythemia Vera (PV), essential Thrombocythemia (ET), and essential myelofibrosis (PMF). Recurrent somatic point mutations in the pseudokinase domain of the Janus kinase 2 (JAK 2) gene were found in most patients with these diseases in 2005 (see, e.g., levine, r. Et al, 2005,Cancer Cell 7:387;James,C. Et al, 2005,Nature 434:1144, which is incorporated herein by reference in its entirety). Specifically, in patients with PV, ET and PMF, the frequency of occurrence of mutations that activate JAK2V617F is 81-99%, 41-72% and 39-57%, respectively (see, e.g., levine, r.l. ET al, 2007,Nat.Rev.Cancer 7:673, which is incorporated herein by reference in its entirety). Furthermore, over-activation of JAK/STAT signaling in a subset of patients that do not carry JAK mutations has been described (see, e.g., quinthas-cartanam a. Et al, 2013,Clinical Cancer Res.Doi:10.1158/1078-0432.Ccr-12-0284, which is incorporated herein by reference in its entirety). In summary, evidence to date supports targeting JAK/STAT pathways, particularly JAK2, in patients with various MPNs.

In various embodiments, the invention relates to a method of treating a myeloproliferative disease or disorder.

In various embodiments, the invention relates to a method for treating a disease or disorder characterized by a single point mutation associated with a myeloproliferative disease or disorder. In some embodiments, the single point mutation is a JAK 2V 617F mutation.

In one aspect, the invention relates to a method of treating a disease or disorder characterized by a JAK 2V 617F mutation, comprising administering an effective amount of a composition comprising megakaryocyte-derived extracellular vesicles disclosed herein, the megakaryocyte-derived extracellular vesicles comprising cargo comprising one or more therapeutic agents capable of treating a myeloproliferative disease or disorder. In some embodiments, the cargo comprises one or more agents capable of treating MPN. In some embodiments, the agent is a therapeutic agent useful for treating a myeloproliferative disease or disorder, such as MPN. In some embodiments, the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes surrounding a lumen and are derived from human pluripotent stem cells, wherein the megakaryocyte-derived extracellular vesicle lumen comprises cargo. In some embodiments, the cargo is associated with the vesicle surface in addition to or as a substitute for cargo located in the lumen of megakaryocyte-derived extracellular vesicles. In some embodiments, the cargo comprises one or more selected from the group consisting of RNA, DNA, proteins, carbohydrates, lipids, biomolecules, and small molecules. In some embodiments, the cargo comprises one or more agents, such as therapeutic agents.

In another aspect, the invention relates to a method of treating a disease or disorder characterized by a JAK 2V 617F mutation, comprising administering an effective amount of a composition comprising megakaryocyte-derived extracellular vesicles disclosed herein, the megakaryocyte-derived extracellular vesicles comprising cargo comprising one or more agents capable of treating a myeloproliferative disease or disorder, such as MPN. In some embodiments, the agent is a therapeutic agent capable of treating MPN. In some embodiments, the composition comprises megakaryocyte-derived extracellular vesicles comprising a nucleic acid encoding a functional Janus kinase 2 (JAK 2) gene or protein product thereof, or a nucleic acid encoding a gene editing protein capable of producing a functional JAK2 gene or protein product thereof.

In embodiments, the methods provide a functional Janus kinase (JAK) receptor in a patient. In some embodiments, the JAK receptor is JAK2 receptor.

In embodiments, the gene is a functional Jak2 gene or encodes a gene-editing protein capable of forming a functional Jak2 gene.

In some embodiments, correcting the JAK 2V 617F point mutation back to its non-mutated state is sufficient to reverse the myeloproliferative phenotype. In some embodiments, corrected wild-type HSCs will overcome JAK 2V 617F mutations in vivo, thereby producing a transmission of therapeutic agents that can cure.

In some embodiments, megakaryocyte-derived extracellular vesicles are made from primary human cd34+ cell cultures and are used to correct JAK 2V 617F mutations by delivering gene-targeting nucleic acids (e.g., plasmid DNA, RNA, etc.) to affected bone marrow HSCs.

In some embodiments, colonies may be cultured from the same patient that contain a sufficient number of cells for simultaneous genotyping and phenotyping, allowing direct comparison of phenotypically equivalent mutant and wild-type cells from the same patient, eliminating differences in age, sex, treatment, physical genetic background, and other confounding variables.

In some embodiments, the disease or disorder characterized by a JAK 2V 671F mutation is a myeloproliferative disease or disorder.

In various embodiments, the invention relates to a method of treating a myeloproliferative disease or disorder. Myeloproliferative diseases and disorders include blood disorders in which the presence of a primary disorder at the level of pluripotent hematopoietic stem cells results in increased production of one or more blood cell types.

In one aspect, the invention relates to a method of treating a myeloproliferative disease or disorder, comprising administering an effective amount of a composition comprising megakaryocyte-derived extracellular vesicles disclosed herein, the megakaryocyte-derived extracellular vesicles comprising cargo comprising one or more agents capable of treating a myeloproliferative disease or disorder, such as MPN. In some embodiments, the agent is a therapeutic agent useful for treating a myeloproliferative disease or disorder, such as MPN. In some embodiments, the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes surrounding a lumen and are derived from human pluripotent stem cells, wherein the megakaryocyte-derived extracellular vesicle lumen comprises cargo. In some embodiments, the cargo is associated with the vesicle surface in addition to or as a substitute for cargo located in the lumen of megakaryocyte-derived extracellular vesicles. In some embodiments, the cargo comprises one or more selected from the group consisting of RNA, DNA, proteins, carbohydrates, lipids, biomolecules, and small molecules. In some embodiments, the cargo comprises one or more agents, such as one or more therapeutic agents.

In one aspect, the invention relates to a method of treating a myeloproliferative disease or disorder, comprising administering an effective amount of a composition comprising megakaryocyte-derived extracellular vesicles disclosed herein that comprise cargo comprising a nucleic acid encoding a functional myeloproliferative disease or disorder-associated gene, such as an MPN-associated gene (e.g., but not limited to, a JAK2 gene, e.g., lacking a V617F mutation) or a protein product thereof, or a nucleic acid encoding a gene-editing protein capable of reducing the expression of a product from a mutant gene (e.g., but not limited to, a mutated JAK2 gene, e.g., a JAK 2V 671F mutation) or a protein product thereof. In a non-limiting example, a nucleic acid encoding a gene editing protein capable of reducing the expression of a product expressed by a mutant gene or its protein product is capable of correcting the mutant gene, thereby expressing a functional protein. In another non-limiting example, a nucleic acid encoding a gene editing protein capable of reducing the expression of a product expressed by a mutant gene or protein product thereof may alter the mutant gene such that the mutant protein is no longer expressed.

In another aspect, the invention relates to a method of treating a myeloproliferative disease or disorder, comprising administering an effective amount of a composition comprising a cell in vitro contacted with a composition comprising megakaryocyte-derived extracellular vesicles disclosed herein, wherein the megakaryocyte-derived extracellular vesicles comprise cargo comprising nucleic acid encoding a functional myeloproliferative disease or disorder-associated gene, such as an MPN-associated gene (e.g., but not limited to JAK2 gene, e.g., lacking the V617F mutation) or a protein product thereof, or nucleic acid encoding a gene-editing protein capable of reducing a product expressed by a mutant gene (e.g., but not limited to a mutation in the JAK2 gene, e.g., the 2V 671F mutation) or a protein product thereof.

In another aspect, the invention relates to a method of treating a myeloproliferative disease or disorder, comprising:

(a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles of the present disclosure;

(b) Incubating the plurality of substantially purified megakaryocyte-derived extracellular vesicles with a therapeutic agent such that the therapeutic agent fills the lumen of the megakaryocyte-derived extracellular vesicles and/or associates with the surface of the megakaryocyte-derived extracellular vesicles and produces a deliverable therapeutic agent,

wherein the therapeutic agent is capable of treating a myeloproliferative disease or disorder; and

(c) Administering the deliverable therapeutic agent to a patient or contacting the deliverable therapeutic agent with a biological cell in vitro and administering the contacted biological cell to the patient,

wherein the megakaryocyte-derived extracellular vesicles are substantially purified and comprise a lipid bilayer membrane surrounding a lumen,

the megakaryocyte-derived extracellular vesicle lumen comprises the therapeutic agent and/or associates with the surface of the megakaryocyte-derived extracellular vesicle; and

the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein.

(a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles of the present disclosure; the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane surrounding a lumen, wherein:

The lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein;

wherein the therapeutic agent is capable of gene editing of the V617F mutation in the JAK2 gene of a myeloproliferative tumor cell;

(c) Administering the deliverable therapeutic agent to a patient or contacting the deliverable therapeutic agent with a biological cell in vitro and administering the contacted biological cell to the patient, thereby editing the V617F mutation to treat a myeloproliferative disease or disorder in the patient.

In some embodiments, the present disclosure provides a method of treating a myeloproliferative disease or disorder, comprising administering an effective amount of a composition disclosed herein, wherein the composition comprises megakaryocyte-derived extracellular vesicles comprising cargo comprising a nucleic acid encoding a functional myeloproliferative disease or disorder-associated gene, such as an MPN-associated gene (e.g., without limitation, a JAK2 gene, such as lacking a V617F mutation), or a protein product thereof, or a nucleic acid encoding a gene capable of producing a functional myeloproliferative disease or disorder-associated gene, such as an MPN-associated gene (e.g., without limitation, a JAK2 gene, such as lacking a V617F mutation), or a protein product thereof.

In some embodiments, the present disclosure provides a method of treating a myeloproliferative disease or disorder, comprising administering an effective amount of a composition comprising cells that are in vitro contact with megakaryocyte-derived extracellular vesicles comprising cargo comprising a nucleic acid encoding a functional myeloproliferative disease or disorder-associated gene, such as an MPN-associated gene (e.g., without limitation, a JAK2 gene, such as lacking a V617F mutation), or a protein product thereof, or a nucleic acid encoding a gene capable of producing a functional myeloproliferative disease or disorder-associated gene, such as an MPN-associated gene (e.g., without limitation, a JAK2 gene, such as lacking a V617F mutation), or a protein product thereof.

In some embodiments, the myeloproliferative disease or disorder is selected from myeloproliferative neoplasms (MPNs), polycythemia vera, thrombocythemia, primary thrombocythemia, idiopathic myelofibrosis, acute myelogenous leukemia, systemic Mastocytosis (SM), chronic Neutrophilic Leukemia (CNL), and myelodysplastic syndrome (MDS). In some embodiments, the myelofibrosis is selected from primary myelofibrosis, secondary myelofibrosis, myelofibrosis following primary thrombocytosis, myelofibrosis following polycythemia vera, myelogenesis with myelofibrosis, chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia, eosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mastocytosis.

In some embodiments, the methods avoid the need for Hematopoietic Stem Cell (HSC) transplantation, including allogeneic HSC transplantation and/or myeloablative chemotherapy.

In some embodiments, the method reduces the occurrence of graft versus host disease, vascular disease (including thrombosis), coronary heart disease, arteriosclerosis, cerebral ischemia, cerebral infarction, thrombotic events, vascular complications, splenomegaly, progressive cytopenia, and/or hypercellular bone marrow in a patient.

In some embodiments, the method reduces the severity of a myeloproliferative disease or disorder.

In some embodiments, the method reduces the white blood cell count of the patient. In some embodiments, the method reduces neutrophil count in the patient. In some embodiments, the method reduces reticulocyte count of the patient. In some embodiments, the method reduces platelet count in the patient. In some embodiments, the method results in a reduction in spleen size in the patient.

In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to a non-diseased level. In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to between about 40% and about 50% of non-diseased levels. In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to between about 50% and about 60% of non-diseased levels. In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to between about 60% and about 70% of non-diseased levels. In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to between about 70% and about 80% of non-diseased levels. In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to between about 80% and about 90% of non-diseased levels. In some embodiments, the functional Jak2 gene restores white blood cell count, neutrophil count, reticulocyte count, platelet count, and/or spleen size to between about 90% and about 100% of non-diseased levels.

In some embodiments, a therapeutic agent comprising megakaryocyte-derived extracellular vesicles is administered in combination with a therapeutic agent selected from one or more of a Janus kinase (JAK) inhibitor and a phosphatidylinositol 3-kinase (PI 3K) inhibitor to a patient suffering from a myeloproliferative disease or disorder.

Non-limiting examples of JAK inhibitors include ruxotinib (ruxolitinib), phenanthroline Zhuo Tini (fedratinib), tofacitinib (tofacitinib), baratinib (baricitinib), letatinib (lebatinib), paretinib (pamitinib), XL019, AZD1480, INCB039110, LY 2784244, BMS911543, NS018, or N- (cyanomethyl) -4- [2- (4-morpholinanilino) pyrimidin-4-yl ] benzamide; or a pharmaceutically acceptable salt thereof.

Non-limiting examples of PI3K inhibitors include L147, BKM120, GDC-0941, BAY80-6946, PX-866, CH5132799, XL756, BEZ235 and GDC-0980, wortmannin, LY294002, PI3K II, TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499, OXY111A, IPI-145, IPI-443, GSK2636771, BAY 10824391, bupanix (buparlist), BYL719, RG7604, n1117, WX-037, AEZS-129, PA799, ZSTK474, AS252424, TGX221, TG100115, IC87114, (S) -2- (1- ((9H-purin-6-yl) amino) propyl) -5-fluoro-3-phenyl-4 (3H) -one, (S) -2- (1H-purin-6-yl) propyl) -5-fluoro-3-phenyl-4 (3H) -quinazolin-4- (3H) -fluoro-6-methyl) -amino (3-chloro-quinazolin-4-yl) amino ([ 3, 3H ] -4-fluoro-4-methyl ] -amino; or a pharmaceutically acceptable salt thereof.

Method for gene editing of cells using megakaryocyte-derived extracellular vesicles

In various embodiments, the invention relates to gene editing of cells using megakaryocyte-derived extracellular vesicles of the present disclosure.

In some embodiments, the method for gene editing of a cell comprises:

(a) Contacting the cells with a composition comprising a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising a lipid bilayer membrane surrounding a lumen,

wherein:

the megakaryocyte-derived extracellular vesicle lumen comprises cargo comprising an agent suitable for gene editing of a cell and/or cargo associated with a surface of a megakaryocyte-derived extracellular vesicle comprising an agent suitable for gene editing of a cell; and

the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein, and

(b) The cells were subjected to gene editing to alter mutations in the JAK2 gene therein.

In some embodiments, the cell comprises the JAK2 gene comprising SEQ ID No. 1, which refers to the wild-type JAK2 gene sequence or fragment thereof:

TTGTATCCTCATCTATAGTCATGCTGAAAGTAGGAGAAAGTGCATCTTTATTATGGCAGAGAGAATTTTCTGAACTATTTATGGACAACAGTCAAACAACAATTCTTTGTACTTTTTTTTTTCCTTAGTCTTTCTTTGAAGCAGCAAGTATGATGAGCAAGCTTTCTCACAAGCATTTGGTTTTAAATTATGGAGTATGTGTCTGTGGAGACGAGAGTAAGTAAAACTACAGGCTTTCTAATGCCTTTCTCAGAGCATCTGTTTTTGTTTATATAGAAAATTCAGTTTCAGGATCACAGCTAGGTGTCAGTGTAAACTATAATTTAACAGGAGTTAAGTATTTTTGAAACTGAAAACACTGTAGGACTATTCAGTTATATCTTGTGAAAAAGGAAAGCAAT(SEQ ID NO:1)。

in some embodiments, SEQ ID NO. 1 contains one or more mutations that result in the aberrant production of the JAK2 protein. In some embodiments, the mutation is a single point mutation. Non-limiting examples of such mutations include the V617F mutation, which has been identified in myeloproliferative neoplasms (MPN) patients. In one aspect, the methods of the present disclosure may be used to edit one or more mutations in the JAK2 gene, including the V617F mutation, to provide a functional JAK2 gene. In some embodiments, the mutation in the JAK2 gene comprises a V617F mutation.

Any agent suitable for gene editing and/or capable of altering a JAK2 gene mutation in a cell is contemplated by the present disclosure. In some embodiments, cargo containing a suitable agent for gene editing of cells is located in the lumen and/or associated with the surface of megakaryocyte-derived extracellular vesicles.

In some embodiments, the cargo is one or more therapeutic agents. In some embodiments, the agent is one or more therapeutic agents suitable for gene editing of a cell and/or capable of altering mutations in the JAK2 gene. In some embodiments, the therapeutic agent is a nucleic acid therapeutic agent. In some embodiments, the nucleic acid therapeutic agent is selected from one or more non-autologous and/or recombinant nucleic acid constructs selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, non-coding and coding RNAs, linear DNA, DNA fragments, or DNA plasmids. In some embodiments, the nucleic acid therapeutic agent is mRNA, and optionally: is transcribed in vitro or synthesized and/or comprises one or more non-canonical nucleotides, optionally selected from pseudouridine and 5-methoxyuridine. In some embodiments, the nucleic acid therapeutic encodes a functional protein. In embodiments, the nucleic acid therapeutic encodes a gene-editing protein and/or related elements for gene-editing function. In embodiments, the gene editing protein is selected from Zinc Finger (ZF), transcription activator-like effector (TALE), meganuclease, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -associated proteins. In embodiments, the CRISPR-associated protein is selected from Cas9, casX, casY, cpf1 and the gRNA complex thereof. In some embodiments, the CRISPR-associated protein is selected from Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof. In some embodiments, the CRISPR-associated protein is Cas9.

In some embodiments, in a population of cells comprising a JAK2 gene mutation, the gene editing alters and/or corrects the mutation in greater than about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the cells in the population. In some embodiments, the mutation is a V617F mutation.

In some embodiments, the composition comprising megakaryocyte-derived extracellular vesicles is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient or carrier.

Goods of megakaryocyte-derived extracellular vesicles

Megakaryocyte-derived extracellular vesicles may contain a variety of cargo including molecules, such as mRNA, micrornas, and cytokines, capable of treating myeloproliferative diseases or disorders such as MPN. Megakaryocyte-derived extracellular vesicles are capable of transferring their cargo to alter the function of target cells. They exert effects on target cells through surface receptor signaling, plasma membrane fusion and internalization. By loading cargo containing biological or therapeutic molecules for the treatment of myeloproliferative diseases or disorders into megakaryocytes or megakaryocyte-derived extracellular vesicles, megakaryocyte-derived extracellular vesicles can be further used as delivery vehicles to achieve targeted therapeutic effects for the treatment of myeloproliferative diseases or disorders. So far, small RNAs (siRNA and miRNA), small linear DNA and plasmid DNA have been successfully loaded into megakaryocyte-derived extracellular vesicles for various delivery applications. Megakaryocyte-derived extracellular vesicle targeting is defined by their surface protein complement and can be further engineered to express or remove specific biomarkers of interest to improve biodistribution and cell-cell recognition. For example, megakaryocyte-derived extracellular vesicles of the invention have their unique biomarker profile, particularly useful for delivering payloads, e.g., for therapy for treating myeloproliferative diseases or disorders such as MPN.

In embodiments, megakaryocyte-derived extracellular vesicles are suitable for loading cargo into a lumen. In some embodiments, the cargo comprises one or more agents, such as one or more therapeutic agents suitable for use in a myeloproliferative disease or disorder. In some embodiments, the cargo comprises one or more of RNA, DNA, proteins, carbohydrates, lipids, biomolecules, and small molecules. In some embodiments, the cargo comprises a biologically produced component. In some embodiments, the cargo comprises synthetically produced components. In some embodiments, the cargo is preloaded into megakaryocyte-derived extracellular vesicles. In some embodiments, the biological component is overexpressed in megakaryocytes, such that the resulting megakaryocyte-derived extracellular vesicles comprise the biological component. In some embodiments, the cargo is post-loaded into megakaryocyte-derived extracellular vesicles. In some embodiments, purified megakaryocyte-derived extracellular vesicles are mixed with cargo to produce cargo-loaded megakaryocyte-derived extracellular vesicles. In some embodiments, the cargo is hydrophobic. In some embodiments, the cargo is hydrophilic. In some embodiments, the cargo is incorporated into the lipid bilayer of megakaryocyte-derived extracellular vesicles. In some embodiments, the cargo is located in the lumen of megakaryocyte-derived extracellular vesicles.

In some embodiments, the cargo is associated with megakaryocyte-derived extracellular vesicles in addition to or as a substitute for cargo located in the lumen of megakaryocyte-derived extracellular vesicles. In some embodiments, the cargo associates with the surface and/or exterior of megakaryocyte-derived extracellular vesicles. Non-limiting examples of cargo associated with megakaryocyte-derived extracellular vesicles include cargo covalently conjugated to the surface of the vesicle, or cargo associated with the surface by electrostatic interactions. As will be appreciated by those of ordinary skill in the art, cargo associated with megakaryocyte-derived extracellular vesicles may be transported even when not loaded into the vesicle lumen.

In some embodiments, cargo is loaded into megakaryocyte-derived extracellular vesicles using a physically-induced and/or chemically-induced active loading strategy. In some embodiments, the active load strategy is physically induced. In some embodiments, the physically induced active loading strategy comprises mechanically or physically disrupting the megakaryocyte-derived extracellular vesicle lipid bilayer by external forces such as electroporation, sonication, freeze-thawing cycles, and extrusion. In some embodiments, electroporation involves the use of an electric field to induce spontaneous pore formation in megakaryocyte-derived extracellular vesicles lipid bilayers, wherein the presence of the electric field disrupts the lipid bilayers, and removal of the electric field enables the pores to close and reform the lipid layers after the cargo is absorbed by the megakaryocyte-derived extracellular vesicles. In some embodiments, the sonication involves applying ultrasonic energy through an ultrasonic probe to reduce the rigidity of megakaryocyte-derived extracellular vesicle lipid bilayers, thereby effecting cargo diffusion. In some embodiments, the freeze-thaw cycle uses thermal energy to promote megakaryocyte-derived extracellular vesicle cargo loading. In some embodiments, extrusion is performed according to established protocols for forming synthetic liposomes, wherein megakaryocyte-derived extracellular vesicles are mixed with free cargo and passed through a membrane containing nanoscale pores, wherein shear forces disrupt lipid bilayers, allowing exogenous cargo to enter the megakaryocyte-derived extracellular vesicles.

In some embodiments, the active loading strategy is chemically induced. In some embodiments, the chemically induced active loading strategy includes the use of a chemical agent, such as a saponin or transfection agent, to bypass the megakaryocyte-derived extracellular vesicle lipid bilayer. In some embodiments, the chemical agent is a detergent, such as a saponin. In some embodiments, saponins are used to selectively remove cholesterol from megakaryocyte-derived extracellular vesicle lipid bilayers, thereby opening pores in the lipid bilayer. In some embodiments, the chemical agent is a transfection agent. In some embodiments, transfection agents are used to deliver nucleic acids into megakaryocyte-derived extracellular vesicles by utilizing cationic species that promote interactions with lipid bilayers and subsequent internalization. In some embodiments, the transfection agent is a cationic liposome and/or a lipid-based agent.

In some embodiments, the nucleic acid loading ratio (i.e., the number of copies of nucleic acid per vesicle) of megakaryocyte-derived extracellular vesicles entering the present disclosure is from about 1 to about 1000, from about 1 to about 500, from about 1 to about 100, from about 10 to about 1000, from about 100 to about 1000, from about 500 to about 1000, from about 100 to about 500,000, from about 1000 to about 300,000, from about 100,000 to about 300,000, from about 1000 to about 10,000, or from about 1000 to about 5000. In some embodiments, the nucleic acid loading ratio into megakaryocyte-derived extracellular vesicles of the present disclosure is from about 100 to about 1000, from about 600 to about 700, from about 1000 to about 10,000, from about 5000 to about 10,000, from about 2000 to about 4000, from about 5000 to about 7000, from about 8000 to about 9000, or from about 8000 to about 8500. In some embodiments, the nucleic acid loading ratio into megakaryocyte-derived extracellular vesicles of the present disclosure is 500, about 3000, about 6000, or about 8300. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is plasmid DNA.

In some embodiments, the loading efficiency for loading cargo, such as cargo comprising nucleic acid, into megakaryocyte-derived extracellular vesicles of the present disclosure is from about 1% to about 99%, from about 10% to about 90%, from about 30% to about 70%, from about 40% to about 60%, from about 40% to about 50%, or from about 50% to about 60%. In some embodiments, the cargo comprises a nucleic acid. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is plasmid DNA. In some embodiments, the load efficiency is calculated using the following equation:

load efficiency (%) = cargo + mv#/total mv#

In some embodiments, the surface of megakaryocyte-derived extracellular vesicles is modified to affect the biodistribution and targeting ability of megakaryocyte-derived extracellular vesicles. In some embodiments, the surface ligand is added to megakaryocyte-derived extracellular vesicles by genetic engineering. In some embodiments, the megakaryocyte-derived extracellular vesicles produced express the fusion protein in their lipid bilayer. In some embodiments, the endogenous proteins in the megakaryocyte-derived extracellular vesicle lipid bilayer are fused to the targeting ligand by cellular engineering.

In embodiments, the cargo comprises one or more therapeutic agents useful for treating a myeloproliferative disease or disorder, such as MPN. In embodiments, the therapeutic agent is a nucleic acid therapeutic agent. In embodiments, the nucleic acid therapeutic encodes a functional protein.

In embodiments, the nucleic acid therapeutic agent is selected from one or more non-autologous and/or recombinant nucleic acid constructs selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, non-coding and coding RNAs, linear DNA, DNA fragments, or DNA plasmids. In some embodiments, the nucleic acid therapeutic agent is selected from one or more of mRNA, miRNA, siRNA and snoRNA.

In embodiments, the nucleic acid therapeutic encodes a wild-type gene that is defective in the patient. In embodiments, the nucleic acid therapeutic agent is mRNA, and optionally: is transcribed in vitro or synthesized and/or comprises one or more non-canonical nucleotides, optionally selected from pseudouridine and 5-methoxyuridine.

In some embodiments of the present invention, in some embodiments, the one or more non-canonical nucleotides are selected from the group consisting of 2-thiouridine, 5-azauridine, pseudouridine, 4-thiouridine, 5-methyluridine, 5-methylpseudouridine, 5-aminouridine, 5-aminopseudouridine, 5-hydroxyuridine, 5-hydroxypseudouridine, 5-methoxyuridine, 5-methoxypseudouridine, 5-ethoxyuridine, 5-ethoxypseudouridine, 5-hydroxymethyl uridine, 5-hydroxymethyl pseudouridine, 5-carboxyuridine, 5-carboxypseudouridine, 5-formyluridine, 5-formylpseudouridine, 5-methyl-5-azauridine 5-amino-5-azauridine, 5-hydroxy-5-azauridine, 5-methyl pseudouridine, 5-amino-pseudouridine, 5-hydroxy-pseudouridine, 4-thio-5-azauridine, 4-thio-pseudouridine, 4-thio-5-methyluridine, 4-thio-5-amino-uridine, 4-thio-5-hydroxy-uridine, 4-thio-5-methyl-5-azauridine, 4-thio-5-amino-5-azauridine, 4-thio-5-hydroxy-5-azauridine, 4-thio-5-methyl-pseudouridine, 4-thio-5-amino-pseudouridine, 4-thio-5-hydroxy-pseudouridine, 2-thiocytidine, 5-azacytidine, pseudoisocytidine, N4-methylcytidine, N4-aminocytidine, N4-hydroxycytidine, 5-methylcytidine, 5-aminocytidine, 5-hydroxycytidine, 5-methoxycytidine, 5-ethoxycytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methyl-5-azacytidine, 5-amino-5-azacytidine, 5-hydroxy-5-azacytidine, 5-methylisocytidine, 5-aminopseudoisocytidine, 5-hydroxy-pseudoisocytidine, N4-methyl-5-azacytidine, N4-methylpseudoisocytidine 2-thio-5-azacytidine, 2-thio-pseudoisocytidine, 2-thio-N4-methylcytidine, 2-thio-N4-aminocytidine, 2-thio-N4-hydroxycytidine, 2-thio-5-methylcytidine, 2-thio-5-aminocytidine, 2-thio-5-hydroxycytidine, 2-thio-5-methyl-5-azacytidine, 2-thio-5-amino-5-azacytidine, 2-thio-5-hydroxy-5-azacytidine, 2-thio-5-methylpseudoisocytidine, 2-thio-5-aminopseudoisocytidine, 2-thio-5-hydroxy-pseudoisocytidine, 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methyl pseudoisocytidine, N4-methyl-5-methylcytidine, N4-methyl-5-aminocytidine, N4-methyl-5-hydroxycytidine, N4-methyl-5-azacytidine, N4-methyl-5-amino-5-azacytidine, N4-methyl-5-hydroxy-5-azacytidine, N4-methyl-5-methyl pseudoisocytidine, N4-methyl-5-amino pseudoisocytidine, N4-methyl-5-hydroxy pseudoisocytidine, N4-amino-5-azacytidine N4-amino-pseudoisocytosine, N4-amino-5-methylcytidine, N4-amino-5-aminocytidine, N4-amino-5-hydroxycytidine, N4-amino-5-methyl-5-azacytidine, N4-amino-5-azacytidine, N4-amino-5-hydroxy-5-azacytidine, N4-amino-5-methylpseudoisocytidine, N4-amino-5-amino-pseudoisocytidine, N4-amino-5-hydroxy-pseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxy-5-methylcytidine, N4-hydroxy-5-aminocytidine, N4-hydroxy-5-hydroxycytidine, N4-hydroxy-5-methyl-5-azacytidine, N4-hydroxy-5-amino-5-azacytidine, N4-hydroxy-5-azacytidine, N4-hydroxy-5-methylpseudoisocytidine, N4-hydroxy-5-aminopseudoisocytidine, N4-hydroxy-5-hydroxy-pseudoisocytidine, 2-thio-N4-methyl-5-methylcytidine, 2-thio-N4-methyl-5-aminocytidine, 2-thio-N4-methyl-5-hydroxycytidine 2-thio-N4-methyl-5-azacytidine, 2-thio-N4-methyl-5-amino-5-azacytidine, 2-thio-N4-methyl-5-hydroxy-5-azacytidine, 2-thio-N4-methyl-5-methyl pseudoisocytidine, 2-thio-N4-methyl-5-amino pseudoisocytidine, 2-thio-N4-methyl-5-hydroxy pseudoisocytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-amino pseudoisocytidine, 2-thio-N4-amino-5-methyl cytidine, 2-thio-N4-amino-5-aminocytidine, 2-thio-N4-amino-5-hydroxycytidine, 2-thio-N4-amino-5-methyl-5-azacytidine, 2-thio-N4-amino-5-azacytidine, 2-thio-N4-amino-5-hydroxy-5-azacytidine, 2-thio-N4-amino-5-methylpseudoisocytidine, 2-thio-N4-amino-5-aminopseudoisocytidine, 2-thio-N4-amino-5-hydroxy-pseudoisocytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-methylcytidine, N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-hydroxy-cytidine, 2-thio-N4-hydroxy-5-methylcytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-azacytidine, 2-thio-N4-hydroxy-5-aminopseudoisocytosine, 2-thio-N4-hydroxy-5-hydroxypseudoisocytosine, N6-methyladenosine, N6-aminoadenosine, N6-hydroxyadenosine, 7-deazaadenosine, 8-azaadenosine, N6-methyl-7-deazaadenosine, N6-methyl-8-azaadenosine, 7-deaza-8-azaadenosine, N6-methyl-7-deaza-8-azaadenosine, N6-amino-7-deazaadenosine, N6-amino-8-azaadenosine, N6-amino-7-deaza-8-azaadenosine, N6-hydroxy-7-deazaadenosine, N6-hydroxy-8-deazaadenosine, 6-thioguanosine, 7-deazaguanosine, 8-deazaguanosine, 6-thioguanosine, 7-deazaguanosine and thioguanosine.

In some embodiments, the methods of the invention comprise gene editing and/or gene correction. In some embodiments, the methods of the invention comprise synthetic RNA-based gene editing and/or gene correction, e.g., using RNA comprising non-canonical nucleotides, e.g., RNA encoding one or more of the following: nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases, meganucleases, nicking enzymes, clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) related proteins, DNA repair proteins, DNA modification proteins, base modification proteins, DNA methyltransferases, proteins that cause DNA demethylation, DNA-substrate enzymes or natural or engineered variants, family members, orthologs, fragments or fusion constructs thereof. In some embodiments, the efficiency of gene editing and/or gene correction is high, e.g., higher than DNA-based gene editing and/or gene correction. In some embodiments, the gene editing and/or gene correction of the invention is sufficient for in vivo applications. In some embodiments, the gene editing and/or gene correction methods of the invention are sufficiently effective that cell selection (e.g., selecting cells that have been edited) is not required. In some embodiments, the gene editing efficiency of the methods of the invention is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%. In some embodiments, the gene correction efficiency of the methods of the invention is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 100%.

In some embodiments, the methods of the invention comprise a high efficiency gene editing protein comprising an engineered nuclease cleavage or DNA modification domain. In some embodiments, the methods comprise a high efficiency gene editing protein comprising an engineered nuclease cleavage or DNA modification domain. In some embodiments, the methods of the invention comprise a high fidelity gene editing protein comprising an engineered DNA binding domain. In some embodiments, the high fidelity gene editing protein comprises an engineered DNA binding domain. In some embodiments, the method comprises a gene-editing protein comprising an engineered repeat sequence. In some embodiments, the methods comprise a gene-editing protein comprising one or more CRISPR-associated family members. In some embodiments, the method comprises altering the DNA sequence of the cell by transfecting the cell with a gene-editing protein or inducing the cell to express the gene-editing protein. In some embodiments, the method comprises altering the DNA sequence of a cell present in an in vitro culture. In some embodiments, the method comprises altering the DNA sequence of a cell present in vivo.

In some embodiments, the methods include one or more fixatives and/or one or more antioxidants in the transfection medium, which may increase in vivo transfection efficiency, in vivo reprogramming efficiency, and in vivo gene editing efficiency. In some embodiments, the method comprises contacting the cell or patient with a glucocorticoid, such as hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, or betamethasone. In some embodiments, the methods comprise inducing the cells to express the protein of interest by contacting the cells with a steroid-containing medium and contacting the cells with one or more nucleic acid molecules. In some embodiments, the nucleic acid molecule comprises synthetic RNA. In some embodiments, the steroid is hydrocortisone. In some embodiments, hydrocortisone is present in the medium at a concentration of about 0.1uM to about 10uM or about 1 uM. In some embodiments, the methods comprise inducing the cells to express the protein of interest by contacting the cells with a culture medium comprising an antioxidant and contacting the cells with one or more nucleic acid molecules. In some embodiments, the antioxidant is ascorbic acid or ascorbic acid-2-phosphate. In some embodiments, the ascorbic acid or ascorbic acid-2-phosphate is present in the medium at a concentration of about 0.5mg/L to about 500mg/L, including about 50 mg/L. In some embodiments, the method comprises reprogramming and/or gene editing cells in vivo by contacting the cells with a culture medium comprising a steroid and/or an antioxidant and contacting the cells with one or more nucleic acid molecules, wherein the one or more nucleic acid molecules encode one or more reprogramming and/or gene editing proteins. In some embodiments, the cell is present in an organism, and the steroid and/or antioxidant is delivered to the organism.

In embodiments, the nucleic acid therapeutic agents may be used to treat a myeloproliferative disease or disorder. In some embodiments, the nucleic acid therapeutic encodes a gene-editing protein and/or related elements for gene-editing functions that can be used to edit a cell to alter a mutation in a gene associated with a myeloproliferative disease or disorder (e.g., the JAK2 gene). In embodiments, the gene editing protein is selected from Zinc Finger (ZF), transcription activator-like effector (TALE), meganuclease, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -associated proteins. In embodiments, the CRISPR-associated protein is selected from Cas9, casX, casY, cpf1 and the gRNA complex thereof. In some embodiments, the CRISPR-associated protein is selected from Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

In embodiments, the therapeutic agent is a biologic therapeutic useful for treating a myeloproliferative disease or disorder. In embodiments, the biologic therapeutic is a protein. In some embodiments, the biologic therapeutic is an interferon, a monoclonal antibody, and/or an interleukin. In some embodiments, the biologic therapeutic is used to achieve an immunotherapy selected from one or more of specific active immunotherapy, non-specific active immunotherapy, passive immunotherapy, and cytotoxic therapy.

In embodiments, the therapeutic agent is a recombinant protein useful for treating a myeloproliferative disease or disorder.

In embodiments, the therapeutic agent is a virus useful for treating a myeloproliferative disease or disorder.

In embodiments, the biologic therapeutic is one of an antibody or antibody fragment, fusion protein, gene-editing protein, cytokine, antigen, and peptide useful for treating a myeloproliferative disease or disorder.

In embodiments, the therapeutic agent is a small molecule therapeutic agent useful for treating a myeloproliferative disease or disorder. In some embodiments, the small molecule therapeutic is one or more of a drug, an inhibitor, or a cofactor. In some embodiments, the medicament is for use in cancer treatment. In some embodiments, the inhibitor is one or more of a kinase inhibitor, a proteasome inhibitor, and an inhibitor that targets apoptosis.

In embodiments, the therapeutic agent is a vaccine and/or immunogenic antigen useful for treating a myeloproliferative disease or disorder.

Megakaryocyte-derived extracellular vesicles

In one aspect, the present disclosure provides megakaryocyte-derived extracellular vesicles useful for the treatment of myeloproliferative diseases or disorders, such as MPN. In some embodiments, megakaryocyte-derived extracellular vesicles comprise cargo useful for treating myeloproliferative diseases or disorders, such as MPN.

Megakaryocyte-derived extracellular vesicles are relatively immunosilent and can be repeatedly administered; has obvious advantages compared with an immunogenic viral vector. In some aspects, megakaryocyte-derived extracellular vesicles are useful in vivo genomic drugs that do not require conditioning therapy, so one can accept them in an outpatient setting. This platform is an important paradigm shift for ex vivo to in vivo delivery of gene therapy, which would lead to the popularization of gene therapy by reducing treatment time and cost.

In one aspect, the invention relates to a composition comprising: a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising a lipid bilayer membrane surrounding a lumen, and the lipid bilayer membrane comprising one or more proteins associated therewith or embedded therein. In some embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells. The megakaryocyte-derived extracellular vesicle lumen comprises one or more megakaryocyte-derived nucleic acid molecules selected from the group consisting of: mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs. In some embodiments, the megakaryocyte-derived extracellular vesicles comprise cargo suitable for gene editing of cells and/or cargo associated with the surface of the megakaryocyte-derived extracellular vesicles. In some embodiments, in addition to or as an alternative to cargo located in the lumen of megakaryocyte-derived extracellular vesicles, cargo is loaded into megakaryocytes for packaging into extracellular vesicles. In some embodiments, the cargo is loaded directly into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to cargo located in the lumen of the megakaryocyte-derived extracellular vesicles.

In another aspect, the invention relates to a composition comprising: a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising a lipid bilayer membrane surrounding a lumen, and the lipid bilayer membrane comprising one or more proteins associated therewith or embedded therein. In some embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells. In some embodiments, the megakaryocyte-derived extracellular vesicles comprise one or more nucleic acid molecules selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs associated with the vesicle surface, and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein. In some embodiments, the nucleic acid molecule is exogenous. In some embodiments, the megakaryocyte-derived extracellular vesicles comprise cargo suitable for gene editing of cells and/or cargo associated with the surface of the megakaryocyte-derived extracellular vesicles. In some embodiments, in addition to or as an alternative to cargo located in the lumen of megakaryocyte-derived extracellular vesicles, cargo is loaded into megakaryocytes for packaging into extracellular vesicles. In some embodiments, the cargo is loaded directly into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to cargo located in the lumen of the megakaryocyte-derived extracellular vesicles.

In one aspect, the invention relates to a composition comprising: a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising a lipid bilayer membrane surrounding a lumen, and the lipid bilayer membrane comprising one or more proteins associated therewith or embedded therein. In some embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells. In some embodiments, megakaryocyte-derived extracellular vesicles are suitable for loading cargo into a lumen. In some embodiments, the cargo comprises one or more agents. In some embodiments, the agent is one or more therapeutic agents, including the therapeutic agents described herein. In some embodiments, the cargo comprises one or more megakaryocyte-derived nucleic acid molecules selected from the group consisting of mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs. In some embodiments, in addition to or as an alternative to cargo located in the lumen of megakaryocyte-derived extracellular vesicles, cargo is loaded into megakaryocytes for packaging into extracellular vesicles. In some embodiments, the cargo is loaded directly into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to cargo located in the lumen of the megakaryocyte-derived extracellular vesicles. In some embodiments, megakaryocyte-derived extracellular vesicles are suitable for carrying cargo associated with the surface of megakaryocyte-derived extracellular vesicles.

In another aspect, the invention relates to a composition comprising: a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising lipid bilayer membranes surrounding a lumen and derived from human pluripotent stem cells, wherein: the extracellular vesicle lumen of megakaryocyte origin contains cargo, and the lipid bilayer membrane contains one or more proteins associated therewith or embedded therein. In some embodiments, the cargo comprises one or more agents. In some embodiments, the agent is one or more therapeutic agents, including the therapeutic agents described herein. In some embodiments, the cargo comprises one or more megakaryocyte-derived nucleic acid molecules selected from the group consisting of mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs. In some embodiments, in addition to or as an alternative to cargo located in the lumen of megakaryocyte-derived extracellular vesicles, cargo is loaded into megakaryocytes for packaging into extracellular vesicles. In some embodiments, the cargo is loaded directly into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to cargo located in the lumen of the megakaryocyte-derived extracellular vesicles. In some embodiments, megakaryocyte-derived extracellular vesicles are suitable for carrying cargo associated with the surface of megakaryocyte-derived extracellular vesicles.

In one aspect, the present invention relates to a composition comprising: a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising lipid bilayer membranes surrounding a lumen and derived from human pluripotent stem cells, wherein: the megakaryocyte-derived extracellular vesicle lumen comprises one or more megakaryocyte-derived nucleic acid molecules selected from the group consisting of mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs, and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein.

In another aspect, the invention relates to a pharmaceutical composition comprising a composition comprising megakaryocyte-derived extracellular vesicles disclosed herein and/or a plurality of megakaryocyte-derived extracellular vesicles and a pharmaceutically acceptable excipient or carrier.

In another aspect, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) Obtaining a megakaryocyte-derived extracellular vesicle of a composition comprising a megakaryocyte-derived extracellular vesicle and/or a plurality of megakaryocyte-derived extracellular vesicles as disclosed herein; (b) Incubating the megakaryocyte-derived extracellular vesicles with the therapeutic agent such that the therapeutic agent fills the lumen of the megakaryocyte-derived extracellular vesicles and produces a deliverable therapeutic agent; and (c) administering the deliverable therapeutic agent to the patient or contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In another aspect, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) Obtaining megakaryocyte-derived extracellular vesicles as disclosed herein; (b) Incubating megakaryocyte-derived extracellular vesicles with a therapeutic agent to cause the therapeutic agent to fill the lumen of and/or associate with the surface of the megakaryocyte-derived extracellular vesicles and produce a deliverable therapeutic agent; and (c) administering the deliverable therapeutic agent to the patient or contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In another aspect, the invention relates to a method of producing megakaryocyte-derived extracellular vesicles disclosed herein, comprising: (a) Obtaining human pluripotent stem cells, which are primary cd34+ hematopoietic stem cells derived from peripheral blood or umbilical cord blood or bone marrow; (b) Differentiating human pluripotent stem cells into megakaryocytes without addition of erythropoietin and without addition of thrombopoietin; and (c) isolating megakaryocyte-derived extracellular vesicles from the megakaryocytes.

In another aspect, the invention relates to a method of treating a myeloproliferative disease or disorder, such as MPN, with megakaryocyte-derived extracellular vesicles of the present disclosure.

Biomarker profile or fingerprint

In various embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are characterized by having unique biomarker profiles or fingerprints that distinguish them from, for example, naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles. In various embodiments, the megakaryocyte-derived extracellular vesicles of the present disclosure are characterized by a biomarker profile or fingerprint that includes an identity (e.g., presence or absence) or amount (e.g., the substantial presence or substantial absence of a biomarker in a population of megakaryocyte-derived extracellular vesicles); or the presence or absence of most megakaryocyte-derived extracellular vesicles in the population; or the percentage of megakaryocyte-derived extracellular vesicles with biomarkers).

In some embodiments, the substantially purified megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane surrounding a lumen and are derived from human pluripotent stem cells, wherein the lipid bilayer membrane comprises one or more proteins (i.e., biomarkers) associated therewith or embedded therein.

In some embodiments, the substantially purified megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane surrounding a lumen, wherein the lipid bilayer membrane comprises one or more proteins (i.e., biomarkers) associated therewith or embedded therein. In some embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells.

In embodiments, the lipid bilayer membrane comprises a protein selected from the group consisting of CD54, CD18, CD43, CD11b, CD62P, CD, CD61, CD21, CD51, phosphatidylserine (PS), CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a, and GPVI.

In embodiments, the lipid bilayer membrane comprises phosphatidylserine, such as, but not limited to, as determined by testing annexin V.

In embodiments, the lipid bilayer membrane comprises one or more proteins selected from CD62P, CD41 and CD61.

In embodiments, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% of megakaryocyte-derived extracellular vesicles comprising a lipid bilayer membrane comprising CD41 also comprise CD61 in the lipid bilayer membrane.

In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the expression and/or presence of one or more of CD54, CD18, CD43, CD11b, CD62P, CD41, CD61, CD21, CD51 and CLEC-2. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the expression and/or presence of one or more of PS, CD62P, LAMP-1 (CD 107 a), CD42b, CD9, CD43, CD31 and CD11 b. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the expression and/or presence of one or more of PS, CD61, CD62P, LAMP-1 (CD 107 a), CLEC-2 and CD 63. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the expression and/or presence of one or more of PS, CD62P, CLEC-2, CD9, CD31, CD147, CD32a and GPVI. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the expression and/or presence of one or more of PS, CD62P, LAMP-1 (CD 107 a), CLEC-2, CD9 and CD 31. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the expression and/or presence of one or more of CD62P, CD41 and CD61. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the basal expression and/or presence of one or more of CD54, CD18, CD43, CD11b, CD62P, CD41, CD61, CD21, CD51 and CLEC-2. In embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by the basal expression and/or presence of one or more of CD62P, CD41 and CD61. In some embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by not expressing and/or comprising a substantial amount of DRAQ5. In some embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by being substantially free of DRAQ5.

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 62P.

In embodiments, the megakaryocyte-derived extracellular vesicles are free or substantially free of CD62P.

In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are characterized by the expression and/or presence of CD62P over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD62P content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are characterized by the expression and/or presence of CD62P lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD62P content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the amount of CD62P of megakaryocyte-derived extracellular vesicles is about 4-fold to about 32-fold or about 8-fold to about 16-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the amount of CD62P of megakaryocyte-derived extracellular vesicles is about 15-fold or about 16-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the amount of CD62P of megakaryocyte-derived extracellular vesicles is about 32-fold to about 128-fold, about 50-fold to about 75-fold, or about 60-fold to about 70-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the amount of CD62P of megakaryocyte-derived extracellular vesicles is about 60-fold, about 64-fold, or about 70-fold lower than that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 41.

In embodiments, the megakaryocyte-derived extracellular vesicles comprise CD41.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD41 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD41 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD41 less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD41 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD41/CD61 content that is about 2-fold to about 8-fold, or about 2-fold to about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD41/CD61 content that is about 2-fold, about 3-fold, or about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD41/CD61 content that is about 1.1-fold to about 2-fold higher than platelet-derived extracellular vesicles (PLT EV). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD41/CD61 content that is about 1.1-fold or about 1.2-fold higher than platelet-derived extracellular vesicles (PLT EV). In embodiments, the megakaryocyte-derived extracellular vesicles have substantially the same CD41/CD61 content as platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 80% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 85% to about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 61.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD61 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD61 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD61 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD61 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD61 content that is about 2-fold to about 8-fold or about 2-fold to about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD61 content that is about 2-fold, about 3-fold, or about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD61 content that is about 1.1-fold to about 2-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD61 content that is about 1.1-fold or about 1.2-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD61 content of megakaryocyte-derived extracellular vesicles is substantially the same as that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 4. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 54.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD54 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD54 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the CD54 content of megakaryocyte-derived extracellular vesicles is about 2-fold to about 10-fold or about 2-fold to about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD54 content of megakaryocyte-derived extracellular vesicles is about 3-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD54 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold to about 4-fold or about 1.1-fold to about 2-fold higher than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD54 content of megakaryocyte-derived extracellular vesicles is about 1.5 times higher than that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD54 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD54 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 18.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD18 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD18 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD18 content that is about 2-fold to about 10-fold, 8-fold to about 64-fold, or about 16-fold to about 32-fold, or about 16-fold to about 24-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD18 content of megakaryocyte-derived extracellular vesicles is about 20-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD18 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold to about 4-fold or about 1.1-fold to about 2-fold higher than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD18 content that is about 1.5 times higher than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD18 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD18 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 43.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD43 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD43 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD43 content that is about 4-fold to about 64-fold, or about 8-fold to about 32-fold, or about 8-fold to about 16-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD43 content that is about 10-fold or about 12-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD43 content of megakaryocyte-derived extracellular vesicles is about 1.5-fold to about 8-fold or about 2-fold to about 4-fold higher than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD43 content that is about 3-fold or about 4-fold higher than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD43 less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD43 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD11 b.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD11b higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD11b content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD11b content that is about 2-fold to about 8-fold, or about 2-fold to about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD11b content that is about 3-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD11b content that is about 1.1-fold to about 4-fold or about 1.1-fold to about 2-fold higher than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD11b content that is about 1.5 times higher than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD11b less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD11b content that is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 21.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD21 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD21 content that is about 2-fold to about 64-fold, about 4-fold to about 32-fold, or about 8-fold to about 16-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD21 content that is about 10-fold or about 12-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD21 content that is about 2-fold to about 8-fold, or about 4-fold to about 8-fold higher than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD21 content that is about 4-fold or about 5-fold higher than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD21 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD21 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 51.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD51 higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD51 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD51 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD51 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD51 content that is about 1.1-fold to about 4-fold, or about 1.1-fold to about 2-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the CD51 content of megakaryocyte-derived extracellular vesicles is about 1.5-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the CD51 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold to about 4-fold, or about 1.1-fold to about 2-fold lower than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD51 content of megakaryocyte-derived extracellular vesicles is about 1.5-fold lower than that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by expression and/or presence of CLEC-2 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CLEC-2 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by expression and/or presence of CLEC-2 less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CLEC-2 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the CLEC-2 content of megakaryocyte-derived extracellular vesicles is about 2-fold to about 16-fold, or about 4-fold to about 8-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the CLEC-2 content of megakaryocyte-derived extracellular vesicles is about 4-fold or about 5-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the CLEC-2 content of megakaryocyte-derived extracellular vesicles is about 4-fold to about 32-fold, or about 8-fold to about 16-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CLEC-2 content of megakaryocyte-derived extracellular vesicles is about 10-fold or about 12-fold lower than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

In embodiments, megakaryocyte-derived extracellular vesicles are free or substantially free of LAMP-1 (CD 107A).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of LAMP-1 (CD 107A) higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the LAMP-1 (CD 107A) content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of LAMP-1 (CD 107A) lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the LAMP-1 (CD 107A) content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a LAMP-1 (CD 107A) content that is about 1-fold to about 2-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the amount of LAMP-1 (CD 107A) of megakaryocyte-derived extracellular vesicles is substantially the same as Platelet Free Plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a LAMP-1 (CD 107A) content that is about 2-fold to about 8-fold, or about 2-fold to about 4-fold lower than the platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a LAMP-1 (CD 107A) content that is about 3-fold or about 4-fold lower than that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 63.

In some embodiments, about 1% to about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 5% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 10% to about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In some embodiments, about 13% to about 19% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD63 higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD63 content that is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD63 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD63 content that is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD63 content that is about 2-fold to about 8-fold, or about 2-fold to about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD63 content that is about 2-fold or about 3-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD63 content that is about 1.1-fold to about 2-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD63 content that is about 1.1-fold or about 1.2-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD63 content of megakaryocyte-derived extracellular vesicles is substantially the same as that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 80% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 90% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD42 b.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD42b higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD42b content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD42b less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD42b content that is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD42b content that is about 8-fold to about 32-fold, or about 10-fold to about 20-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD42b content that is about 16-fold or about 20-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD42b content that is about 64-fold to about 128-fold, or about 50-fold to about 75-fold lower than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD42b content that is about 64-fold or about 70-fold lower than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 20% to about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 35% to about 55% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 9.

In some embodiments, about 50% to about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 60% to about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 62% to about 68% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In some embodiments, about 65% to about 66% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD9 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD9 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is about 1.5-fold to about 4-fold, or about 2-fold to about 4-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is about 2-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold to about 2-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold or about 1.2-fold lower than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD9 content of megakaryocyte-derived extracellular vesicles is substantially the same as platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 31.

In some embodiments, about 5% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 10% to about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 10% to about 35% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In some embodiments, about 13% to about 31% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD31 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD31 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD31 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD31 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD31 content that is about 1.1-fold to about 4-fold, or about 1.1-fold to about 2-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD31 content that is about 1.5 times lower than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD31 content that is about 2-fold to about 4-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD31 content that is about 2-fold or about 3-fold lower than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 70% or less of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 47.

In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 10% to about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 20% to about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In some embodiments, about 25% to about 35% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD47 higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD47 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression of CD47 and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD47 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD47 content that is about 128-fold to about 512-fold, or about 256-fold to about 512-fold, or about 250-fold to about 300-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD47 content that is about 256-fold or about 300-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD47 content that is about 1.1-fold to about 2-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD47 content that is about 1.1-fold or about 1.5-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the amount of CD47 of megakaryocyte-derived extracellular vesicles is substantially the same as platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 147.

In some embodiments, about 1% to about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 3% to about 8% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In some embodiments, about 4% to about 7% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD147 over naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD147 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD147 less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD147 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD147 content that is about 2-fold to about 8-fold, or about 2-fold to about 4-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the CD147 content of megakaryocyte-derived extracellular vesicles is about 2-fold or about 3-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the CD147 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold to about 2-fold lower than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the CD147 content of megakaryocyte-derived extracellular vesicles is about 1.1-fold or about 1.2-fold lower than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the amount of CD147 of megakaryocyte-derived extracellular vesicles is substantially the same as platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD32 a.

In embodiments, the megakaryocyte-derived extracellular vesicles are free or substantially free of CD32a.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD32a higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the CD32a content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD32a less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD32a content that is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the megakaryocyte-derived extracellular vesicles have a CD32a content from about 50-fold to about 100-fold, 128-fold to about 512-fold, or about 256-fold to about 512-fold, or about 250-fold to about 300-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD32a content that is about 250-fold or about 256-fold lower than platelet-free plasma (PFP) MkEV. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD32a content that is about 250-fold to about 400-fold, or about 256-fold to about 512-fold lower than platelet-derived extracellular vesicles (PLT EVs). In embodiments, the megakaryocyte-derived extracellular vesicles have a CD32a content that is about 256-fold or about 300-fold lower than platelet-derived extracellular vesicles (PLT EVs).

In embodiments, greater than about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In some embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In some embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In some embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In some embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In some embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In some embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, greater than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI.

In embodiments, about 50% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI.

In some embodiments, less than about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 30% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 25% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 20% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 15% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 10% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, less than about 1% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI.

In some embodiments, about 1% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 1% to about 25% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 1% to about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 1% to about 2% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 50% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 75% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 90% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In some embodiments, about 95% to about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI.

In some embodiments of the present invention, in some embodiments, less than about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by expression of GPVI and/or the presence of higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the GPVI content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by expression of GPVI and/or the presence of less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the GPVI content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, the GPVI content of megakaryocyte-derived extracellular vesicles is about 8-fold to about 64-fold, or about 16-fold to about 32-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the GPVI content of megakaryocyte-derived extracellular vesicles is about 30-fold or about 32-fold higher than platelet-free plasma (PFP) MkEV. In embodiments, the GPVI content of megakaryocyte-derived extracellular vesicles is about 2-fold to about 16-fold, or about 4-fold to about 8-fold lower than that of platelet-derived extracellular vesicles (PLT EVs). In embodiments, the GPVI content of megakaryocyte-derived extracellular vesicles is about 4-fold or about 5-fold lower than that of platelet-derived extracellular vesicles (PLT EVs).

In embodiments, megakaryocyte-derived extracellular vesicles are free or substantially free of LAMP-1 (CD 107A). In embodiments, megakaryocyte-derived extracellular vesicles have fewer LAMP-1 (CD 107A) than naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles.

In embodiments, less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by having CD62P and being free or substantially free of LAMP-1 (CD 107A).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by a population of megakaryocyte-derived extracellular vesicles, wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising LAMP-1 (CD 107A), and greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P.

In some embodiments, less than about 70%, or less than about 60%, or less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising Phosphatidylserine (PS).

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by expression and/or presence of Phosphatidylserine (PS) less than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In embodiments, the Phosphatidylserine (PS) content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EVs), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by being free or substantially free of Phosphatidylserine (PS).

In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by a population of megakaryocyte-derived extracellular vesicles, wherein less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising Phosphatidylserine (PS), and greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by a population of megakaryocyte-derived extracellular vesicles, wherein about 20% to about 40% of the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes that are positive for Phosphatidylserine (PS) and/or Phosphatidylserine (PS), about 80% to about 99%, or about 85% to about 99% of the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD61, and about 25% to about 55%, or about 35% to about 55% of the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CD 9.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by expression and/or presence of CD41 over naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles. In embodiments, the CD41 content of megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are characterized by the expression and/or presence of CD41 less than naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles have a CD41 content that is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold lower than naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles contain full length filamin a.

In embodiments, the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising phosphatidylserine. In embodiments, the megakaryocyte-derived extracellular vesicles are characterized by a population of megakaryocyte-derived extracellular vesicles, wherein greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, greater than about 95%, or greater than about 99% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising phosphatidylserine.

In embodiments, the megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes that are annexin V positive. For example, annexin V, which interacts with Phosphatidylserine (PS), can be used as a surrogate for phosphatidylserine expression and/or the presence or absence. In embodiments, megakaryocyte-derived extracellular vesicles are characterized by a population of megakaryocyte-derived extracellular vesicles, wherein greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% of the megakaryocyte-derived extracellular vesicles are PS positive.

In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by a population of megakaryocyte-derived extracellular vesicles, wherein about 20% to about 40% comprise phosphatidylserine and/or phosphatidylserine-positive lipid bilayer membranes.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2, 3, 4, 5, 6, 7, or 8 of Phosphatidylserine (PS), CD62P, LAMP-1 (CD 107 a), CD42b, CD9, CD43, CD31, and CD11 b. In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2, 3, or 4 of PS, CD62P, CD, and CD11 b. In embodiments, the level of one or more of Phosphatidylserine (PS), CD62P, LAMP-1 (CD 107 a), CD42b, CD9, CD43, CD31, and CD11b in megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized as being substantially free of DRAQ5.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2, 3, 4, 5, or 6 of Phosphatidylserine (PS), CD61, CD62P, LAMP-1 (CD 107 a), CLEC-2, and CD 63. In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2 or 3 of PS, CD61, and CD 63. In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise Phosphatidylserine (PS) and CD61. In embodiments, the level of one or more of Phosphatidylserine (PS), CD61, CD62P, LAMP-1 (CD 107 a), CLEC-2, and CD63 in megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold, or about 1000-fold higher than that of naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized as being substantially free of DRAQ5.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2, 3, 4, 5, 6, 7, or 8 of Phosphatidylserine (PS), CD62P, CLEC-2, CD9, CD31, CD147, CD32a, and GPVI. In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2, 3, or 4 of Phosphatidylserine (PS), CD9, CD31, and CD 147. In embodiments, the level of one or more of Phosphatidylserine (PS), CD62P, CLEC-2, CD9, CD31, CD147, CD32a, and GPVI in megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold, or about 1000-fold higher than naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized as being substantially free of DRAQ5.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2, 3, 4, 5, or 6 of Phosphatidylserine (PS), CD62P, LAMP-1 (CD 107 a), CLEC-2, CD9, and CD 31. In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2 or 3 of Phosphatidylserine (PS), CD62P, and CD9. In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise PS and CD9. In embodiments, the level of one or more of Phosphatidylserine (PS), CD62P, LAMP-1 (CD 107 a), CLEC-2, CD9, and CD31 in megakaryocyte-derived extracellular vesicles is about 2-fold, or about 10-fold, or about 50-fold, or about 100-fold, or about 300-fold, or about 500-fold, or about 1000-fold higher than that of naturally occurring megakaryocyte-derived extracellular vesicles, platelet-derived vesicles or extracellular vesicles, such as platelet-derived extracellular vesicles (PLT EV), and/or platelet-free plasma (PPF) megakaryocyte-derived extracellular vesicles. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In some embodiments, the megakaryocyte-derived extracellular vesicles are characterized as being substantially free of DRAQ5.

In some embodiments, the megakaryocyte-derived extracellular vesicles and/or the plurality of megakaryocyte-derived extracellular vesicles and/or the population of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane, wherein

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD54, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD18, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD43, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11b, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD62P, and/or

Greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, greater than about 95%, or greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD41, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD21, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD51, and/or

Greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, greater than about 95%, or greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD61, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD147, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD31, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD47, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32a, and/or

Greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, greater than about 95%, or greater than about 99% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD9, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise CLEC-2-containing lipid bilayer membranes, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising LAMP-1 (CD 107 a), and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD24b, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising GVPI, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD63, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising Phosphatidylserine (PS). In some embodiments, greater than about 40%, greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, greater than about 95%, or greater than about 99% of the megakaryocyte-derived extracellular vesicles and/or the plurality of megakaryocyte-derived extracellular vesicles and/or the population of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41.

Size spectrum or fingerprint

In various embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are characterized by having a unique size (e.g., blastula diameter) profile or fingerprint that distinguishes them from, for example, naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles. In various embodiments, the megakaryocyte-derived extracellular vesicles of the present disclosure are characterized by their size spectra or fingerprints that facilitate the formation of larger particles, e.g., that render them more carrier-competent than naturally occurring megakaryocyte-derived extracellular vesicles and/or platelet-derived vesicles or extracellular vesicles.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles from about 30nm to about 100 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles from about 30nm to about 400 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles of about 100nm to about 200 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles of about 100nm to about 300 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles from about 100nm to about 500 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles from about 100nm to about 600 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles having an average diameter of about 200 nm.

In various embodiments, megakaryocyte-derived extracellular vesicles of the invention are characterized by a bias towards the formation of particles having an average diameter of about 250 nm.

In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially less than about 100nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 30nm and about 300 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 30nm and about 400 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 100nm and about 300 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 200nm and about 300 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 300nm and about 400 nm. In embodiments, the megakaryocyte-derived extracellular vesicles have a diameter substantially between about 400nm and about 500 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 500nm and about 600 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 600nm and about 700 nm. In embodiments, the megakaryocyte-derived extracellular vesicles have a diameter substantially between about 700nm and about 800 nm. In embodiments, megakaryocyte-derived extracellular vesicles have diameters substantially between about 800nm and about 900 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 900nm and about 1000 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 500nm and about 1000 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 600nm and about 1000 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 100nm and about 500 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 100nm and about 600 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 150nm and about 500 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 100nm and about 200 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 100nm and about 200 nm. In embodiments, megakaryocyte-derived extracellular vesicles have a diameter substantially between about 200nm and about 600 nm. In some embodiments, the megakaryocyte-derived extracellular vesicles have a diameter substantially between about 30nm and 100nm, or between about 30nm and 400nm, or between about 100nm and about 200nm, or between about 100nm and about 500nm, or between about 200nm and about 350nm, or between about 400nm and about 600 nm.

In embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more, of the megakaryocyte-derived extracellular vesicles have a diameter between about 30 and 100 nm.

In embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more, of the megakaryocyte-derived extracellular vesicles have a diameter between about 30 and 400 nm.

In embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more, of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 200 nm.

In embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more, of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 300 nm.

In some embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of megakaryocyte-derived extracellular vesicles have a diameter between about 200nm and about 350 nm.

In some embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 600 nm.

In some embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of megakaryocyte-derived extracellular vesicles have a diameter between about 400nm and about 600 nm.

In some embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of megakaryocyte-derived extracellular vesicles have a diameter between about 200nm and about 600 nm.

In some embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more, of the megakaryocyte-derived extracellular vesicles have a diameter of between about 30 and about 100nm, and/or between about 30 and about 400nm, and/or between about 100 and about 200nm, and/or between about 100 and about 300nm, and/or between about 200 and about 350nm, and/or between about 400 and about 600 nm.

In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise various subsets of vesicles of different diameters. For example, in embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise one or more (e.g., one, or two, or three, or four) of the following: a sub-population of about 50nm in diameter, a sub-population of about 150nm in diameter, a sub-population of about 200nm in diameter, a sub-population of about 250nm in diameter, a sub-population of about 300nm in diameter, a sub-population of about 400nm in diameter, a sub-population of about 500nm in diameter, and a sub-population of about 600nm in diameter. In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise one or more (e.g., one, or two, or three, or four) of the following: a sub-population of about 45nm in diameter, a sub-population of about 135nm in diameter, a sub-population of about 285nm in diameter, and a sub-population of about 525nm in diameter.

In some embodiments, about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more, of the megakaryocyte-derived extracellular vesicles have a diameter of about 50nm, and/or a diameter of about 150nm, and/or a diameter of about 300nm, and/or a diameter of about 500nm.

In some embodiments, the megakaryocyte-derived extracellular vesicle population exhibits the following characteristics:

a) About 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles in the population are substantially free of nuclei;

b) About 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of megakaryocyte-derived extracellular vesicles have diameters between about 100nm and about 600 nm;

c) About 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more of the megakaryocyte-derived extracellular vesicles in the population contain CD41; and

d) The population comprises about 1x10 ⁷ Or more, about 1.5x10 ⁷ Or more, about 5x10 ⁷ Or more, about 1x10 ⁸ Or more, about 1.5x10 ⁸ Or more, about 5x10 ⁸ Or more, about 1x10 ⁹ Or more, about 5x10 ⁹ Or more, about 1x10 ¹⁰ Or more, or about 1x10 ¹⁰ Or more megakaryocyte-derived extracellular vesicles.

c) About 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, or about 90% or more of the megakaryocyte-derived extracellular vesicles in the population contain CD61; and

The present disclosure is intended to encompass any method for determining the nuclear content in a megakaryocyte-derived extracellular vesicle population. Non-limiting examples of methods include staining megakaryocyte-derived extracellular vesicles with a nuclear stain such as DRAQ5, wherein an under staining indicates that the megakaryocyte-derived extracellular vesicles are substantially free of nuclei.

Sources and characterization of megakaryocyte-derived extracellular vesicles

Megakaryocytes are large polyploid cells derived from hematopoietic stem and progenitor cells, contained in CD34 ⁺ A cell compartment. In embodiments, megakaryocytes are characterized by the expression and/or presence of one or more of CD41, CD62P, GPVI, CLEC-2, CD42b and CD 61. In embodiments, the megakaryocyte is one or more of cd42b+, cd61+, and dna+. One morphological feature of mature megakaryocytes is the development of large multilobal nuclei. Mature megakaryocytes can stop proliferating, but continue to increase their DNA content by intracellular mitosis while the cell size increases in parallel.

In some embodiments, megakaryocytes can shed pre-platelets and primitive platelets as well as platelet-like particles in addition to extracellular vesicles. These shed portions can mature into platelets. In some embodiments, the pre-platelets and the original platelets as well as the platelet-like particles are distinct products, which can be distinguished by size, morphology, biomarker expression and/or presence, and function.

Megakaryocytes are derived from multipotent Hematopoietic Stem Cell (HSC) precursors. HSCs are produced primarily by the liver, kidneys, spleen and bone marrow, and are capable of producing a variety of blood cells based on the signals they receive.

Thrombopoietin (TPO) is the primary signal that induces HSC differentiation into megakaryocytes. Other molecular signals for inducing megakaryocyte differentiation include granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, IL-11, SCF, fms-like tyrosine kinase 3 ligand (FLT 3L), interleukin 9 (IL-9), and the like. The details of the generation are also described elsewhere herein.

In some embodiments, the substantially purified megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells.

In embodiments, the human pluripotent stem cells are primary cd34+ hematopoietic stem cells. In embodiments, the primary cd34+ hematopoietic stem cells are derived from peripheral blood or umbilical cord blood. In embodiments, the peripheral blood is adult peripheral blood (mPB) mobilized by granulocyte colony stimulating factor. In some embodiments, the human pluripotent stem cells are HSCs produced by the liver, kidney, spleen, or bone marrow. In some embodiments, the HSCs are produced by the liver. In some embodiments, the HSCs are produced by the kidneys. In some embodiments, the HSCs are produced by the spleen. In some embodiments, the HSCs are produced from bone marrow. In some embodiments, HSCs are induced to differentiate into megakaryocytes by receiving a molecular signal selected from one or more of the following: TPO, GM-CSF, IL-3, IL-6, IL-11, SCF, flt3L, IL-9, and the like. In some embodiments, the molecular signal is TPO. In some embodiments, the molecular signal is GM-CSF. In some embodiments, the molecular signal is IL-3. In some embodiments, the molecular signal is IL-6. In some embodiments, the molecular signal is IL-11. In some embodiments, the molecular signal is SCF. In some embodiments, the molecular signal is Flt3L. In some embodiments, the molecular signal is IL-9.

In some embodiments, the molecular signal is a chemokine.

In some embodiments, the molecular signal promotes cell fate decisions toward megakaryocytogenesis.

In some embodiments, the molecular signal lacks Erythropoietin (EPO).

In embodiments, the human pluripotent stem cells are Embryonic Stem Cells (ESCs). ESCs have the ability to form cells from all three germ layers of the body, regardless of the method by which the embryonic stem cells are obtained. Embryonic stem cells are functional stem cells that may have one or more of the following characteristics: (a) Capable of inducing teratomas when transplanted into immunodeficient mice; (b) Capable of differentiating cell types of all three germ layers (i.e., ectodermal, mesodermal, and endodermal cell types); (c) One or more markers (e.g., oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, SSEA-5 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.) are expressed in embryonic stem cells.

In embodiments, the human pluripotent stem cells are induced pluripotent stem cells (iPC). Mature differentiated cells can be reprogrammed and dedifferentiated into embryonic-like cells with embryonic stem-like properties. ipscs may be generated using fetal, postnatal, neonatal, juvenile or adult somatic cells. Fibroblasts may be reversed to multipotency by retroviral transduction of, for example, certain transcription factors, thereby producing iPS. In some embodiments, iPS is produced from a variety of tissues, including fibroblasts, keratinocytes, melanocyte blood cells, bone marrow cells, adipocytes, and tissue resident progenitor cells. In some embodiments, the iPSC is produced by one or more reprogramming or mountain (Yamanaka) factors, such as Oct3/4, sox2, klf4, and c-Myc. In certain embodiments, at least two, three, or four reprogramming factors are expressed in the somatic cells to reprogram the somatic cells.

Once the pluripotent cell has completed differentiation and becomes a mature megakaryocyte, it begins the process of producing platelets, which do not contain nuclei, and may be about 1-3um in diameter. Megakaryocytes also produce extracellular vesicles.

In embodiments, megakaryocytes of the present invention are induced to facilitate production of megakaryocyte-derived extracellular vesicles rather than platelets. That is, in embodiments, megakaryocytes of the present invention produce substantially more extracellular vesicles of megakaryocyte origin than platelets. In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of platelets. In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure contain less than about 10%, or less than about 7%, or less than about 5%, or less than about 3%, or less than about 2%, or less than about 1% platelets.

In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of platelet-derived extracellular vesicles. In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure contain less than about 10%, or less than about 7%, or less than about 5%, or less than about 3%, or less than about 2%, or less than about 1% of platelet-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of organelles. Non-limiting examples of contaminating organelles include, but are not limited to, mitochondria and nuclei. In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of mitochondria. In some embodiments, the formulation comprising megakaryocyte-derived extracellular vesicles of the present disclosure is substantially free of exosomes. In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise organelles.

In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of nuclei. In some embodiments, from about 80% to about 100%, from about 85% to about 100%, from about 90% to about 100%, or from about 95% to about 100% of megakaryocyte-derived extracellular vesicles in the population are substantially free of nuclei. In some embodiments, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, greater than about 99%, or about 100% of the megakaryocyte-derived extracellular vesicles in the population are substantially free of nuclei.

Target finding

Megakaryocyte-derived extracellular vesicles can home to a range of target cells. When megakaryocyte-derived extracellular vesicles bind to target cells, they can release their cargo through various mechanisms of target cell internalization of megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vivo. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vitro. In some embodiments, megakaryocyte-derived extracellular vesicles home to bone marrow in vivo with about 2-fold, or about 3-fold, or about 4-fold, or about 5-fold, or about 6-fold, or about 7-fold, or about 8-fold, or about 9-fold, or about 10-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types.

In some embodiments, megakaryocyte-derived extracellular vesicles home to one or more myelogenous cells in bone marrow in vivo. In some embodiments of the present invention, in some embodiments, one or more myeloblasts selected from the group consisting of myeloblasts, promyelocytes, neutrophils, eosinophils, and combinations thereof neutrophils, eosinophils, lobular nuclear neutrophils, lobular nuclear eosinophils, lobular nuclear basophils and mast cells. In some embodiments, megakaryocyte-derived extracellular vesicles home to one or more erythropoietic cells in the bone marrow in vivo. In some embodiments, the one or more erythropoietic cells are selected from the group consisting of orthoerythroblasts, basophils, multi-chromatic erythroblasts, and orthochromatic erythroblasts. In some embodiments, megakaryocyte-derived extracellular vesicles home to one or more of plasma cells, reticulocytes, lymphocytes, monocytes, and megakaryocytes in vivo.

In some embodiments, megakaryocyte-derived extracellular vesicles home to one or more hematopoietic cells in the bone marrow in vivo. In some embodiments, megakaryocyte-derived extracellular vesicles home to one or more hematopoietic cells, such as thrombogenic cells, in the bone marrow.

In some embodiments, megakaryocyte-derived extracellular vesicles home to one or more hematopoietic stem cells in the bone marrow in vivo.

In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to HSCs in vivo. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to HSCs in vitro. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 2-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 3-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 4-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 5-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 6-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 7-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 8-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 9-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 10-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types.

In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vivo. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vitro. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 2-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 3-fold higher specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 4-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 5-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 6-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 7-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 8-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 9-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes in vivo with about 10-fold greater specificity than for another cell type, or for another organ, or for all other combined cell types.

In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vivo. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vitro. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 2-fold higher specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 3-fold higher specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 4-fold higher specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 5-fold greater specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 6-fold greater specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 7-fold greater specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 8-fold higher specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 9-fold higher specificity than to another cell type, or to another organ, or to all other combined cell types. In some embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 10-fold greater specificity than to another cell type, or to another organ, or to all other combined cell types.

In one aspect, the present disclosure provides methods of treating a myeloproliferative disease or disorder, including methods of transferring a deliverable therapeutic agent.

In some embodiments, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) obtaining megakaryocyte-derived extracellular vesicles; (b) Incubating megakaryocyte-derived extracellular vesicles with a therapeutic agent capable of treating a myeloproliferative disease or disorder, such that the therapeutic agent fills the lumen of the megakaryocyte-derived extracellular vesicles and produces a deliverable therapeutic agent; and (c) administering the deliverable therapeutic agent to the patient or contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In some embodiments, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) obtaining megakaryocyte-derived extracellular vesicles; (b) Incubating megakaryocyte-derived extracellular vesicles with a therapeutic agent capable of treating a myeloproliferative disease or disorder, such that the therapeutic agent associates with the surface of the megakaryocyte-derived extracellular vesicles and produces a deliverable therapeutic agent; and (c) administering the deliverable therapeutic agent to the patient or contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In one aspect, the present disclosure provides ex vivo methods of transferring deliverable therapeutic agents useful in the treatment of myeloproliferative diseases or disorders. In some embodiments, the method comprises: (a) obtaining megakaryocyte-derived extracellular vesicles; (b) Incubating megakaryocyte-derived extracellular vesicles with a therapeutic agent capable of treating a myeloproliferative disease or disorder, such that the therapeutic agent fills the lumen of the megakaryocyte-derived extracellular vesicles and produces a deliverable therapeutic agent; (c) obtaining biological cells from the patient; and (d) contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In one aspect, the present disclosure provides in vivo methods for transferring a deliverable therapeutic agent. In some embodiments, the method comprises: (a) obtaining megakaryocyte-derived extracellular vesicles; (b) Incubating megakaryocyte-derived extracellular vesicles with a therapeutic agent capable of treating a myeloproliferative disease or disorder, such that the therapeutic agent associates with the surface of the megakaryocyte-derived extracellular vesicles and produces a deliverable therapeutic agent; (c) obtaining biological cells from the patient; and (d) contacting the deliverable therapeutic agent with the biological cells in vitro and administering the contacted biological cells to the patient.

In some embodiments, contacting the deliverable therapeutic agent capable of treating the myeloproliferative disease or disorder with the biological cell comprises co-culturing the deliverable therapeutic agent with the biological cell to provide transfer of cargo from the deliverable therapeutic agent to the biological cell.

In embodiments, megakaryocyte-derived extracellular vesicles bind to cell surface receptors on patient cells. In embodiments, megakaryocyte-derived extracellular vesicles bind to cell surface receptors on biological cells following the contacting of step (c). In some embodiments, the biological cell is one or more of a cancer cell, a tumor cell, a virus-infected cell, an epithelial cell, an endothelial cell, a nerve cell, a muscle cell, a connective tissue cell, a healthy cell, a diseased cell, a differentiated cell, and a pluripotent cell.

In embodiments, megakaryocyte-derived extracellular vesicles are fused with the extracellular membrane of patient cells. In embodiments, megakaryocyte-derived extracellular vesicles are fused to the extracellular membrane of the biological cell of step (c). In some embodiments, the biological cell is one or more of a cancer cell, a tumor cell, a virus-infected cell, an epithelial cell, an endothelial cell, a nerve cell, a muscle cell, a connective tissue cell, a healthy cell, a diseased cell, a differentiated cell, and a pluripotent cell.

In embodiments, megakaryocyte-derived extracellular vesicles are endocytosed by cells of the patient. In embodiments, the megakaryocyte-derived extracellular vesicles are endocytosed by the biological cells of step (c). In some embodiments, the biological cell is one or more of a cancer cell, a tumor cell, a virus-infected cell, an epithelial cell, an endothelial cell, a nerve cell, a muscle cell, a connective tissue cell, a healthy cell, a diseased cell, a differentiated cell, and a pluripotent cell.

Method for producing megakaryocyte-derived extracellular vesicles

In some embodiments, the cell culture process is used to generate allogeneic megakaryocyte-derived extracellular vesicles from primary human peripheral blood cd34+ HSCs. In some embodiments, megakaryocyte-derived extracellular vesicles are produced by a method comprising obtaining primary human peripheral blood cd34+ HSCs from a commercial vendor and transitioning from a stem cell maintenance medium to a HSC expansion medium. In some embodiments, megakaryocyte-derived extracellular vesicles are produced by a method comprising obtaining primary human cord blood cd34+ HSCs. In some embodiments, megakaryocyte-derived extracellular vesicles are produced by a method comprising obtaining primary human bone marrow cd34+ HSCs. In embodiments, the method further comprises placing the HSC culture in megakaryocyte differentiation medium and collecting megakaryocyte-derived extracellular vesicles from the culture supernatant. Thus, in embodiments, megakaryocyte-derived extracellular vesicles of the invention are produced from starting cd34+ HSCs.

In some embodiments, megakaryocyte differentiation is demonstrated by biomarker expression and/or presence of one or more of CD41, CD61, CD42b, megakaryocyte-specific cytoskeletal protein β1-tubulin, alpha particle components (e.g., platelet factor 4 and von willebrand factor), secretory granules, and ultrastructural features (e.g., invagination membrane system, dense tube system, multivesicular body).

In some embodiments, megakaryocytes produce from about 10 to about 3000, from about 50 to about 2600, from about 80 to about 500, from about 500 to about 2600, or from about 500 to about 1500 megakaryocyte-derived extracellular vesicles/cells.

In some embodiments, nanoparticle analysis, electron microscopy, flow cytometry, and/or western blotting are used to confirm biomarker expression and/or presence and composition of megakaryocyte-derived extracellular vesicles.

In embodiments, megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced without the addition of erythropoietin. In embodiments, megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced with the addition of thrombopoietin.

In some embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of nucleic acids. In some embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of autologous nucleic acids. In some embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of RNA. In some embodiments, the megakaryocyte-derived extracellular vesicles comprise nucleic acids. In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise autologous nucleic acids. In some embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise autologous RNA. Non-limiting examples of RNAs include rRNA, siRNA, microrna, regulatory RNA, and/or non-coding and coding RNAs. In some embodiments, megakaryocyte-derived extracellular vesicles are substantially free of RNA from cells from which the vesicles are derived. In non-limiting examples, megakaryocyte-derived extracellular vesicles are free of RNA due to the method of preparing the vesicles and/or due to the use of RNase to remove native RNA.

In some embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA. In some embodiments, megakaryocyte-derived extracellular vesicles are substantially free of DNA from the cells from which the vesicles are derived. In non-limiting examples, megakaryocyte-derived extracellular vesicles are free of DNA due to the method of preparing the vesicles and/or due to the use of dnase to remove native DNA. In embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of one or more of the following: (a) megakaryocytes, (b) platelets derived from megakaryocytes, and (c) extracellular vesicles derived from platelets.

In some embodiments, human peripheral blood cd34+ cells mobilized by frozen granulocyte colony-stimulating factor (G-CSF) are obtained and cultured to megakaryocytes, followed by enrichment of the cd41+ cells (megakaryocytes) prior to culturing, and then measuring CD41 expression and/or presence and analyzing the concentration of megakaryocyte-derived extracellular vesicles in the cell culture by flow cytometry or nanoparticles. In some embodiments, megakaryocyte-derived extracellular vesicles are produced by a series of centrifugation, e.g., at increasing speeds/forces. In some embodiments, megakaryocyte-derived extracellular vesicles are produced by the following method: (a) Removing cells from the culture medium, for example, by centrifugation at about 150 Xg, for example, about 10 minutes; (b) Platelet-like particles (PLP) and cell debris are removed by centrifugation at, for example, about 1000×g for, for example, about 10 minutes; (c) Megakaryocyte-derived extracellular vesicles are enriched from the supernatant by ultracentrifugation, e.g., at about 25,000rpm (38000×g), e.g., at about 4 ℃ for about 1 hour.

In some embodiments, a pH and pO with different pH are used ₂ Or pCO ₂ And a multi-stage culture process of a mixture of different cytokines to greatly increase megakaryocyte production.

In some embodiments, megakaryocytes are produced by the following method: (a) Culturing cd34+ HSCs with a molecular signal/factor/cytokine mixture that promotes megakaryocyte progenitor cell production; (b) The cells are transferred to different conditions to expand mature megakaryocytes from the progenitor cells. In some embodiments, commercial media is used. In some embodiments, serum-free medium is used. In some embodiments, the pH is changed to increase megakaryocyte production. In some embodiments, the CO is altered ₂ Percentage to increase megakaryocyte production. In some embodiments, the properties of the molecular signal/factor/cytokine are altered to increase megakaryocyte production. In embodiments, the molecular signal/factor/cytokine mixture comprises one or more of TPO, GM-CSF, IL-3, IL-6, IL-11, SCF, flt3L, IL-9, and the like.

In embodiments, the present production method further involves the step of characterizing one or more of CD54, CD18, CD43, CD11b, CD62P, CD41, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a, and GPVI of the resulting megakaryocyte-derived extracellular vesicles, for example, but not limited to, by nanoparticle analysis, electron microscopy, flow cytometry, and/or western blot analysis. In embodiments, the production methods of the invention further involve the step of phosphatidylserine characterization of the resulting megakaryocyte-derived extracellular vesicles, such as, but not limited to, by testing annexin V, such as, but not limited to, by nanoparticle analysis, electron microscopy, flow cytometry, and/or western blot analysis.

In some embodiments, megakaryocyte-derived extracellular vesicles are produced from mature megakaryocytes. In some embodiments, megakaryocyte-derived extracellular vesicles are produced from immature megakaryocytes.

In some embodiments, the method of producing megakaryocyte-derived extracellular vesicles is standardized to achieve large scale production.

In some embodiments, the process of the invention for producing megakaryocyte-derived extracellular vesicles varies by less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% between batches/donors. In some embodiments, methods of producing megakaryocyte-derived extracellular vesicles have been developed such that the batch-to-batch/donor-to-donor variability is less than 12.5%. In some embodiments, methods of producing megakaryocyte-derived extracellular vesicles have been developed such that the batch-to-batch/donor-to-donor variability is less than 10%. In some embodiments, methods of producing megakaryocyte-derived extracellular vesicles have been developed such that the batch-to-batch/donor-to-donor variability is less than 7.5%. In some embodiments, methods of producing megakaryocyte-derived extracellular vesicles have been developed such that the batch-to-batch/donor-to-donor variability is less than 5%. In some embodiments, methods of producing megakaryocyte-derived extracellular vesicles have been developed such that the batch-to-batch/donor-to-donor variability is less than 2.5%.

In some embodiments, the population comprises about 1x10 ⁷ Or more, about 1.5x10 ⁷ Or more, about 5x10 ⁷ Or more, 1x10 ⁸ Or more, about 1.5x10 ⁸ Or more, about 5x10 ⁸ Or more, about 1x10 ⁹ Or more, about 5x10 ⁹ Or more, about 1x10 ¹⁰ Or more, or about 1x10 ¹⁰ Or more megakaryocyte-derived extracellular vesicles.

In some embodiments, the population comprises about 2x10 ¹⁰ Up to about 1x10 ¹¹ About 4x10 ¹⁰ Up to about 9x10 ¹¹ Or about 5x10 ¹⁰ To about 8.5x10 ¹¹ Extracellular vesicles derived from megakaryocytes.

In some embodiments, megakaryocyte-derived extracellular vesicles are isolated as a population. In some embodiments, the population of megakaryocyte-derived extracellular vesicles is substantially homogeneous.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD54. In some embodiments, from about 0% to about 5%, from about 0% to about 10%, from about 15% to about 90%, from about 30% to about 80%, or from about 50% to about 70% of the megakaryocyte-derived extracellular vesicles in the population comprise CD54. In some embodiments, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD54. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise CD54. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD54.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD18. In some embodiments, from about 0% to about 5%, from about 0% to about 10%, from about 15% to about 90%, from about 30% to about 80%, or from about 50% to about 70% of the megakaryocyte-derived extracellular vesicles in the population comprise CD18. In some embodiments, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD18. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise CD18. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD18.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD43. In some embodiments, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, or about 1% to about 15%, about 0% to about 5%, or about 0% to about 10% of the megakaryocyte-derived extracellular vesicles in the population comprise CD43. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise CD43. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD43.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD11b. In some embodiments, from about 0% to about 5%, from about 0% to about 10%, from about 1% to about 50%, from about 5% to about 40%, or from about 10% to about 35% of the megakaryocyte-derived extracellular vesicles in the population comprise CD11b. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles in the population comprise CD11b. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD11b.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD62P. In some embodiments, from about 0% to about 40%, from about 0% to about 30%, from about 0% to about 20%, from about 0% to about 10%, or from about 0% to about 5% of the megakaryocyte-derived extracellular vesicles in the population comprise CD62P. In some embodiments, less than about 40%, less than about 30%, less than about 20%, less than about 10% of megakaryocyte-derived extracellular vesicles in the population comprise CD62P. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD62P.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD41. In some embodiments, about 15% to about 90%, about 30% to about 80%, or about 50% to about 70% of megakaryocyte-derived extracellular vesicles in the population comprise CD41. In some embodiments, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD41.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD61. In some embodiments, about 40% to about 100%, about 60% to about 100%, or about 85% to about 10% of megakaryocyte-derived extracellular vesicles in the population comprise CD61. In some embodiments, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 85%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD61.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD21. In some embodiments, from about 0% to about 10%, from about 0% to about 5%, from about 15% to about 90%, from about 30% to about 80%, or from about 50% to about 70% of the megakaryocyte-derived extracellular vesicles in the population comprise CD21. In some embodiments, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD21. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles in the population comprise CD21. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD21.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD51. In some embodiments, from about 0% to about 10%, from about 0% to about 5%, from about 15% to about 90%, from about 30% to about 80%, or from about 50% to about 70% of the megakaryocyte-derived extracellular vesicles in the population comprise CD51. In some embodiments, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD51. In some embodiments, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles in the population comprise CD51. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD51.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CLEC-2. In some embodiments, from about 0% to about 10%, from about 0% to about 5%, or from about 0% to about 12% of megakaryocyte-derived extracellular vesicles in the population comprise CLEC-2. In some embodiments, less than about 10%, less than about 5%, or less than about 2% of the megakaryocyte-derived extracellular vesicles in the population comprise CLEC-2. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CLEC-2.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise LAMP-1 (CD 107 a). In some embodiments, about 0% to about 20%, about 1% to about 15%, about 2% to about 10%, about 0% to about 5%, or about 0% to about 5% of megakaryocyte-derived extracellular vesicles in the population comprise LAMP-1 (CD 107 a). In some embodiments, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of megakaryocyte-derived extracellular vesicles in a population comprise LAMP-1 (CD 107 a). In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of LAMP-1 (CD 107 a).

In some embodiments, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of the population of cd41+ megakaryocyte-derived extracellular vesicles comprises LAMP-1 (CD 107 a).

In some embodiments, megakaryocyte-derived extracellular vesicles in the population are substantially free of DRAQ5. In some embodiments, from about 0% to about 20%, from about 0% to about 15%, from about 0% to about 10%, or from about 0% to about 5% of the megakaryocyte-derived extracellular vesicles in the population comprise DRAQ5. In some embodiments, less than about 20%, less than about 15%, less than about 10%, or less than about 5% of megakaryocyte-derived extracellular vesicles in a population comprise DRAQ5.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD63. In some embodiments, about 1% to about 20%, about 1% to about 15%, or about 1% to about 10% of megakaryocyte-derived extracellular vesicles in the population comprise CD63. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles in the population comprise CD63. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD63.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD42b. In some embodiments, from about 0% to about 20%, from about 0% to about 15%, from about 0% to about 10%, or from about 0% to about 5% of the megakaryocyte-derived extracellular vesicles in the population comprise CD42b. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles in the population comprise CD42b. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD42b

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD9. In some embodiments, about 40% to about 100%, about 50% to about 80%, or about 60% to about 70% of megakaryocyte-derived extracellular vesicles in the population comprise CD9. In some embodiments, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise CD9.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD31. In some embodiments, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, or about 1% to about 15% of the megakaryocyte-derived extracellular vesicles in the population comprise CD31. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise CD31. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD31.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD47. In some embodiments, about 1% to about 40%, about 1% to about 35%, about 1% to about 20%, about 20% to about 30%, about 30% to about 40%, or about 1% to about 15% of the megakaryocyte-derived extracellular vesicles in the population comprise CD47. In some embodiments, less than about 40%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise CD47. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD47.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD147. In some embodiments, about 1% to about 30%, about 1% to about 25%, about 1% to about 20%, about 20% to about 30%, or about 1% to about 15% of the megakaryocyte-derived extracellular vesicles in the population comprise CD147. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise CD147. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD147.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD32a. In some embodiments, from about 0% to about 20%, from about 1% to about 15%, or from about 1% to about 10% of megakaryocyte-derived extracellular vesicles in the population comprise CD32a. In some embodiments, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of megakaryocyte-derived extracellular vesicles in the population comprise CD32a. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD32a.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In some embodiments, from about 0% to about 5%, from about 0% to about 10%, from about 0% to about 30%, from about 0% to about 15%, or from about 0% to about 10% of the megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of GVPI.

In some embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise phosphatidylserine. In some embodiments, about 15% to about 90%, about 30% to about 80%, or about 50% to about 70% of megakaryocyte-derived extracellular vesicles in the population comprise phosphatidylserine. In some embodiments, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95% or greater than about 99% of the megakaryocyte-derived extracellular vesicles in the population comprise phosphatidylserine. In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In some embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of phosphatidylserine.

In some embodiments, megakaryocyte-derived extracellular vesicles are produced by the following method: (a) Obtaining human pluripotent stem cells, which are primary cd34+ HSCs derived from peripheral blood or umbilical cord blood; (b) Differentiating human pluripotent stem cells into megakaryocytes without the addition of EPO and without the addition of TPO; and (c) isolating megakaryocyte-derived extracellular vesicles from the megakaryocytes.

In embodiments, the method is an in vivo method. In embodiments, the method is an ex vivo method.

In embodiments, the cd34+ HSCs derived from peripheral blood are multipotent stem cells derived from volunteers that are mobilized into the blood stream by administration of mobilizing agents such as granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF).

In embodiments, the cord blood comprises pluripotent stem cells derived from blood left in the placenta and attached umbilical cord after delivery.

In embodiments, the megakaryocyte-derived extracellular vesicles are autologous to the patient. In some embodiments, human pluripotent stem cells are extracted from a patient and used to produce megakaryocytes, from which megakaryocyte-derived extracellular vesicles comprising the selected cargo are produced, and then administered to the patient. In some embodiments, differentiated cells are extracted from a patient and used to produce ipscs, which in turn are used to produce megakaryocytes, from which extracellular vesicles derived from megakaryocytes comprising the selected cargo are produced, and then administered to the patient.

In embodiments, the megakaryocyte-derived extracellular vesicles are allogeneic to the patient. In some embodiments, human pluripotent stem cells are extracted from a human subject that is not a patient and used to produce megakaryocytes, from which extracellular vesicles derived from megakaryocytes comprising the selected cargo are produced, and then administered to the patient. In some embodiments, differentiated cells are extracted from a human subject that is not a patient and used to produce ipscs, which in turn are used to produce megakaryocytes, from which extracellular vesicles derived from megakaryocytes comprising the selected cargo are produced, and then administered to the patient.

In embodiments, the megakaryocyte-derived extracellular vesicles are allogenic with the patient. In some embodiments, pluripotent stem cells are extracted from a non-human subject and used to generate megakaryocytes, from which megakaryocyte-derived extracellular vesicles comprising the selected cargo are generated, and then administered to a patient. In some embodiments, differentiated cells are extracted from a non-human subject and used to produce ipscs, which in turn are used to produce megakaryocytes, from which megakaryocyte-derived extracellular vesicles comprising the selected cargo are produced, and then administered to a patient.

In embodiments, incubating includes one or more of sonication, saponin permeabilization, mechanical vibration, hypotonic dialysis, extrusion through a porous membrane, cholesterol conjugation, application of an electric current, and combinations thereof. In embodiments, incubating includes one or more of electroporation, transformation, transfection, and microinjection.

In embodiments, the method further comprises (d) contacting megakaryocyte-derived extracellular vesicles with radiation. In embodiments, the radiation is gamma radiation. In embodiments, the amount of gamma radiation is greater than 12kGy, 25kGy or 50kGy. In some embodiments, the amount of gamma radiation is between about 12kGy and 15 kGy. In some embodiments, the amount of gamma radiation is between about 15kGy and 20 kGy. In some embodiments, the amount of gamma radiation is between about 20kGy and 25 kGy. In some embodiments, the amount of gamma radiation is between about 25kGy and 30 kGy. In some embodiments, the amount of gamma radiation is between about 30kGy and 35 kGy. In some embodiments, the amount of gamma radiation is between about 35kGy and 40 kGy. In some embodiments, the amount of gamma radiation is between about 40kGy and 45 kGy. In some embodiments, the amount of gamma radiation is between about 45kGy and 50kGy. In some embodiments, the amount of gamma radiation is between about 50kGy and 55 kGy. In some embodiments, the amount of gamma radiation is between about 55kGy and 60 kGy.

In embodiments, the method is substantially serum-free. In some embodiments, the method is greater than 60% serum-free. In some embodiments, the method is greater than 70% serum-free. In some embodiments, the method is greater than 80% serum-free. In some embodiments, the method is greater than 90% serum-free.

In various embodiments, the megakaryocyte-derived extracellular vesicles of the present disclosure are substantially purified megakaryocyte-derived extracellular vesicles. In embodiments, substantially purifying is synonymous with biologically pure. In embodiments, the substantially purified megakaryocyte-derived extracellular vesicles are largely free to a varying extent of components that are typically associated with them found in their native state. "isolated" means separated from the original source or environment. In embodiments, the substantially purified megakaryocyte-derived extracellular vesicles are sufficiently free of other materials that any impurities do not substantially affect the biological properties of the megakaryocyte-derived extracellular vesicles or cause other adverse consequences. In embodiments, the substantially purified megakaryocyte-derived extracellular vesicles are substantially free of cellular material, viral material, or culture medium that may be required for production. Purity and homogeneity are typically determined using biochemical techniques known in the art. In some embodiments, megakaryocyte-derived extracellular vesicles are purified using size exclusion filtration. In some embodiments, the filter has a pore size of about 650 nm. In some embodiments, megakaryocyte-derived extracellular vesicles are purified using size exclusion filtration. In some embodiments, the filter has a pore size of about 50nm to about 600 nm. In some embodiments, the filter has a pore size of at least 50 nm. In some embodiments, the filter has a pore size of about 600 nm.

Pharmaceutical composition

In one aspect, the present disclosure provides compositions useful for treating myeloproliferative diseases or disorders, such as MPN, wherein the compositions comprise megakaryocyte-derived extracellular vesicles of the present disclosure. In another aspect, the present disclosure provides a composition useful for treating a myeloproliferative disease or disorder, such as MPN, comprising a plurality of substantially purified megakaryocyte-derived extracellular vesicles comprising a lipid bilayer membrane surrounding a lumen, wherein: the megakaryocyte-derived extracellular vesicle lumen comprises cargo and/or cargo associates with the surface of the megakaryocyte-derived extracellular vesicle; and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein. In some embodiments, the cargo comprises one or more agents useful in the treatment of myeloproliferative diseases or disorders, such as MPN. In some embodiments, the agent is one or more therapeutic agents, including therapeutic agents useful in the treatment of myeloproliferative diseases or disorders, such as MPN.

Therapeutic treatment involves the use of one or more routes of administration and one or more formulations intended to achieve a therapeutic effect at an effective dose while minimizing toxicity to the patient to whom the treatment is administered.

In some embodiments, the effective dose is an amount that substantially avoids cytotoxicity in vivo. In various embodiments, an effective dose is an amount that substantially avoids an immune response in a human patient. For example, the immune response may be an immune response mediated by the innate immune system. Markers known in the art (e.g., cytokines, interferons, TLRs) can be used to monitor immune responses. In some embodiments, an effective dose eliminates the need to treat a human patient with an immunosuppressant for alleviating residual toxicity.

When formulated, the solution is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount as described herein. The formulations are readily administered in a variety of dosage forms, such as injection solutions. For example, for parenteral administration in aqueous solution, the solution is typically suitably buffered and the liquid diluent is first rendered isotonic with, for example, sufficient saline or glucose. Such aqueous solutions may be used for intravenous, intramuscular, subcutaneous and intraperitoneal administration, for example. Preferably, a sterile aqueous medium known to those skilled in the art is used.

The pharmaceutical formulation may additionally comprise a delivery agent (also known as a "transfection agent", also known as a "vehicle", also known as a "delivery vehicle") and/or an excipient. Pharmaceutically acceptable delivery agents, excipients, and methods of making and using the same, including methods of making and administering pharmaceutical formulations to patients, are well known in the art and are described in numerous publications, including, for example, U.S. patent application No. US 2008/0213377, which is incorporated herein by reference in its entirety. In various aspects, the invention relates to a pharmaceutical composition comprising a composition disclosed herein and a pharmaceutically acceptable excipient or carrier.

For example, the pharmaceutical composition may be in the form of a pharmaceutically acceptable salt. Such salts include those listed, for example, in the following: pharma. Sci.66,2-19 (1977) and The Handbook of Pharmaceutical Salts; properties, selection, and use.P.H.Stahl and C.G.Wermuth (eds.), verlag, zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety. Non-limiting examples of pharmaceutically acceptable salts include: sulfate, citrate, acetate, oxalate, hydrochloride, hydrobromide, hydroiodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, camphorsulfonate, pamoate, phenylacetate, trifluoroacetate, acrylate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, and process for preparing a film methylbenzates, o-acetoxybenzoates, naphthalene-2-benzoates, isobutyrates, phenylbutyrates, alpha-hydroxybutyrates, butyne-1, 4-dicarboxylic acid salts, hexyne-1, 4-dicarboxylic acid salts, decanoates, octanoates, cinnamic acid salts, glycolates, hippurates, malates, hydroxymaleates, malonates, mandelic acid salts, methanesulfonates, nicotinates, phthalates, terephthalates, propiolates, propionates, phenylpropionates, sebacates, suberate, p-bromobenzenesulfonates, chlorobenzenesulfonates, ethanesulfonates, 2-hydroxyethanesulfonates, methylsulfonates, naphthalene-1-sulfonates, naphthalene-2-sulfonates, naphthalene-1, 5-sulfonates, xylenesulfonates, tartrates, alkali metals such as sodium, hydroxides of potassium and lithium; hydroxides of alkaline earth metals such as calcium and magnesium; hydroxides of other metals such as aluminum and zinc; ammonia and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-or trialkylamines, dicyclohexylamines; tributylamine; pyridine; n-methylamine, N-ethylamine; diethylamine; triethylamine; mono-, di-, or tri- (2-OH-lower alkylamines), such as mono-, di-, or tri- (2-hydroxyethyl) amine, 2-hydroxy tert-butylamine, or tris- (hydroxymethyl) methylamine, N-di-lower alkyl-N- (hydroxy-lower alkyl) -amine, such as N, N-dimethyl-N- (2-hydroxyethyl) amine, or tri- (2-hydroxyethyl) amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like.

The pharmaceutical compositions of the invention may comprise excipients, which include liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients may be, for example, saline, acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used. In some embodiments, the pharmaceutically acceptable excipient is sterile when administered to a patient. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any of the agents described herein may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

In some embodiments, the pharmaceutical composition is formulated for topical, intrathecal, intralesional, intracoronary, intravenous (IV), intra-articular, intramuscular, intranasal, and intrabronchial administration and administration by one or more of intrapancreatic intravascular injection, intramedullary, lumbar puncture, intramyocardial, endocardial, intra-fistula, intramedullary gap, intranasal, and epidural space injection.

In some embodiments, the pharmaceutical composition is formulated for infusion. In some embodiments, the pharmaceutical composition is formulated for infusion, wherein the pharmaceutical composition is delivered to the patient's blood stream through a peripheral line, a central line, a tunneled line, an implantable port, and/or a catheter through a needle in a vein of the patient. In some embodiments, the patient may also receive supportive medications or treatments, such as hydration, via infusion. In some embodiments, the pharmaceutical composition is formulated for intravenous infusion. In some embodiments, the infusion is a continuous infusion, a secondary intravenous therapy (IV) and/or an IV bolus. In some embodiments, infusion of the pharmaceutical composition may be administered by using a device selected from one or more of an infusion pump, a hypodermic needle, a drip chamber, a peripheral cannula, and a pressure bag.

In embodiments, the pharmaceutical composition is introduced into or onto the skin in the form of a cosmeceutical, for example, for intradermal, intradermal or subcutaneous administration (see, e.g., epstein, h., clin. Dermotol.27 (5): 453-460 (2009)). In embodiments, the pharmaceutical composition is in the form of a cream, lotion, ointment, gel, spray, solution, or the like. In embodiments, the pharmaceutical composition further includes a penetration enhancer such as, but not limited to, surfactants, fatty acids, bile salts, chelating agents, non-chelating non-surfactants, and the like. In embodiments, the pharmaceutical composition may further comprise a fragrance, a colorant, a sunscreen, an antimicrobial, and/or a humectant.

In order that the invention disclosed herein may be more effectively understood, the following examples are provided. It should be understood that these examples are for illustrative purposes only and should not be construed as limiting the invention in any way.

Examples

Example 1: megakaryocyte-derived extracellular vesicle production

Allogeneic megakaryocyte-derived extracellular vesicles were generated from primary human peripheral blood cd34+ Hematopoietic Stem Cells (HSCs) using a cell culture process (fig. 1A).

Primary human cd34+ HSCs from commercial suppliers were thawed and transferred from stem cell maintenance medium to HSC expansion medium. During this time, HSCs significantly proliferate. These cultures were then placed in megakaryocyte differentiation medium and megakaryocyte-derived fines were collected from the culture supernatantExtracellular vesicles. Biomarker expression of CD41, CD61, CD42b, megakaryocyte-specific cytoskeletal protein β1-tubulin, alpha-particle component (platelet factor 4 and von willebrand factor), secretory particles and ultrastructural features (invagination membrane system, dense tube system, multivesicular body) confirm megakaryocyte differentiation. Megakaryocytes produce 500-1500 megakaryocyte-derived extracellular vesicles/cells with diameters between 30-600nm, 100-300nm, DNA-, CD41+. Megakaryocyte-derived extracellular vesicles are further isolated/concentrated by tangential flow filtration and purified at 1.5x10 ⁸ Megakaryocyte-derived extracellular vesicles per mL of targeted concentration packaging. Megakaryocyte-derived extracellular vesicles exhibit robust expression of megakaryocyte and platelet-specific biomarkers, RNAs, and cytoplasmic proteins.

Nanoparticle analysis, flow cytometry and freeze transmission electron microscopy confirmed the expression and composition of biomarkers.

It was found that the yield of MkEV increased over time during megakaryocyte (Mk) differentiation in vitro (fig. 1B). The phenotype of mkevs in culture was assessed (fig. 1C) and representative histograms of cell surface marker expression were generated along with microscopic images of megakaryocytes and harvested mkevs.

MkEV biomarker expression was examined. The surface marker expression of mkevs of the present disclosure was compared with Platelet Free Plasma (PFP) mkevs and platelet derived EVs (PLT EVs) (fig. 2A-2E). A representative diagram showing the following is shown: flow cytometry gating strategies (fig. 2A-2B), marker profiles of cd4+mkev, cd4+pfp MKEV, and cd4+plt EV of the present disclosure (fig. 2C) and fold-change in marker expression between MKEV and PFP MKEV of the present disclosure (fig. 2D), and fold-change in marker expression between MKEV and PLT EV of the present disclosure (fig. 2E). The data show that the mkevs of the present disclosure exhibit different surface marker expression compared to PFP mkevs and PLT EVs, and establish a marker profile of the mkevs of the present invention relative to PFP mkevs and PLT EVs. Minimal presence of DRAQ5 positive events indicated no cellular contamination (fig. 2F).

The size and morphology of the mkevs of the present disclosure are characterized. Frozen EM images of the mkevs of the present disclosure were made of CD41 (fig. 3A) and phosphatidylserine (fig. 3B) immunogold-labeled. Measurement of MkEV in frozen EM images showed that MkEV size ranged from 100 to 300nm with an average diameter of about 250nm. FIG. 3C is an image of MKEV isolated from PFP plasma, with CD41 (large dots) and PS (small dots) co-stained (see Brisson et al, platelets28:263-271 (2017), which is incorporated herein by reference in its entirety). With respect to organelle content, preliminary analysis showed no evidence of mitochondrial evidence in MkEV, as assessed by (1) electron microscopy and (2) mitochondrial respiration analysis (Agilent Seahorse). Genomic analysis was performed by sequencing coding RNA and non-coding miRNA. Proteomic analysis was performed using mass spectrometry, and proteomic data validated flow and EM surface markers.

The mkevs of the present disclosure were compared to PFP mkevs in size using flow cytometry analysis and frozen EM analysis labeled with cd41+ immunogold. The size distribution of the mkevs of the present disclosure overlaps with but differs from that of PFP mkevs and platelet-derived EVs (fig. 4A to 4K). (FIG. 4C is adapted from Arraud et al Journal of Thrombosis and Haemostasis 12:614-627 (2014); FIGS. 4D and 4E are found in Brisson et al Platelets28:263-271 (2017), all of which are incorporated herein by reference in their entirety).

Purification of MkEV was also examined and size exclusion filtration was found to be effective in removing aggregates in the unfiltered product. For example, post-harvest filtration using a 650nm size exclusion filter was found to be able to successfully remove large aggregate material (observed by EM in frozen MkEV samples) compared to unfiltered MkEV products (fig. 5A).

Example 2: MV manufacturing process and product release for in vivo gene delivery

This example relates to the process of standard and large-scale manufacturing and isolation of mkevs from primary human cd34+ HSCs. Mkevs were characterized and tested for batch-to-batch variability and release. The gene load and transfection efficiency of mkevs were determined, which enabled tracking of in vivo biodistribution and efficacy and determination of product parameters for gene delivery applications.

For clinical access, mkEV manufacturing must meet release criteria, including standardization of organizational procurement, manufacturing, production, testing, and storage. The MkEV quality and batch-to-batch variability with respect to identity, purity, efficacy and yield are defined and used to define product release criteria. Mkevs meet or exceed minimum quality and storage requirements.

MkEV made from primary human CD34+ cells was used for batch culture of about 400mL (about 1200cm ² Corresponding to about 5x t 225) to produce about 8e10MkEV per batch. Mkevs were tested to assess identity and purity (biomarker expression, composition%) and yield (total MkEV events per lot). Table 1 shows an example of MkEV release specifications.

Table 1: mkEV release Specification instance

Testing	Method	Specification of specification
			Identity/purity
Size of the device	Nanoparticle analyzer	≥95％100-600nm
			DNA	High sensitivity flow cytometry	Negative of 95% DRAQ5 or more
CD41	High sensitivity flow cytometry	More than or equal to 50 percent of positive
			Yield rate
MV event	Nanoparticle analyzer	Each batch is not less than 1e10

Standardized and scalable procedure for making and isolating mkevs from primary human cd34+ HSCs: primary human cd34+ Hematopoietic Stem Cells (HSCs) were used. Initial isolation, enrichment, and storage of HSCs (90-95% purity) was performed and met using a series of assays according to FDA guidelines to demonstrate identity, sterility, viability, and storage stability. HSCs were mobilized from donor bone marrow to blood by granulocyte colony stimulating factor and collected from peripheral blood by apheresis and tested for Chagas, CMV, hepB, hepC, HIV-1/HIV-2Plus O, HTLV I/II, syphilis, HBV, HCV and WNV (including the covd-19 test) prior to storage. HSC vials were cryopreserved in clinically approved media prior to shipping and exhibited viability after thawing. In a non-limiting example, HSC vials are cryopreserved in clinically approved media prior to shipping and exhibit viability after thawing.

The process flow of the MkEV production in the initial stage comprises the following steps: MV is manufactured from HSCs using scalable cGMP-compatible processes. MkEV production is divided into 2 discrete parts: (A) HSC expansion, megakaryocyte differentiation and MkEV production, and (B) MV separation/concentration by tangential flow filtration and vial filling (1.5e8mv/mL). The MkEV vials were frozen for storage. Centralized manufacturing is intended for HSC expansion and MkEV production/processing/filling.

Part A: primary human cd34+ HSCs underwent about 30-fold biomass expansion during cell culture at 5e6 cells/batch, yielding about 1.5e8 megakaryocytes/batch. Differentiation of CD34+ HSC into megakaryocyte progenitor cells occurs over a period of 7-9 days. Each megakaryocyte produces 500-1500 MVs, resulting in a total batch yield of about 7.5e10 MkEV per batch prior to harvest from the supernatant.

Part B: the MkEV was isolated/concentrated by tangential flow filtration (differential centrifugation as an alternative if necessary) to reduce the volume to about 500mL. The MkEV was packaged at a concentration of about 1.5e8 MkEV/mL to yield about 500 bottles/batch.

Example 3: characterization of MkEV, batch-to-batch variability and performance of release tests

Mkevs were collected from batch processing. High sensitivity flow cytometry was used to determine surface biomarker expression (CD 41, CD62P, CLEC-2, LAMP-1 (CD 107A)), organelle content (mitochondria), and phospholipid composition (phosphatidylserine), in combination with nuclear dye (DRAQ 5) to distinguish from nucleated cells. The total fluorescence intensity was calculated after subtracting the fluorophore conjugated IgG antibody specific control. Forward and side light scattering of mkevs were examined to evaluate size distribution, purity, and aggregation. The sized nanoparticles were used as gating control. The MkEV size and total batch yield were determined using a nanoparticle analyzer (Nanosight, malvern Instruments). MkEV protein content (Alix and TSG 101) DNA content was determined and measured by ELISA to estimate potential contamination by cell debris and nuclei. MkEV integrity and purity were confirmed by freeze electron microscopy and immunogold labeling, and allowed for further determination of surface molecules (CD 41, phosphatidylserine). For multiple independent MkEV batches, these experiments were repeated multiple times per batch. In one non-limiting embodiment, the experiment is repeated at least 3 times per lot for a minimum of 3 independent MkEV batches. MkEV/PEV from human whole blood was used as a positive control.

Mkevs were collected or generated from megakaryocytes and platelets, respectively, and cd4+ expression was quantified using nanoparticle tracking analysis in combination with immunogold labeling and electron microscopy characterization. Human cd34+ derived megakaryocytes produced 500-1500 mkevs per megakaryocyte (fig. 6A), with similar average sizes, approximately 200nm/MkEV, between murine bone marrow and fetal hepatocyte culture control (fig. 6B). Although the percentage of CD41+ MKEV from human CD34+ -derived megakaryocyte cultures was comparable to murine bone marrow-derived MKEV, human MKEV had more CD 41-binding gold particles as examined by immunogold electron microscopy (FIGS. 6C-6D). Human platelets activated with conventional agonists (thrombin and collagen) and inflammatory stimuli (LPS, mimicking an in vivo model) produced similar numbers of EV/platelets (FIG. 6E) and were larger in size than MKEV (FIG. 6F) (see French et al, blood Advances,4:3011-3023 (2020), incorporated herein by reference in its entirety for FIGS. 6A-6F). Platelet-derived EVs may also contain mitochondria and other organelles (unlike mkevs) because of their larger size. The percentage of CD41+PEV and the relative expression of CD 41-bound gold particles by PEV were compared to human and murine MKEV controls (FIGS. 6G-6H).

To assess lot-to-lot consistency, mkevs were collected or generated from megakaryocytes and characterized using flow cytometry to quantify cd41+ expression. There was minimal batch-to-batch variability in the total number/mL of mkevs and the total number of mkevs produced per batch (fig. 7A) and surface marker expression on the fabricated mkevs (fig. 7B).

Example 4: correction of JAK2-V617F mutations as a treatment for myeloproliferative neoplasms (MPNs) using gene editing methods A method of manufacturing the same.

To treat JAK2-V617F mutation positive MPN, a gene editing construct targeting the JAK2-V617F mutation was designed and constructed. The JAK2-V617F point mutation was corrected using the Homology Directed Repair (HDR) method. The editing construct consists of Cas9 and sequence-specific guide RNAs (sgrnas). To facilitate Homology Directed Repair (HDR), the editing construct was accompanied by a single stranded DNA template (ssODN). To verify the editability of these novel constructs, HEL cells (a leukemic cell line containing multiple copies of the JAK2-V617F mutant allele) were electroporated with Ribonucleoprotein (RNP) and ssODN. GFP-tagged Cas9 was used to form RNPs, enabling selection of cells that successfully internalize the RNP complex. 24-48 hours after electroporation, gfp+ cells were sorted and isolated by Fluorescence Activated Cell Sorting (FACS), and genomic DNA was isolated and assessed for correction of JAK2-V617F mutation (fig. 8A). Evaluation of genomic DNA after simultaneous electroporation of RNP complex and ssODN revealed successful correction of JAK2 mutations in gfp+ cells (fig. 8B). Next Generation Sequencing (NGS) of edited cells (gfp+ cell fraction) revealed 41% editing efficiency and successful T > G substitution, restoring wild-type JAK2 (fig. 9A and 9B). The base ratios of uncorrected (HEL cell line) and RNP transfected HEL cells (corrected HEL cell line) at the V617F point mutation locus (fig. 9B) and the ratio of insertions and deletions in uncorrected and RNP transfected HEL cells (fig. 9C) are shown. Briefly, RNP and ssODN mediated correction reduced V617F loading to 56% with 41% of reads carrying wild-type T > G substitutions (fig. 9B). In addition, 33% of mapped reads contained insertions or deletions at the V617F point mutation locus (fig. 9C).

Next, HEL cells were HDR using simultaneous transfection of RNP and ssODN and expanded for 24 and 48 hours prior to FACS-mediated RNP-containing cell selection. After 24 and 48 hours of incubation following electroporation, the genomic DNA of HEL cells was successfully edited, as shown in fig. 10A and 10B, wild-type and V617F-specific amplification of genomic DNA revealed correction of JAK2 mutant allele at two time points, with cells amplified for 24 hours showing highest levels of wild-type JAK2 (JAK 2-WT; fig. 10B) and lowest JAK2-V617F burden (fig. 10A).

Next, the editing efficacy of the editing construct was verified using an alternative method. Briefly, plasmid DNA (pDNA) sequences were constructed to express sgrnas, cas9, and ZsGreen reporter. HEL cells were transfected simultaneously with the pDNA construct and the single ssODN template, thereby selecting two pDNA doses. Cells were GFP-positively sorted 24 hours and 48 hours after electroporation. Genomic DNA was extracted and its gene editing was evaluated. Similar to the preformed RNP complex, cells transfected with pDNA-encoded editing complex and ssODN template alone were successfully edited as shown by significant increase in wild-type JAK2 amplification and reduced JAK2-V617F burden (fig. 11A and 11B).

Example 5: gene load and transfection efficiency defining MKEVRate of

To define gene loading efficiency, mkevs were electroporated with approximately 8300bp pDNA encoding MPN editing constructs. MkEV was treated with dnase to remove non-internalized pDNA. pDNA was loaded into MKV, isolated and quantified by qPCR. The control samples included pDNA incubated with MkEV without electroporation ± adding dnase. As shown in fig. 12A and 12B, loading of pDNA was achieved using 4D-Nucelofector (Lonza Wakersville, inc) with more than 50-fold increase in pDNA successful internalization into MkEV compared to non-electroporated controls.

To define gene loading efficiencies, approximately 500bp, 3,000bp, and 6,000bp plasmid DNA was conjugated to Cy5 fluorescent labels using Label IT Tracker Cy (Mirus); 4-10 marker molecules per plasmid, as described previously. DNA of MKEV labeled with Cy5+ was used at 250X10 using MaxCyte VLX ³ (DNA/MV) ratio electroporation was performed in 100. Mu.L (15 min, 37C), maxCyte VLX is a scalable cGMP-compliant electroporation system that can transfect up to 2000 hundred million cells per batch for commercial manufacturing. The MkEV was washed to improve the MkEV aggregated nucleic acid and incubated on ice for 20 minutes for recovery, followed by centrifugation to remove large aggregates generated during electroporation. MkEV was washed in PBS and resuspended in co-culture medium for transfection studies. To define pDNA copy number, pDNA was purified from the loaded MkEV using QIAprep Spin Miniprep Kit (Qiagen) and its concentration was quantified using Qubit dsDNA HS Assay Kit (Invitrogen).

Load efficiency (%) =cy 5+mv#/total wv#

pDNA copy # = [ supported pDNA (ng) # 10≡9/molecular weight ]. Avogaldel Cy5 refers to the number of Cy5 positive megakaryocyte vesicles; MV# refers to the number of megakaryocyte vesicles; the loaded pDNA refers to the amount of pDNA loaded into the MV; the molecular weight refers to the molecular weight of pDNA.

The pDNA copy number was confirmed by quantitative PCR amplification of the plasmid DNA fraction and gel electrophoresis of the amplicons. To determine transfection efficiency in vitro, mkevs were co-cultured with cd34+ HSCs at a ratio of 25, 50, 100 mkevs per HSC and centrifuged at 600xg for 30 min at 37 ℃ using the methods previously described (Kao and papout sakis, science advance 4:1-11 (2018), which is incorporated herein by reference in its entirety). The percentage of Cy5+ HSC was quantified by flow cytometry at 24, 48 and 72 hours. To determine the nuclear transfection efficiency, the nuclei of HSCs were isolated at 24 hours and the percentage of Cy5+ nuclei was quantified by flow cytometry as described previously.

The load efficiency of each MkEV is expected to be proportional to the pDNA size; and about 50-60% transfection efficiency. It is expected that the loading efficiency and capacity of DNA in EVs depends on DNA size, and linear DNA molecules less than 1000bp in length associate more efficiently with mkevs than larger linear DNA and plasmid DNA using this method. If the pDNA loading efficiency is limited, these studies will be repeated using linear DNA and the results compared to historical studies of other MKEVs. Other non-limiting methods of loading genetic material into mkevs include sonication, saponin permeabilization, hypotonic dialysis, cholesterol conjugation, and megakaryocyte microinjection/transfection. Transfection efficiency studies provide information for in vivo dosing strategies.

To define protein loading efficiency, mkevs were electroporated with Cas9 protein. Following electroporation, MKEV was treated with proteinase K to digest any non-internalized protein cargo, then lysed and subjected to western blot analysis to quantify Cas9 loading. Controls included MkEV plus Cas9 without electroporation ± proteinase K. As shown in fig. 13, cas9 is completely digested by proteinase K without electroporation. When mkevs were electroporated with Cas9, cas9 persisted after exposure to proteinase K, indicating that mkevs successfully protected Cas9 protein cargo.

Example 6: use of megakaryocyte-derived extracellular vesicles (MKEV) as myeloproliferative neoplasms (MPNs) Therapeutic delivery vehicles.

To test the ability to load mkevs into Hematopoietic Stem and Progenitor Cells (HSPCs) in vitro, mkevs were loaded with MPN-targeted Ribonucleoproteins (RNPs) and passed through a 300KDa filter to remove any non-loaded proteins. Primary bone marrow cells were harvested from mice carrying the cDNA sequence of human JAK2 with V617F mutation, lineage positive cells (B cells, T cells, erythrocytes and granulocyte populations) were depleted, and the remaining lineage negative cells (HSPC enriched populations) were co-cultured in vitro with MkEV loaded with GFP-tagged Cas9 by electroporation for 4 hours. The doses were 80, 155 and 465 MKEV/cell. Controls included unloaded cells, cells co-cultured with unloaded MkEV, and cells co-cultured with GFP-tagged RNP alone, treated in parallel with MkEV (i.e., subjected to 300KDa filtration). Flow cytometry showed a distinct gfp+ cell population, indicating successful MkEV uptake/association with HSPCs (fig. 14A). In contrast, cells that underwent RNP (filtration) alone showed no GFP positivity (fig. 14A). The percentage of gfp+ cells increased with increasing dose (fig. 14B). Figure 14C shows Median Fluorescence Intensity (MFI) in gfp+ cell populations. Cargo-loaded mkevs were co-cultured with lineage negative cells at the same three doses for 14 hours instead of 4 hours (fig. 15A-15C), resulting in successful uptake of mkevs as demonstrated by gfp+ cells (fig. 15A, 15B), and demonstrated an increase in median fluorescence intensity in gfp+ cells with increasing dose of mkevs (fig. 15C). The prolonged incubation time increased the proportion of gfp+ cells to >2%. After 18 hours in culture, mkEV was associated even more with lineage negative cells, 17% of which were positive for GFP expression due to cargo-loaded MkEV-mediated delivery (fig. 16A, 16B). MkEV: an increase in the cell ratio resulted in a proportional increase in the proportion of gfp+ cells and median fluorescence intensity (fig. 16B, fig. 16C). Overall, these results indicate MkEV-mediated RNP delivery to primary lineage depleted bone marrow cells.

Mkevs were also found to preferentially target primary HSPCs (fig. 17A-17C). Lineage depleted cells co-cultured for 18 hours with RNP-loaded MKEVs were stained for HSPC specific markers (c-Kit and Sca-1) to determine the phenotype of cells targeted by the MKEV. Flow analysis of lineage negative/c-kit+/Sca-1+ (LSK) cells (a primary HSPC population) was performed on GFP+ and GFP-cell fractions (FIG. 17A). MkEV: an increase in the cell ratio was accompanied by an increase in the proportion of LSK cells in the gfp+ fraction (fig. 17A). The 600 MkEV/cell dose was sufficient to target most LSK cells (fig. 17A, 17B), data indicating that mkevs selectively target the first tested HSPC compartments in vitro. Confocal microscopy of gfp+ cells after co-culture at 600 MkEV/cell confirmed MkEV-mediated delivery of RNP complexes to target cells (fig. 17C). Imaging revealed internalization of GFP signal in target cells (fig. 17C).

To determine the efficacy of MkEV-mediated delivery of RNP complexes to HSPCs, RNP-loaded mkevs were supplemented to primary LSK cells in vitro (fig. 18A, 18B, 19A, 19B). For this, LSK cells were freshly isolated from mice harboring Heterozygote (HET) and Homozygote (HOM) JAK2-V617F mutations. Cells (20,000 cells for HOM and 15,000 cells for HET) were seeded and incubated for 24 hours, followed by co-culture with RNP-loaded MkEV for 14 hours. HOM JAK2-V617F cells were co-cultured with RNP-loaded mkevs at two different doses (670 MkEV/cell and 2000 MkEV/cell). In addition, in the absence of electroporation, mkev+rnp provided a dose of 670 MkEV/cell. The control sample consisted of filtered RNP without supplementing MkEV. JAK2-V617F HOM LSK cells were co-cultured with loaded mkevs to deliver RNPs to 19% and 36% LSK cells, respectively, in a dose-dependent manner (fig. 18A, 18B). MkEV was incubated with RNP without electroporation, resulting in gfp+ cells being detected. While not wishing to be bound by any particular theory, the results indicate that mkevs promote uptake of RNPs and/or association with target cells. HET JAK2-V617F LSK cells were co-cultured with RNP-loaded MKEV at two doses: 890 MKV/cell and 2570 MKV/cell. Consistent with the increase in MkEV/cell ratio, the proportion of gfp+lsk cells increased to 28% and 45%, respectively (fig. 19A, 19B). As shown in FIGS. 18A-18B, the co-culture with RNP-associated MKEV in the absence of electroporation resulted in 23% GFP positivity in LSK. Taken together, these data demonstrate that mkevs promote efficient association/uptake of therapeutic RNP complexes by primary HSPCs carrying JAK2-V617F mutations.

MKEV was tested for its ability to correct V617F mutation in vitro: MK EV was developed to target JAK2V617F mutation in vitro. Assays were performed in the patient-derived JAK2V617F mutant cell line (HEL) and compared to the gold standard (viral vector delivery) currently in the field to determine baseline efficiencies. Primary cell cultures of mouse HSCs isolated from JAK2V617F mouse model and wild type littermates were subjected to in vitro gene targeting tests using the established assay previously disclosed. See, for example, shepherd et al, "Single-cell approaches identify the molecular network driving malignant hemato poietic stem cell self-renewal," Blood 132:791-803 (2018), which is incorporated herein by reference in its entirety.

In vivo testing of JAK2V617F mouse model: the JAK2V617F mouse model has a robust and traceable phenotype that can be observed from 4 weeks of age (90% hematocrit, red hand/foot, excessive erythrocyte progenitors, EPO-independent, stem cell deficiency). See Li and Kent et al, "JAK2V617F homozygosity drives a phenotypic switch in myeloproliferative neoplasms, but is insufficient to sustain disease," Blood 123:3139-3153 (2014), which is incorporated herein by reference in its entirety. These mice were given MK EV developed to target the JAK2V617F mutation and followed the blood cell phenotype. The inherent stem cell defect dominates the gene corrected cells, thus enhancing the impact of potentially low gene correction efficiency.

Verification of gene targeting in HSC of primary patient: JAK 2V 617F mutant patient samples were grown in a set of single cell assays to determine the growth and differentiation potential and mutation status of individual HSCs. See Ortmann and Kent et al, "Effect of Mutation Order on Myeloproliferative Neoplasms," N.Engl. J.Med.372:601-612 (2015), which is incorporated herein by reference in its entirety. This allows for robust measurement of gene correction efficiency and impact on primary patient HSC function, which can be used in preclinical experiments in patients.

Example 7: cancer model of MPN

JAK 2V 617F was targeted in vitro by designing the targeting construct. Cargo is loaded into MK EVs that transduce both the mutant cell line and primary mouse stem/progenitor cells. Recombination efficiency was then assessed.

Patient samples were then validated in vitro. Primary patient samples (e.g., from cambridge blood stem cell bio-pool (Cambridge Blood Stem Cell Biobank)) were obtained. Human cd34+ stem/progenitor cells are transduced and cell function is assessed in vitro (e.g., EPO-independent).

After in vitro validation of patient samples, JAK 2V 617F was targeted in vivo. The mouse JAK V617 knock-in model was used (see, e.g., li et al, blood.2010;116 (9): 1528-38, and blood.2014;123 (20): 3139-51, both of which are incorporated herein by reference in their entirety), and the human JAK 2V 617F mutation (a known stem cell defect in mutant cells) was knocked in to the mouse locus. Mice were treated in vivo with MKEV and cell function (robust traceable phenotype in erythrocytes) was assessed in vivo using a homozygous humanized JAK2-V617F mouse model. MkEV cargo loaded with MPN-targeted gene editor was injected intravenously into JAK2-V617F mutant mice and at discrete time points after Intravenous (IV) administration, bone marrow was isolated and the editing efficiency of bone marrow cell subsets was analyzed by qPCR and NGS. Mice were analyzed for phenotypes, including peripheral blood whole blood counts and differences. An erythropoietin (Epo) -independent growth assay was performed to quantify the correction of non-dependent Epo-dependent growth in the edited cells. Edited HSPCs were isolated and tested for stem cell capacity in a bone marrow transplant assay.

Successful in vivo gene correction was demonstrated in a patient-derived xenograft (PDX) model. PDX in immunodeficient mice was used. The founder: xenografts of the mouse blood system were treated in vivo with MKEV. The persistence of the gene correction and the effect on the molecular/cellular biology of the transplanted cells were evaluated.

Preclinical safety and quality control tests were established. Whole genome sequencing and transcriptional profiling of the gene corrected cells were examined. In vivo experiments for persistent gene/disease correction were monitored.

Equivalent scheme

While the application has been described in connection with specific embodiments thereof, it will be understood that the application is capable of additional modifications and this application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed by the scope of the appended claims.

Incorporated by reference

All patents and publications mentioned herein are hereby incorporated by reference in their entirety.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application.

As used herein, all headings are for organization only and are not intended to limit the disclosure in any way. The contents of any individual portion may be equally applicable to all portions.

Sequence listing

<110> St biological Co (STRM. Bio Incorporated)

J, sony (Thon, jonathan)

<120> compositions and methods relating to megakaryocyte-derived extracellular vesicles for the treatment of myeloproliferative neoplasms

<130> STRM-004PC/126555-5004

<150> US 63/104,769

<151> 2020-10-23

<150> US 63/173,735

<151> 2021-04-12

<150> 63/209,084

<151> 2021-06-10

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 401

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<220>

<223> synthetic sequence

<400> 1

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taggtgtcag tgtaaactat aatttaacag gagttaagta tttttgaaac tgaaaacact 360

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Claims

1. A method for gene editing of cells, comprising

wherein:

the megakaryocyte-derived extracellular vesicle lumen comprises cargo comprising an agent suitable for gene editing of the cell and/or cargo comprising an agent suitable for gene editing of the cell is associated with the surface of the megakaryocyte-derived extracellular vesicle; and

2. The method of claim 1, wherein the lipid bilayer membrane comprises one or more proteins selected from CD54, CD18, CD43, CD11b, CD62P, CD, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a and GPVI, and/or the lipid bilayer membrane comprises phosphatidylserine.

3. The method of claim 2, wherein:

less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD62P, and/or

Greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD41, and/or

Greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD61, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD147, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32a, and/or

Greater than about 40%, or greater than about 50%, or greater than about 60%, or greater than about 70%, or greater than about 80%, or greater than about 90%, or greater than about 95% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD9, and/or

Less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

4. The method of claim 2 or 3, wherein less than about 70%, or less than about 60%, less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising Phosphatidylserine (PS).

5. The method of any one of claims 2-4, wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising LAMP-1 (CD 107A).

6. The method of any one of claims 1-5, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 600 nm.

7. The method of any one of claims 1-5, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 30nm to about 100 nm.

8. The method of any one of claims 1-6, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 300 nm.

9. The method of any one of claims 1-6, wherein about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 600 nm.

10. The method of any one of claims 1-6, wherein about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 300 nm.

11. The method of any one of claims 1-10, wherein the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA.

12. The method of any one of claims 1-11, wherein the megakaryocyte-derived extracellular vesicles are substantially free of:

(a) Megakaryocytes, and/or

(b) Platelets.

13. The method of any one of claims 1-12, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to hematopoietic stem cells in vivo and/or in vitro.

14. The method of any one of claims 1-13, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vivo and/or in vitro.

15. The method of claim 14, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vivo and/or in vitro.

16. The method of claim 15, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vivo and/or in vitro.

17. The method of any one of claims 1-16, wherein the cargo is located in the lumen and/or the cargo is associated with a surface of the megakaryocyte-derived extracellular vesicle.

18. The method of claim 17, wherein the agent is one or more therapeutic agents.

19. The method of claim 18, wherein the therapeutic agent is a nucleic acid therapeutic agent.

20. The method of claim 19, wherein the nucleic acid therapeutic agent is selected from one or more non-autologous and/or recombinant nucleic acid constructs selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, non-coding and coding RNAs, linear DNA, DNA fragments, or DNA plasmids.

21. The method of claim 20, wherein the nucleic acid therapeutic agent is mRNA, and optionally: is transcribed in vitro or synthesized and/or comprises one or more non-canonical nucleotides, optionally selected from pseudouridine and 5-methoxyuridine.

22. The method of claim 20, wherein the nucleic acid therapeutic encodes a functional protein.

23. The method of any one of claims 20-22, wherein the nucleic acid therapeutic encodes a gene-editing protein and/or a related element for gene-editing function.

24. The method of claim 23, wherein the gene editing protein is selected from Zinc Finger (ZF), transcription activator-like effector (TALE), meganuclease, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -associated protein.

25. The method of claim 24, wherein the CRISPR-associated protein is selected from Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

26. The method of any one of claims 1-25, wherein the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells, optionally wherein the human pluripotent stem cells are primary cd34+ hematopoietic stem cells.

27. The method of claim 26, wherein the primary cd34+ hematopoietic stem cells are derived from peripheral blood or umbilical cord blood.

28. The method of claim 27, wherein the peripheral blood is adult peripheral blood (mPB) mobilized by granulocyte colony-stimulating factor.

29. The method of any one of claims 1-28, wherein the human pluripotent stem cell is an Embryonic Stem Cell (ESC).

30. The method of any one of claims 1-28, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPS).

31. The method of any one of claims 1-30, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the absence of added erythropoietin.

32. The method of any one of claims 1-31, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the presence of added thrombopoietin.

33. The method of any one of claims 1-31, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient or carrier.

34. The method of any one of claims 1-33, wherein the mutation comprises a V617F mutation.

35. A method of treating a myeloproliferative disease or disorder comprising

(a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles;

the megakaryocyte-derived extracellular vesicle lumen comprises the therapeutic agent and/or the therapeutic agent associates with the surface of the megakaryocyte-derived extracellular vesicle; and

36. The method of claim 35, wherein the lipid bilayer membrane comprises one or more proteins selected from CD54, CD18, CD43, CD11b, CD62P, CD, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a and GPVI, and/or the lipid bilayer membrane comprises phosphatidylserine.

37. The method of claim 36, wherein:

38. The method of claim 36 or 37, wherein less than about 70%, or less than about 60%, less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising Phosphatidylserine (PS).

39. The method of any one of claims 36-38, wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising LAMP-1 (CD 107A).

40. The method of any one of claims 35-39, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 600 nm.

41. The method of any one of claims 35-39, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 30nm to about 100 nm.

42. The method of any one of claims 35-40, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 300 nm.

43. The method of any one of claims 35-40, wherein about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 600 nm.

44. The method of any one of claims 35-40, wherein about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 300 nm.

45. The method of any one of claims 35-41, wherein the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA.

46. The method of any one of claims 35-41, wherein the megakaryocyte-derived extracellular vesicles are substantially free of:

(a) Megakaryocytes, and/or

(b) Platelets.

47. The method of any one of claims 35-46, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to hematopoietic stem cells in vivo and/or in vitro.

48. The method of any one of claims 35-47, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vivo and/or in vitro.

49. The method of claim 48, wherein said megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vivo and/or in vitro.

50. The method of claim 15, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vivo and/or in vitro.

51. The method of any one of claims 35-50, wherein the megakaryocyte-derived extracellular vesicles are suitable for loading the therapeutic into the lumen and/or loading the therapeutic by association with the surface of the megakaryocyte-derived extracellular vesicles.

52. The method of claim 35, wherein the incubating is performed in vivo.

53. The method of claim 35, wherein the incubating is performed ex vivo.

54. The method of claim 53, wherein the method further comprises obtaining biological cells from the patient.

55. The method of claim 53 or 54, wherein contacting the deliverable therapeutic agent with the biological cell comprises co-culturing the deliverable therapeutic agent with the biological cell.

56. The method of any one of claims 35-55, wherein the megakaryocyte-derived extracellular vesicles are autologous to the patient.

57. The method of any one of claims 35-55, wherein the megakaryocyte-derived extracellular vesicles are allogeneic to the patient.

58. The method of any one of claims 35-55, wherein the megakaryocyte-derived extracellular vesicles are allogenic with the patient.

59. The method of any one of claims 35-58, wherein the therapeutic agent is a nucleic acid therapeutic agent.

60. The method of claim 59, wherein the nucleic acid therapeutic agent is selected from one or more non-autologous and/or recombinant nucleic acid constructs selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, non-coding and coding RNAs, linear DNA, DNA fragments or DNA plasmids.

61. The method of claim 59 or 60, wherein said nucleic acid therapeutic encodes a wild-type gene that is defective in said patient.

62. The method of any one of claims 59-61, wherein the nucleic acid therapeutic encodes a gene-editing protein and/or a related element for gene-editing function.

63. The method of claim 62, wherein the gene editing protein is selected from the group consisting of Zinc Fingers (ZF), transcription activator-like effectors (TALE), meganucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -related proteins.

64. The method of claim 63, wherein the CRISPR-associated protein is selected from Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

65. The method of any one of claims 35-58, wherein the therapeutic agent is a biologic therapeutic, optionally a virus.

66. The method of claim 65, wherein the biologic therapeutic is a protein.

67. The method of claim 66, wherein the therapeutic agent is a recombinant protein.

68. The method of claim 66 or 67, wherein the therapeutic agent is one or more of an antibody or antibody fragment, a fusion protein, a gene editing protein, a cytokine, an antigen, and a peptide.

69. The method of any one of claims 51-58, wherein the therapeutic agent is a small molecule therapeutic agent.

70. The method of any one of claims 35-69, wherein the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells, optionally wherein the human pluripotent stem cells are primary cd34+ hematopoietic stem cells.

71. The method of claim 70, wherein the primary cd34+ hematopoietic stem cells are derived from peripheral blood or umbilical cord blood.

72. The method of claim 71, wherein the peripheral blood is adult peripheral blood (mPB) mobilized by granulocyte colony-stimulating factor.

73. The method of any one of claims 35-72, wherein the human pluripotent stem cells are Embryonic Stem Cells (ESCs).

74. The method of any one of claims 35-72, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPS).

75. The method of any one of claims 35-74, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the absence of added erythropoietin.

76. The method of any one of claims 35-74, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the presence of added thrombopoietin.

77. The method of any one of claims 35-69, wherein the incubating comprises one or more of sonication, saponin permeabilization, mechanical vibration, hypotonic dialysis, extrusion through a porous membrane, cholesterol conjugation, application of an electric current, and combinations thereof.

78. The method of any one of claims 35-77, wherein the incubating comprises one or more of electroporation, transformation, transfection, and microinjection.

79. The method of any one of claims 35-78, wherein the megakaryocyte-derived extracellular vesicles bind to a cell surface receptor on a cell of the patient.

80. The method of any one of claims 35-79, wherein the megakaryocyte-derived extracellular vesicles bind to cell surface receptors on the contacted biological cells of step (c).

81. The method of any one of claims 35-80, wherein the megakaryocyte-derived extracellular vesicles are fused with the extracellular membrane of cells of the patient.

82. The method of any one of claims 35-81, wherein the megakaryocyte-derived extracellular vesicles are fused to the extracellular membrane of the biological cell of step (c).

83. The method of any one of claims 35-82, wherein the megakaryocyte-derived extracellular vesicles are endocytosed by cells of the patient.

84. The method of any one of claims 35-81, wherein the megakaryocyte-derived extracellular vesicles are endocytosed by the biological cells of step (c).

85. A method of treating a myeloproliferative neoplasm comprising

(a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles; the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane surrounding a lumen, wherein:

(c) Administering the deliverable therapeutic agent to a patient or contacting the deliverable therapeutic agent with a biological cell in vitro and administering the contacted biological cell to a patient, thereby editing the V617F mutation to treat a myeloproliferative disease or disorder in the patient.

86. The method of claim 85, wherein the lipid bilayer membrane comprises one or more proteins selected from the group consisting of CD54, CD18, CD43, CD11b, CD62P, CD, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a and GPVI, and/or the lipid bilayer membrane comprises phosphatidylserine.

87. The method of claim 86, wherein:

88. The method of claim 86 or 87, wherein less than about 70%, or less than about 60%, less than about 50%, or less than about 40%, or less than about 30%, or less than about 20%, or less than about 10%, or less than about 5%, or less than about 1% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising Phosphatidylserine (PS).

89. The method of any one of claims 86-88, wherein less than about 20%, or less than about 15%, or less than about 10%, or less than about 5% of the megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising LAMP-1 (CD 107A).

90. The method of any one of claims 86-89, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 600 nm.

91. The method of any one of claims 86-89, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 30nm to about 100 nm.

92. The method of any one of claims 86-90, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 300 nm.

93. The method of any one of claims 86-90, wherein about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 600 nm.

94. The method of any one of claims 86-90, wherein about 90% or more, or about 95% or more, or about 97% or more, or about 99% or more of the megakaryocyte-derived extracellular vesicles have a diameter between about 100nm and about 300 nm.

95. The method of any one of claims 85-94, wherein the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA.

96. The method of any one of claims 85-95, wherein the megakaryocyte-derived extracellular vesicles are substantially free of:

(a) Megakaryocytes, and/or

(b) Platelets.

97. The method of any one of claims 85-96, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to hematopoietic stem cells in vivo and/or in vitro.

98. The method of any one of claims 85-96, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vivo and/or in vitro.

99. The method of claim 98, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vivo and/or in vitro.

100. The method of claim 99, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vivo and/or in vitro.

101. The method of any one of claims 85-100, wherein the megakaryocyte-derived extracellular vesicle is suitable for loading the therapeutic into the lumen and/or loading the therapeutic in association with the surface of the megakaryocyte-derived extracellular vesicle.

102. The method of claim 101, wherein the therapeutic agent is a nucleic acid therapeutic agent.

103. The method of claim 102, wherein the nucleic acid therapeutic agent is selected from one or more non-autologous and/or recombinant nucleic acid constructs selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, non-coding and coding RNAs, linear DNA, DNA fragments, or DNA plasmids.

104. The method of claim 103, wherein the nucleic acid therapeutic agent is mRNA, and optionally: is transcribed in vitro or synthesized and/or comprises one or more non-canonical nucleotides, optionally selected from pseudouridine and 5-methoxyuridine.

105. The method of claim 103, wherein the nucleic acid therapeutic encodes a functional protein.

106. The method of any one of claims 103-105, wherein the nucleic acid therapeutic encodes a gene-editing protein and/or a related element for gene-editing function.

107. The method of claim 106, wherein the gene editing protein is selected from Zinc Finger (ZF), transcription activator-like effector (TALE), meganuclease, and Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) -associated protein.

108. The method of claim 107, wherein the CRISPR-associated protein is selected from Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

109. The method of any one of claims 85-108, wherein the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells, optionally wherein the human pluripotent stem cells are primary cd34+ hematopoietic stem cells.

110. The method of claim 109, wherein the primary cd34+ hematopoietic stem cells are derived from peripheral blood or umbilical cord blood.

111. The method of claim 110, wherein the peripheral blood is adult peripheral blood (mPB) mobilized by granulocyte colony-stimulating factor.

112. The method of any one of claims 85-111, wherein the human pluripotent stem cells are Embryonic Stem Cells (ESCs).

113. The method of any one of claims 85-111, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPS).

114. The method of any one of claims 85-113, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the absence of added erythropoietin.

115. The method of any one of claims 85-114, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the presence of added thrombopoietin.

116. The method of any one of claims 85-115, wherein the myeloproliferative disease or disorder is selected from the group consisting of myeloproliferative neoplasm (MPN), polycythemia vera, thrombocythemia, primary thrombocythemia, idiopathic myelofibrosis, acute myelogenous leukemia, systemic Mastocytosis (SM), chronic Neutrophilic Leukemia (CNL), and myelodysplastic syndrome (MDS).

117. The method of claim 116, wherein the myelofibrosis is selected from primary myelofibrosis, secondary myelofibrosis, post-primary thrombocytosis myelofibrosis, post-polycythemia vera myelofibrosis, myelometaplasia accompanied by myelofibrosis, chronic Myelogenous Leukemia (CML), chronic myelomonocytic leukemia, eosinophilic syndrome, juvenile myelomonocytic leukemia, and systemic mastocytosis.

118. The method of any one of claims 85-117, wherein the method provides a functional JAK2 receptor in the patient.

119. The method of claim 118, wherein the gene is a functional Jak2 gene or encodes a gene-editing protein capable of forming a functional Jak2 gene.

120. The method of claim 119, wherein the gene editing protein is selected from Zinc Fingers (ZF), transcription activator-like effectors (TALEs), meganucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated proteins.

121. The method of claim 120, wherein the CRISPR-associated protein is selected from Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

122. The method of any one of claims 85-121, wherein the method causes a decrease in one or more of white blood cell count, neutrophil count, reticulocyte count, platelet count, and spleen size of the patient.

123. The method of any one of claims 85-122, wherein the method reduces the occurrence of graft-versus-host disease, vascular disease including thrombosis, coronary heart disease, arteriosclerosis, cerebral ischemia, cerebral infarction, thrombotic events, vascular complications, splenomegaly, progressive cytopenia, and/or hypercellular bone marrow in the patient.

124. The method of any one of claims 85-123, wherein the method eliminates the need for Hematopoietic Stem Cell (HSC) transplantation and/or myeloablative chemotherapy.