CN117500513A

CN117500513A - Compositions and methods relating to megakaryocyte-derived extracellular vesicles for fanconi anemia

Info

Publication number: CN117500513A
Application number: CN202280030795.3A
Authority: CN
Inventors: J·索恩; D·赖泽尔; L·高柏
Original assignee: Strom Biology
Current assignee: Strom Biology
Priority date: 2021-04-26
Filing date: 2022-04-26
Publication date: 2024-02-02

Abstract

Disclosed herein are compositions and methods relating to megakaryocyte-derived extracellular vesicles derived from human pluripotent stem cells, wherein the megakaryocyte-derived extracellular vesicles are useful for treating Fanconi Anemia (FA).

Description

Compositions and methods relating to megakaryocyte-derived extracellular vesicles for fanconi anemia

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional patent application Ser. No. 63/179,808, filed on 26, 4, 2021, and 63/209,085, filed on 10, 6, 2021, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to compositions and methods relating to megakaryocyte-derived extracellular vesicles derived from human pluripotent stem cells for the treatment of Fanconi Anemia (FA).

Background

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. In addition to target non-specific and inefficient unloading of therapeutic agents, liposomes can 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.

Fanconi Anemia (FA) is an autosomal recessive genetic disorder characterized by congenital abnormalities, bone marrow failure and a propensity for malignancy, including myelodysplastic syndrome and acute myelogenous leukemia. Most patients develop bone marrow failure at mid-age five years. Progressive whole blood cytopenia and congenital malformations, including short stature, radial hypoplasia, abnormal urinary tract, hyperpigmentation and bradykinin are common symptoms. Vanconi anemia is associated with an increased susceptibility to cancer, particularly acute myelogenous leukemia, and an increased risk of developing solid tumors.

The FA gene product plays an important role in protecting the integrity of the human genome; any mutation of the FA gene can result in genomic instability due to failure to repair DNA damage. Over the last decade, the role of fanconi anemia gene products in DNA repair has been established. However, sources and chemicals that lead to excessive instability of the genome in FA patients with dysplasia, BMF and a malignant phenotype have been elusive, and FA treatment has focused mainly on symptoms rather than healing.

Thus, there is a need for delivery vehicles that can be mass produced in a cost-effective manner, that have side effects when administered to patients that are eliminated or reduced, and that can also provide new therapies for FA that are capable of repairing DNA damage and improving chromosome stability.

Disclosure of Invention

Disclosed herein are compositions and methods relating to megakaryocyte-derived extracellular vesicles. In particular, megakaryocyte-derived extracellular vesicles of the invention are useful for drug delivery and treatment of Fanconi Anemia (FA), among others. The methods disclosed herein may be in vivo or ex vivo and may be used, for example, in gene therapy, gene replacement therapy, and gene editing.

In one aspect, the disclosure relates to methods for modifying cells. The disclosed methods comprise (a) contacting a cell with a composition comprising a plurality of substantially purified megakaryocyte-derived extracellular vesicles (mkevs) comprising a lipid bilayer membrane surrounding a lumen, wherein: the MkEV lumen comprises a load comprising an agent suitable for modifying a cell and/or a load comprising an agent suitable for modifying a cell is associated with a surface of the MkEV; and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein, and (b) modifying the cell to provide a functional Fanconi Anemia (FA) related gene and/or to repair a functional FA related gene mutated therein.

In another aspect, the invention relates to a method for treating Fanconi Anemia (FA), the method comprising (a) obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles; (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 FA; and (c) administering the deliverable therapeutic agent to the 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 comprising the therapeutic agent and/or the therapeutic agent 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 method of treating Fanconi Anemia (FA), the method comprising: (a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles; the megakaryocyte-derived extracellular vesicles comprise a lipid layer membrane surrounding a lumen, wherein: the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein; (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 increasing or restoring FA-associated gene expression and/or levels and/or functions of one or more FA-associated proteins; 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, thereby restoring or increasing expression of the FA-associated gene in the patient to a normal level in a patient not suffering from FA.

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 cd41+ MkEV, cd41+ PFP MkEV, and cd41+ PLT EV of the present disclosure. The mkevs of the present disclosure have 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 mkevs and PFP mkevs 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)

Fig. 8 shows confocal microscopy images of HSPCs (lineage depleted cd150+cd48-murine bone marrow cells) co-cultured with mkevs loaded with GFP-tagged Cas9 Ribonucleoprotein (RNP). Cells co-cultured with the loaded mkevs were GFP positive, indicating that the cells ingested GFP-tagged Cas 9-loaded mkevs. In contrast, control samples comprising cells alone and cells co-cultured with MkEV plus RNP (MkEV mixed with RNP but not loaded by electroporation prior to co-culture with cells, (no EP)) showed no GFP positivity. These data indicate that RNP-loaded mkevs have been successfully delivered to HSPCs.

FIGS. 9A-9C show preferential targeting of MKEV to ex vivo hematopoietic stem and progenitor cells. The Cas9 protein loaded with GFP-markers (fig. 9A) or MkEV-labeled with lipophilic fluorescent dye DiD (fig. 9B) was co-cultured with primary whole bone marrow derived from wild-type mice. After 24 hours of co-culture, the cells were analyzed for the percentage of GFP+ or DID+ (i.e., MKEV+) cells by flow cytometry. In addition, the percentage of lineage positive (Lin+), lineage negative (Lin-) and lineage negative/c-kit+/Sca-1+ (LSK) cells was determined simultaneously using fluorescently labeled antibodies against the lineage positive markers, sca-1 and c-Kit cell surface proteins. The percentage of each cell subtype in the heterogeneous whole bone marrow population is shown in fig. 9A. For cells co-cultured with MkEV loaded with GFP-tagged Cas9 (as shown by the bar graph in fig. 9A), while the vast majority (95%) of the cells in culture were lin+ cells (differentiated cells), only up to 23% of these cells were MkEV positive. In contrast, although the proportion of hematopoietic stem and progenitor cells (Lin-cells) was <5%, almost 50% of these cells were MkEV positive at 300EV per cell dose. Finally, LSK cells account for only 0.25% of the cell population, but almost 40% of the cell population is MkEV positive, for the least and most potent hematopoietic stem cells evaluated in these cultures. These data indicate that bone marrow-derived hematopoietic stem and progenitor cells are preferentially targeted ex vivo. Also, as shown in fig. 9B, for whole bone marrow cells co-cultured with DiD-tagged MkEV; only 20% of Lin+ cells were MKEV positive. In contrast, 30% of the more rare Lin-cell populations were MKEV positive. Finally, for the least and most potent hematopoietic stem cells (LSKs) evaluated in these cultures, up to 48% of the cell population were MkEV positive. The percentage of total lin+ and Lin-cells in whole bone marrow cultures did not significantly change compared to controls under all conditions of MkEV co-culture (fig. 9C).

FIGS. 10A-10K show in vivo biodistribution of fluorescence-labeled MKEV following in vivo delivery to wild-type mice. The experimental design is shown in fig. 10A (n=3-5 mice/group). The fluorescently labeled MkEV was intravenously injected into wild-type mice via the tail vein, and tissues were harvested 16 hours post injection and analyzed for fluorescence. Fig. 10B shows fluorescence signals detected by IVIS in the femur dissected from mice. N=5 mice/group. Fluorescence in each homogenized tissue was measured by plate reader and normalized to tissue weight (fig. 10C). Figures 10D and 10E show graphs of experimental data of bone marrow cells stained with antibodies against CD45, lineage markers, CD150, CD201, and CD48, and analyzed by flow cytometry to determine the percentage of MkEV positive hematopoietic cells (cd45+ cells; left panel, figure 10D) and the percentage of very primitive long term hematopoietic stem cells (cd45+/Lin-/cd150+/cd201+/CD 48-cells; right panel, figure 10E). MkEV preferentially targets hematopoietic cells within the bone marrow (fig. 10D), and within this compartment, very rare (less than 0.03% of bone marrow cells) long-term hematopoietic stem cells (fig. 10E). There was no change in peripheral white blood cell count (fig. 10F), hemoglobin (fig. 10G), platelet count (fig. 10H), WBC differential (fig. 10I), or percentage of cd45+ and CD 45-cells (fig. 10J) or percentage of long-term hematopoietic stem cells in bone marrow (fig. 10K) 16 hours after MkEV injection, indicating lack of hematopoietic toxicity after in vivo injection.

Fig. 11A-11B show experimental data demonstrating successful loading of FANCA encoded pDNA (fig. 11A) and Cas9 (fig. 11B) into mkevs. FIG. 11A shows a graph of experimental data demonstrating Electroporation (EP) of 9.8kb pDNA encoding wild-type FANCA into MKEV. After electroporation, all samples were treated with dnase to remove any non-internalized pDNA load prior to DNA extraction. The DNA was then extracted and qPCR was performed. The loaded pDNA was quantified (ng) according to a standard curve run in parallel and pDNA copy number/MkEV was calculated. The experimental data graph shown in fig. 11B demonstrates Cas9 loading into mkevs. MkEV was electroporated with Cas9, treated with proteinase K to remove any uninhibited load (free load and load associated with the vesicle surface), or filtered to remove any free load, and then analyzed by western blot to quantify Cas9. Controls included MkEV plus Cas9, no electroporation ± proteinase K ± filtration. After filtration, cas9 was present in electroporated mkevs, but not in control non-electroporated mkevs, indicating successful vesicle association and/or internalization of protein load. After proteinase K digestion, cas9 was present in electroporated mkevs but not in control non-electroporated mkevs, indicating successful internalization and protection of the loaded protein load following electroporation.

The experimental data shown in fig. 12A-12B demonstrate successful delivery of mkevs loaded with pDNA encoding wild-type FANCA to murine Hematopoietic Stem and Progenitor Cells (HSPCs). Murine myeloid lineage depleted HSPCs from wild-type mice were co-cultured with mkevs, which are loads (drug (DP) -EVs) loaded with pDNA encoding wild-type human FANCA. After 48 hours of co-culture, human FANCA mRNA (amplicon length 413 bp) was quantified by qPCR. Fig. 12A shows a gel indicating that cells co-cultured with DP-EV in duplicate show strong expression of human FANCA from MkEV-mediated pDNA delivery. In contrast, mock controls (cells co-cultured with MkEV treated in parallel but not loaded with pDNA load) did not show human FANCA expression. FIG. 12B shows an optical density reading showing an 18.5-fold increase in FANCA mRNA expression in cells treated with DP-EV as compared to mock-treated cells.

The experimental data shown in fig. 13A-13B demonstrate that mkevs loaded with pDNA encoding wild-type FANCA successfully resulted in FANCA mRNA expression in a dose-dependent manner. Murine myeloid lineage depleted cells from wild type mice were co-cultured with MkEV, a load (drug (DP) -EV) loaded with pDNA encoding wild type human FANCA. After 48 hours of co-culture, human FANCA mRNA (amplicon length 413 bp) was quantified by qPCR. Fig. 13A shows a gel indicating that cells co-cultured with DP-EV show strong expression of human FANCA from MkEV-mediated pDNA delivery. In contrast, mock controls (cells co-cultured with MkEV treated in parallel but not loaded with pDNA load) did not show FANCA expression. FIG. 13B shows optical density readings showing 1.3 and 4.4 fold increases in FANCA mRNA expression in cells treated with low and high doses of DP-EV, respectively, as compared to mock-treated cells.

The experimental data shown in FIGS. 14A-14B demonstrate successful restoration of FANCA mRNA expression in FANCA-deficient murine HSPC. MkEV loaded with pDNA encoding wild-type FANCA can restore FANCA mRNA expression in FANCA-/-Hematopoietic Stem and Progenitor Cells (HSPCs). Murine myeloid lineage depleted cells from FANCA-/-mice were co-cultured with MKEV, a Drug (DP) -EV loaded with pDNA encoding wild-type human FANCA. After 48 hours of co-culture, human FANCA mRNA (amplicon length 413 bp) was quantified by qPCR. Fig. 14A shows a gel indicating that cells co-cultured with DP-EV show strong expression of human FANCA from MkEV-mediated pDNA delivery. In contrast, mock controls (cells co-cultured with MkEV treated in parallel but not loaded with pDNA load) did not show FANCA expression. FIG. 14B shows an optical density reading graph demonstrating a 60-fold increase in FANCA mRNA expression in cells treated with DP-EV as compared to mock-treated cells.

FIG. 15 shows experimental data demonstrating the functional benefit of FANCA-deficient cells co-cultured with FANCA-corrected RNP-loaded MKEV. For these experiments, mkevs were loaded with GFP-tagged Cas9 and grnas targeting the FANCA mutation (drug (DP) -EVs). The RNP construct can induce therapeutic insertion deletions in the mutated FANCA gene. RNP-loaded EV (DP-EV) was co-cultured with FANCA-deficient human cells (e.g., immortalized FA patient-derived lymphoblastic cells). Individual cells and cells treated with MkEV treated in parallel but without RNP loading (mock) served as controls. After 24 hours of co-culture, 100% of cells co-cultured with DP-EV were GFP positive and no change in viability was observed compared to unchanged cells, indicating no toxicity (data not shown). Cells co-cultured with DP-EV showed an increase in cell number after 14 days of culture compared to cells alone and mock control. These data indicate that successful restoration of FANCA expression in DP-EV treated cells results in its proliferative advantage, i.e., the functional benefit of successful restoration of FANCA expression.

Detailed Description

The present disclosure is based in part on the discovery of compositions and methods useful for treating Fanconi Anemia (FA). In embodiments, the treatment of Fanconi Anemia (FA) further includes treating a symptom associated with FA and/or the likelihood of a patient developing one or more diseases and conditions associated with FA. In 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 a load in a lumen for delivery of an agent, such as a therapeutic agent for treating Fanconi Anemia (FA). In 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 disclosure provides compositions and methods for treating Fanconi Anemia (FA) using megakaryocyte-derived extracellular vesicles that are continuously produced, have desirable properties, and carry specific loads-making their therapeutic use for treating Fanconi Anemia (FA) more likely to be successful.

Method for modifying cells using megakaryocyte-derived extracellular vesicles

In one aspect, the present disclosure relates to a method for modifying a cell, the method comprising: (a) Contacting the cells with a composition comprising a plurality of substantially purified megakaryocyte-derived extracellular vesicles (MKEVs) comprising a lipid bilayer membrane surrounding a lumen,

wherein: the MkEV lumen contains a load containing reagents suitable for modifying the cells; and/or the number of the groups of groups,

a load comprising an agent suitable for modifying cells is associated with the surface of the MkEV; and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein, and (b) modifying the cell to provide a functional Fanconi Anemia (FA) -related gene and/or a functional FA-related gene that repairs mutations therein. In embodiments, the megakaryocyte-derived extracellular vesicles are contained in the lumen and a load associated with the surface of the megakaryocyte-derived extracellular vesicles.

Wherein: the MkEV comprises a load comprising an agent suitable for modifying a cell; the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein, and (b) modifying the cell to provide a functional Fanconi Anemia (FA) related gene and/or a functional FA related gene that repairs mutations therein. In embodiments, the megakaryocyte-derived extracellular vesicles comprise a load in the lumen or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles are contained in the lumen and a load associated with the surface of the megakaryocyte-derived extracellular vesicles.

In embodiments, the disclosed methods for modifying a cell are methods for modifying gene expression of a cell.

In embodiments, the agent that loads and/or is suitable for modifying cells comprises one or more therapeutic agents.

In embodiments, the therapeutic agent comprises a FA-associated gene or fragment thereof comprising FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7), and FANCW (RFWD 3) or fragment thereof.

In embodiments, the therapeutic agent increases or restores FA-associated gene expression and/or level and/or function of one or more FA-associated proteins.

In embodiments, the FA-associated protein comprises FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7), and FANCW (RFWD 3).

In embodiments, the therapeutic agent is a small molecule therapeutic or a biologic therapeutic. In embodiments, the therapeutic agent is used in gene therapy. In embodiments, the biotherapeutic agent encodes a functional protein or a recombinant protein. In embodiments, functional or recombinant proteins include wild-type proteins, fusion proteins, cytokines, antigens and peptides, antibodies or antibody fragments. In embodiments, the therapeutic agent is a nucleic acid therapeutic agent. In embodiments, the nucleic acid therapeutic agent expresses a wild-type functional FA gene, and/or comprises a nucleic acid encoding a functional FA-related gene or a protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-related gene or a protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-related gene or a protein product thereof. 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, plasmid DNA or DNA fragments.

In embodiments, one or more non-autologous and/or recombinant nucleic acid constructs are incorporated into the vector.

In embodiments, the vector is a composition of matter that comprises an isolated nucleic acid and is useful for delivering the isolated nucleic acid into the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ionic or amphoteric compounds, plasmids, and viruses. In embodiments, the vector comprises an autonomously replicating plasmid or virus. In embodiments, the vector includes non-plasmid and non-viral compounds that facilitate transfer of the nucleic acid into the cell, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, and the like.

For example, expression of a natural or synthetic nucleic acid encoding a FA-related gene may be achieved by, but is not limited to, the following: the nucleic acid encoding the FA-related gene or a portion thereof is operably linked to a promoter and the construct is incorporated into an expression vector. In embodiments, the vectors used in the present invention are suitable for replication and optionally integration in eukaryotic cells. Typical vectors contain transcriptional and translational terminators, initiation sequences, and promoters useful for regulating expression of the desired nucleic acid sequence.

In embodiments, the vectors used in the present disclosure may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Pat. nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entireties. In embodiments, the present disclosure provides gene therapy vectors.

In embodiments, the nucleic acids of the present disclosure provide functional Fanconi Anemia (FA) -related genes or fragments thereof that can be cloned into various types of vectors. For example, the nucleic acid may be cloned into vectors, including but not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe-generating vectors and sequencing vectors. In addition, the vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York) and other virology and molecular biology manuals. Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors contain an origin of replication that is functional in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

In embodiments, the operably linked sequences include an expression control sequence adjacent to the gene of interest and an expression control sequence that acts in trans or at a distance to control the gene of interest. In embodiments, expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; a sequence that stabilizes cytoplasmic mRNA; sequences that increase translation efficiency (i.e., kozak consensus sequences); a sequence that enhances protein stability; and, when desired, sequences that enhance secretion of the encoded product. Numerous expression control sequences, including natural, constitutive, inducible and/or tissue specific promoters, are known in the art and may be used.

Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, they are located in a region 30-110bp upstream of the start site, although many promoters have recently been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is generally flexible, so that promoter function is preserved when the elements are inverted or moved relative to each other. One example of a suitable promoter is the immediate early Cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence to which it is operably linked. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV 40) early promoter, mouse Mammary Tumor Virus (MMTV), human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, moMuLV promoter, avian leukemia virus promoter, epstein barr virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked thereto when expression is desired or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

Enhancer sequences found on the vector also regulate the expression of the genes contained therein. Typically, enhancers bind to protein factors to increase transcription of a gene. Enhancers may be located upstream or downstream of the gene they regulate. Enhancers may also be tissue-specific to enhance transcription in a particular cell or tissue type. In one embodiment, the vectors of the invention comprise one or more enhancers to enhance transcription of genes present within the vector.

In embodiments, the vector is an expression vector comprising an expression control sequence operably linked to a nucleotide sequence. In embodiments, the vector is a plasmid, phagemid, phage derivative, cosmid or viral vector. In embodiments, the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentiviral vector, a sendai virus vector, a herpes simplex virus vector, a cytomegalovirus vector, or a chimeric virus vector.

In embodiments, the therapeutic agent is any nucleic acid delivery system known in the art that can be used for vaccination. In one embodiment, the nucleic acid delivery system is a vaccine vector, DNA plasmid, or mRNA vaccine. In embodiments, the therapeutic agent is a vaccine and/or an immunogenic antigen.

In embodiments, the nucleic acid therapeutic encodes a gene-editing protein and/or a related element of a gene-editing function. 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, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

Methods of treatment using megakaryocyte-derived extracellular vesicles

In embodiments, the present disclosure relates to methods of treating Fanconi Anemia (FA) with megakaryocyte-derived extracellular vesicles of the invention. In embodiments, the present disclosure relates to methods of treating FA by delivering a therapeutic load, such as a gene therapy load, in or associated with megakaryocyte-derived extracellular vesicles of the invention.

In one aspect, the invention relates to a method for treating Fanconi Anemia (FA). The disclosed method comprises the following steps: (a) Obtaining a plurality of substantially purified megakaryocyte-derived extracellular vesicles; (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 the megakaryocyte-derived extracellular vesicles 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 FA; 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 method for treating Fanconi Anemia (FA). The disclosed method comprises the following steps: (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: the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein; (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 increasing or restoring FA-associated gene expression and/or levels and/or functions of one or more FA-associated proteins; 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, thereby restoring or increasing expression of the FA-associated gene in the patient to a normal level in a patient not suffering from FA.

In embodiments, treatment includes therapeutic treatment for a disease or disorder and prophylactic or inhibitory measures. In embodiments, the term "treatment" and related terms such as "treatment" and "treatment" mean a decrease in the progression, severity and/or duration of a disease condition or at least one symptom thereof. In embodiments, treatment refers to any regimen that may benefit a subject. The treatment may be against an existing disorder or may be prophylactic (prophylactic treatment). Treatment may include therapeutic, palliative or prophylactic action. In embodiments, "therapeutic" and "prophylactic" treatments are considered in their broadest context. In embodiments, the term "therapeutic" does not necessarily mean that the subject is treated until complete recovery. In embodiments, the term "prophylactic" does not necessarily mean that the subject will not ultimately be infected with a disease condition. In embodiments, the term treatment includes administration of an agent either before or after the onset of the disease or disorder, thereby preventing or eliminating all signs of the disease or disorder. In embodiments, the agent is administered after the clinical manifestation of the disease to combat symptoms of the disease including "treatment" of the disease.

In embodiments, treatment may include suppression, inhibition, prevention, treatment, delay of onset, or a combination thereof. Treatment refers in particular to increasing the time to progression, accelerating remission, inducing remission, enhancing remission, accelerating recovery, increasing the efficacy of a replacement therapy or decreasing resistance to a replacement therapy or a combination thereof. In embodiments, "suppressing" or "inhibiting" refers to, inter alia, delaying onset of symptoms, preventing recurrence of a disease, reducing the number or frequency of recurrent episodes, increasing latency between episodes of symptoms, reducing severity of acute episodes of symptoms, reducing the number of symptoms, reducing the incidence of symptoms associated with a disease, reducing latency of symptoms, improving symptoms, reducing secondary infections, prolonging patient survival, or a combination thereof.

In embodiments, the present disclosure relates to a method of treating FA comprising administering an effective amount of a composition disclosed herein, wherein the composition comprises loaded megakaryocyte-derived extracellular vesicles. In 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 a payload. In embodiments, the load is associated with the vesicle surface in addition to or as an alternative to the load located in the lumen of megakaryocyte-derived extracellular vesicles. In embodiments, the load is selected from one or more of RNA, DNA, proteins, carbohydrates, lipids, biomolecules, and small molecules. In embodiments, the load is one or more therapeutic agents.

In embodiments, the present disclosure relates to a method of treating FA comprising administering an effective amount of a composition described herein, wherein the composition comprises megakaryocyte-derived extracellular vesicles comprising a nucleic acid encoding a FA-related gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-related gene or protein product thereof.

In embodiments, the present disclosure relates to a method of treating FA comprising administering an effective amount of a composition comprising a cell contacted in vitro with a composition described herein, wherein the composition comprises megakaryocyte-derived extracellular vesicles comprising a nucleic acid encoding a functional FA-associated gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-associated gene or protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-associated gene or protein product thereof.

In embodiments, the methods of the invention are performed in vivo or ex vivo.

In embodiments, the present disclosure relates to a method of treating FA comprising administering an effective amount of a composition comprising a cell in ex vivo contact with a composition described herein, wherein the composition comprises megakaryocyte-derived extracellular vesicles comprising a nucleic acid encoding a functional FA-associated gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-associated gene or protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-associated gene or protein product thereof.

In embodiments, the present disclosure relates to a method of treating FA comprising administering an effective amount of a composition comprising a cell in contact with an object of the composition described herein, wherein the composition comprises megakaryocyte-derived extracellular vesicles comprising a nucleic acid encoding a functional FA-associated gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-associated gene or protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-associated gene or protein product thereof.

In embodiments, the composition is administered to the subject by injection. In embodiments, the composition is administered to the subject by intravenous injection.

In embodiments, the methods for treating Fanconi Anemia (FA) allow for restoring or increasing FA-related gene expression in a patient to reach normal levels in patients not suffering from FA.

In embodiments, the FA-associated gene expression level in the patient is compared to a reference (i.e., control) expression level of FA-associated gene expression measured in a patient not suffering from FA and/or exhibiting no FA-associated symptoms. For example, a patient not suffering from FA may include a healthy subject. Preferably, the healthy subject is a subject similar to the age, sex, race of the patient receiving FA treatment based on the presently disclosed methods.

In embodiments, the level of FA-associated gene expression in the patient is compared to a reference (i.e., control) level of FA-associated gene expression based on the average or mean level of FA-associated gene expression in a normal population of subjects not suffering from FA and/or not exhibiting any FA-associated symptoms. In embodiments, the mean or average level of FA-associated gene expression is based on values known in the art.

Examples of methods for assessing gene expression levels are known in the art and include, but are not limited to, microarray, PCR, RT-PCR, quantitative PCR, or next generation sequencing techniques.

In embodiments, the method for treating Fanconi Anemia (FA) allows for restoring or increasing FA-related gene expression in a patient to 5% or about 5% of normal levels in patients not suffering from FA; 10% or about 10% of normal levels in patients not suffering from FA; 15% or about 15% of normal levels in patients not suffering from FA; 20% or about 20% of normal levels in patients not suffering from FA; 25% or about 25% of normal levels in patients not suffering from FA; 30% or about 30% of normal levels in patients not suffering from FA; 35% or about 35% of normal levels in patients not suffering from FA; 40% or about 40% of normal levels in patients not suffering from FA; 45% or about 45% of normal levels in patients not suffering from FA; 50% or about 50% of normal levels in patients not suffering from FA; 55% or about 55% of normal levels in patients not suffering from FA; 60% or about 60% of normal levels in patients not suffering from FA; 65% or about 65% of normal levels in patients not suffering from FA; 70% or about 70% of normal levels in patients not suffering from FA; 75% or about 75% of normal levels in patients not suffering from FA; 80% or about 80% of normal levels in patients not suffering from FA; 85% or about 85% of normal levels in patients not suffering from FA; 90% or about 90% of normal levels in patients not suffering from FA; 95% or about 95% of normal levels in patients not suffering from FA; or 100% or about 100% of the normal levels of patients not suffering from FA.

In embodiments, the load and/or the agent suitable for modifying a cell comprises one or more therapeutic agents.

The present disclosure encompasses any FA-related gene. Non-limiting examples of FA-related genes include FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7), and FANCW (RFWD 3).

In embodiments, the FA treatment of the invention provides a gene therapy load in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention, the gene therapy load comprising a nucleic acid encoding a wild-type, unmutated and/or functional FA-related gene selected from FANCA, FANCB, FANCC, FANCD (BRCA 2), fascd 2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), fascp (SLX 4), fascq (ERCC 4), fascr (RAD 51), fascs (BRCA 1), fasct (UBE 2T), fascu (XRCC 2), fascv (REV 7) and fascow (rf3). In embodiments, the gene therapy load is DNA or RNA.

In embodiments, the FA treatment of the invention provides a gene therapy load in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention, the gene therapy load comprising a nucleic acid encoding a wild-type, unmutated and/or functional FA-related gene selected from FANCA, FANCB, FANCC, FANCE, FANCF, FANCG, FANCL, FANCM, FANCD, FANCJ, FANCN and FANCP. In embodiments, the gene therapy load is DNA or RNA.

In embodiments, the FA treatment of the invention provides a gene therapy load in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention, the gene therapy load comprising a gene-editing protein and/or related elements for gene-editing functions against FA-related genes. In embodiments, the FA treatment of the invention provides a gene therapy load in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention, the gene therapy load comprising a gene-editing protein and/or related elements for gene-editing functions for FA-related genes selected from FANCA, FANCB, FANCC, FANCD (BRCA 2), fascd 2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7) and FANCW (RFWD 3). In embodiments, the gene editing proteins provide for the correct editing of functional FA proteins.

In embodiments, the FA treatment of the invention provides a gene therapy load in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention, the gene therapy load comprising a gene-editing protein and/or related elements for gene-editing functions against FA-related genes. In embodiments, the FA treatment of the invention provides a gene therapy load in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention comprising a gene-editing protein and/or related elements for gene-editing functions against FA-related genes selected from FANCA, FANCB, FANCC, FANCF, FANCG, FANCL, FANCM, FANCD, FANCJ, FANCN and FANCP. In embodiments, the gene editing proteins provide for the correct editing of functional FA proteins.

In embodiments, the present disclosure relates to methods of increasing or restoring, for example, the level and/or function of fanconi anemia protein (FANC) in a target cell (e.g., hematopoietic stem cell) by delivering a therapeutic load, such as a gene therapy load, loaded in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention. In embodiments, the level and/or function of one or more FA-associated genes is restored to reach the non-diseased level and/or non-diseased function and/or normal level in a patient not having FA. In embodiments, the level and/or function of the one or more FA-associated genes is restored to a level and/or function that is substantially the same as the non-diseased level and/or non-diseased function and/or normal level of the patient not having FA. In embodiments, the level and/or function of the one or more FA-associated genes is restored to about 1% to about 99%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 70%, about 80% to about 90%, about 90% to about 99% of the level and/or function of the non-diseased and/or normal level of the patient not having FA. In a non-limiting example, the level and/or function of the one or more FA-associated genes is restored to about 5% to about 10% of the non-diseased level and/or non-diseased function and/or normal level of the patient not suffering from FA, resulting in a clinical benefit and/or beneficial risk/benefit condition.

In embodiments, the level and/or function of one or more FA-associated genes improves over time following treatment with megakaryocyte-derived extracellular vesicles (mkevs) of the invention. In embodiments, the level and/or function of one or more FA-associated genes is improved to achieve substantially the same level and/or function as the non-diseased level and/or non-diseased function and/or normal level of a patient not having FA. In embodiments, the level and/or function of one or more FA-associated genes is improved to about 1% to about 99%, about 1% to about 5%, about 5% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 70%, about 80% to about 90%, about 90% to about 99% of the level and/or function of the non-diseased and/or normal level of a patient not having FA. In a non-limiting example, the level and/or function of one or more FA-associated genes is improved to about 5% to about 10% as compared to the non-diseased level and/or non-diseased function and/or normal level of a patient not having FA, resulting in a clinical benefit and/or favorable risk/benefit profile.

In embodiments, the present disclosure relates to methods of increasing or restoring the level and/or function of one or more FA-associated genes, e.g., in a target cell (e.g., hematopoietic stem cell), by delivering a therapeutic load, such as a gene therapy load, loaded in or associated with megakaryocyte-derived extracellular vesicles (mkevs) of the invention. In embodiments, the level and/or function of one or more FA-associated genes is restored to a non-diseased level and/or non-diseased function. In embodiments, the level and/or function of the one or more FA-associated genes is restored to substantially the same level and/or function as the non-diseased level and/or function. In embodiments, the one or more FA-related genes are selected from FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7), and FANCW (RFWD 3).

In embodiments, the FA is caused by a mutation in one or more genes of the FA pathway and/or the subject being treated has a mutation in one or more genes of the FA pathway and/or the method of the invention corrects a mutation in one or more genes of the FA pathway.

In embodiments, the FA is caused by a mutation in one or more genes of the FA core complex and/or the subject being treated has a mutation in one or more genes of the FA core complex and/or the method of the invention corrects a mutation in one or more genes of the FA core complex.

In embodiments, FA is caused by a mutation in one or more of the following genes and/or a subject receiving treatment has a mutation in one or more of the following genes and/or the method of the invention corrects a mutation in one or more of the following genes: FANCA, FANCB, FANCC, FANCD1 (BRCA 2), FANCD2, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (Rad 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7) and FANCW (RFWD 3).

In embodiments, FA is caused by a mutation in one or more of the following genes and/or a subject receiving treatment has a mutation in one or more of the following genes and/or the method of the invention corrects a mutation in one or more of the following genes: FANCD1 (BRCA 2), FANCJ (BRIP 1), FANCB, FANCD2, FANCF, FANCF, FANCI, FANCQ (ERCC 4), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4) and FANCT (UBE 2T).

In embodiments, FA is caused by a mutation in one or more of the following genes and/or a subject receiving treatment has a mutation in one or more of the following genes and/or the method of the invention corrects a mutation in one or more of the following genes: FANCA, FANCB, FANCC, FANCE, FANCF, FANCG and FANCL.

In embodiments, FA is caused by a mutation in one or more of the following genes and/or a subject receiving treatment has a mutation in one or more of the following genes and/or the method of the invention corrects a mutation in one or more of the following genes: FANCM, FANCD1, FANCJ, FANCN, and FANCP.

In embodiments, FA is caused by a mutation in one or more of the following genes and/or a subject receiving treatment has a mutation in one or more of the following genes and/or the method of the invention corrects a mutation in one or more of the following genes: FANCA, FANCC and FANCG.

In embodiments, FA is caused by a mutation in FANCR (Rad 51) and/or the subject receiving treatment has a mutation in FANCR (Rad 51) and/or the method of the invention corrects a mutation in FANCR (Rad 51).

In embodiments, the FA is caused by a mutation in one or more of the following genes or loci and/or the subject being treated has a mutation in one or more of the following genes or loci and/or the present method corrects a mutation in one or more of the following genes or loci: FANCA (16q24.3) FANCB (Xp22.31), FANCC (9q22.3), FANCD1 (BRCA 2) (13q12.3), FANCD2 (3p25.3), FANCE (6p21.3), FANCF (11 p 15), FANCG (X RCC 9) (9 p 13), FANCI (KIAA 1794) (15 q 25), FANCJ (BRIP 1) (17q22.3), FANCL (PHF 9/POG) (2p16.1), FANCM (Hef) (14q21.3) FA NCN (PALB 2) (16p12.1), FANCO (RAD 51C) (17q25.1), FANCP (S LX 4) (16p13.3), FANCQ (XPF/ERCC 4) (16p13.12), FANCR (Rad 51) (15 q 15), FANCS (BRCA 1) (1721.31), FANCT (WD 2) (17q22.3), FANCV (FANCC 2) (16p1.36.37) and FANCV (QC 1.36.37).

In embodiments, the FA is caused by a mutation in one or more of the following genes or loci and/or the subject being treated has a mutation in one or more of the following genes or loci and/or the present method corrects a mutation in one or more of the following genes or loci: FANCA (16q24.3) FANCB (Xp22.31), FANCC (9q22.3), FANCD1 (BRCA 2) (13q12.3), FANCD2 (3p25.3), FANCE (6p21.3), FANCF (11 p 15), FANCG (XRCC 9) (9 p 13), FANCI (KIAA 1794) (15 q 25), FANCJ (BRIP 1) (17q22.3), FANCL (PHF 9/POG) (2p16.1), FANCM (Hef) (14q21.3) FANCN (PALB 2) (16p12.1), FANCO (RAD 51C) (17q25.1), FANCP (SLX 4) (16p13.3), FANCQ (XPF/ERCC 4) (16p13.12).

In embodiments, the FA is caused by one or more mutations in one or more FA-related genes and/or the subject being treated has one or more mutations in one or more FA-related genes and/or the method of the invention corrects one or more mutations in one or more FA-related genes. Non-limiting examples of mutations can be found in US2014/0087397 and the rocfeiler university fanconi anemia mutation database (Rockefeller University Fanconi Anemia Mutation Database) (see rocfeller. In embodiments, the FA is caused by one or more of the following mutations and/or the subject being treated has one or more of the following mutations and/or the method of the invention corrects one or more of the following mutations:

●FANCC c.456+4A>T(IVS4)

●FANCD1 c.6174delT

●FANCA c.3788_3790del

●FANCG c.1077-2A>G

●FANCC c.67delG(322delG)

●FANCG c.1480+1G>C

●FANCA c.2172dupG,4275delT,c.2574C>G,c.890-893del

●FANCA c.2546delC,c.3720_3724del

●FANCC c.456+4A>T

●FANCG c.307+1G>C,c.1066C>T

●FANCA c.2546delC,c.3720_3724del

●FANCG c.307+1G>C,c.1066C>T

●FANCC c.165+1G>T

●FANCA c.1007-？_3066+？del,c.1007-？_1626+？del,c.3398delA

●FANCG c.637_643del

●FANCA c.295C>T

●FANCD2 c.1948-16T>G。

In embodiments, FA subjects, i.e., subjects receiving treatment, are characterized by chromosomal instability.

In embodiments, the FA subject, i.e., the subject receiving treatment, is characterized by autosomal recessive inheritance of one or more of BRCA2, BRIP1, FANCB, FANCD2, FANCE, FANCF, FANCI, ERCC4, FANCL, FANCM, PALB2, RAD51C, SLX4, and UBE 2T.

In embodiments, FA subjects, i.e., subjects receiving treatment, are characterized by X-linked recessive inheritance of FANCB.

In embodiments, FA subjects, i.e., subjects receiving treatment, are characterized by an autosomal dominant inheritance of RAD 51.

In embodiments, the subject is a member of a population known to have a basal mutation associated with FA, such as de jerusalem (FANCC, BRCA2/FANCD 1), north european (FANCC), south african dutch (FANCA), saharan south black race (FANCG) or spanish roman (FANCA).

In embodiments, the therapeutic agent is capable of increasing expression of the functional FA-associated gene or protein product thereof by about 1-fold, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 10-fold, about 15-fold, about 18-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 60-fold, about 100-fold, about 500-fold, or about 1000-fold as compared to expression of the non-functional and/or defective FA-associated gene or protein product.

In embodiments, the methods of treatment of the invention reduce, ameliorate or eliminate various symptoms or manifestations of FA, such as bone marrow dysfunction, blood cell deficiency, or abnormal cell production. In embodiments, the methods of treatment of the present invention reduce, ameliorate or eliminate defects in one or more of patient white blood cell count, neutrophil count, reticulocyte count, platelet count and red blood cell count, bone marrow or normal cell production.

In embodiments, the methods of treatment of the invention reduce, ameliorate or eliminate various symptoms or manifestations of FA, such as anemia, thrombocytopenia, and neutropenia.

In embodiments, the methods of treatment of the invention reduce, ameliorate or eliminate the likelihood of one or more of various secondary complications of FA, such as, but not limited to, headache, dizziness, fatigue, shortness of breath, anemia, thrombocytopenia, neutropenia, myelodysplastic syndrome (MDS), kidney-related diseases, and Acute Myeloid Leukemia (AML) in a patient.

In embodiments, the methods of the invention reduce, improve or eliminate FA, as detected or detectable using one or more of chromosome breakage assays (optionally DEB (butylene dioxide) and/or MMC (mitomycin C)), cell cycle assays in peripheral blood lymphocytes, complete peripheral blood counts, mitomycin C resistance assays and mutation assays (e.g., chromosome, sequencing, etc.), measurement of complete blood counts, and/or restoration or partial restoration of FANC protein production and localization.

In embodiments, the present methods supplement or replace one or more FA therapeutics or therapeutic methods selected from the group consisting of: androgens, hematopoietic growth factors, transfusion support, hematopoietic Stem Cell Transplantation (HSCT) and surgery (e.g., to correct skeletal deformities such as those affecting thumb and forearm bones, heart defects, and gastrointestinal abnormalities, such as tracheoesophageal fistulae or esophageal closure, and anal closure).

In embodiments, the treatment eliminates the need for blood and/or bone marrow transplantation, androgen therapy, synthetic growth factor therapy, chemotherapy, and/or surgery.

In embodiments, the FA treatment of the invention provides gene replacement of any FA-related gene described herein.

Megakaryocyte-derived extracellular vesicles

In one aspect, the present disclosure 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 embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells. In embodiments, 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 embodiments, the megakaryocyte-derived extracellular vesicles comprise a load in the lumen or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles are contained in the lumen and a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles comprise a load suitable for modifying cells and/or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the modified cells include gene therapy, including but not limited to the use of biotherapeutic agents encoding functional or recombinant proteins. In embodiments, the modified cell comprises gene editing. In embodiments, the megakaryocyte-derived extracellular vesicles comprise a load suitable for gene editing of cells and/or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, in addition to or as an alternative to the load located in the lumen of megakaryocyte-derived extracellular vesicles, the load is loaded into megakaryocytes for packaging into extracellular vesicles. In embodiments, the load is directly loaded into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to the load located in the lumen of the megakaryocyte-derived extracellular vesicles.

In another aspect, the present disclosure is directed 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 embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells. In 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 embodiments, the nucleic acid molecule is exogenous. In embodiments, the megakaryocyte-derived extracellular vesicles comprise a load suitable for modifying cells and/or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the modified cells include gene therapy, including but not limited to the use of biotherapeutic agents encoding functional or recombinant proteins. In embodiments, the modified cell comprises gene editing. In embodiments, in addition to or as an alternative to the load located in the lumen of megakaryocyte-derived extracellular vesicles, the load is loaded into megakaryocytes for packaging into extracellular vesicles. In embodiments, the load is directly loaded into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to the load located in the lumen of the megakaryocyte-derived extracellular vesicles.

In one aspect, the present disclosure 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 embodiments, the megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for loading into the lumen. In embodiments, the load comprises one or more agents. In embodiments, the agent is one or more therapeutic agents, including the therapeutic agents described herein. In embodiments, the payload comprises one or more megakaryocyte-derived nucleic acid molecules selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs. In embodiments, the megakaryocyte-derived extracellular vesicles comprise a load in the lumen or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles are contained in the lumen and a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, in addition to or as an alternative to the load located in the lumen of megakaryocyte-derived extracellular vesicles, the load is loaded into megakaryocytes for packaging into extracellular vesicles. In embodiments, the load is directly loaded into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to the load located in the lumen of the megakaryocyte-derived extracellular vesicles. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for carrying a load associated with the surface of megakaryocyte-derived extracellular vesicles.

In another aspect, the present disclosure is directed 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 a load, and the lipid bilayer membrane contains one or more proteins associated therewith or embedded therein. In embodiments, the load comprises one or more agents. In embodiments, the agent is one or more therapeutic agents, including the therapeutic agents described herein. In embodiments, the payload comprises one or more megakaryocyte-derived nucleic acid molecules selected from mRNA, tRNA, rRNA, siRNA, micrornas, regulatory RNAs, and non-coding and coding RNAs. In embodiments, the megakaryocyte-derived extracellular vesicles comprise a load in the lumen or a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, the megakaryocyte-derived extracellular vesicles are contained in the lumen and a load associated with the surface of the megakaryocyte-derived extracellular vesicles. In embodiments, in addition to or as an alternative to the load located in the lumen of megakaryocyte-derived extracellular vesicles, the load is loaded into megakaryocytes for packaging into extracellular vesicles. In embodiments, the load is directly loaded into the megakaryocyte-derived extracellular vesicles in addition to or as an alternative to the load located in the lumen of the megakaryocyte-derived extracellular vesicles. In embodiments, megakaryocyte-derived extracellular vesicles are suitable for carrying a load associated with the surface of megakaryocyte-derived extracellular vesicles.

In one aspect, the present disclosure 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 present disclosure 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 present disclosure is directed 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 a patient suffering from FA, exhibiting FA-related symptoms, and/or likely developing FA.

In another aspect, the present disclosure is directed 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 a patient suffering from FA, exhibiting FA-related symptoms, and/or likely developing FA.

In another aspect, the present disclosure 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, further wherein the megakaryocyte-derived extracellular vesicles produced comprise a load for modifying the cells to provide functional Fanconi Anemia (FA) -related genes and/or to repair functional FA-related genes mutated therein.

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 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 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 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 CD18, CD43, CD11b, CD62P, CD, CD61, CD21, CD51, phosphatidylserine (PS), CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD54, 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 an embodiment, the megakaryocyte-derived extracellular vesicles of the invention are characterized by not expressing and/or comprising a substantial amount of DRAQ5. In an embodiment, the 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 62P.

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

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

In the context of an embodiment of the present invention, 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 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 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 41.

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In 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 embodiments, less than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, less than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 61.

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

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

In the context of an embodiment of the present invention, 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 and/or presence of CD61 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 4. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 54.

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

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

In the context of an embodiment of the present invention, 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 and/or presence of CD54 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 18.

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

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

In the context of an embodiment of the present invention, 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 and/or presence of CD18 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 43.

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD11 b.

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 21.

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

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

In the context of an embodiment of the present invention, 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 and/or presence of CD21 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 51.

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

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

In the context of an embodiment of the present invention, 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 and/or presence of CD51 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising CLEC-2.

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A). In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising LAMP-1 (CD 107A).

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63.

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

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

In the context of an embodiment of the present invention, 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 embodiments, about 1% to about 30% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 5% to about 25% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In embodiments, about 10% to about 20% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 63. In 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 and/or presence of CD63 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, about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD42 b.

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 9.

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

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

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

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

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

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

In the context of an embodiment of the present invention, 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 embodiments, about 5% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In embodiments, about 10% to about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In embodiments, about 10% to about 35% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 31. In 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 and/or presence of CD31 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47.

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

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

In the context of an embodiment of the present invention, 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 embodiments, about 1% to about 50% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 10% to about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In embodiments, about 20% to about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 47. In 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 and/or presence of CD47 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147.

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

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

In the context of an embodiment of the present invention, 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 embodiments, about 1% to about 15% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 1% to about 10% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In embodiments, about 3% to about 8% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD 147. In 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise a lipid bilayer membrane comprising CD32 a.

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

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

In the context of an embodiment of the present invention, 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 embodiments, greater than about 40% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In embodiments, greater than about 60% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In embodiments, greater than about 70% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In embodiments, greater than about 80% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In embodiments, greater than about 90% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GPVI. In embodiments, greater than about 95% of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In 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 embodiments, about 40% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, about 60% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, about 70% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, about 80% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, about 90% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, about 95% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI. In embodiments, about 99% or less of megakaryocyte-derived extracellular vesicles comprise lipid bilayer membranes comprising GVPI.

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

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

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

In 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 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 an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by being substantially free of DRAQ5.

In 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 embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2 or 3 of PS, CD61, and CD 63. In 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 an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by being substantially free of DRAQ5.

In 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 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 an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by being substantially free of DRAQ5.

In 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 embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise 2 or 3 of Phosphatidylserine (PS), CD62P, and CD9. In 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 an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by not expressing a substantial amount of DRAQ5. In an embodiment, the megakaryocyte-derived extracellular vesicles are characterized by being substantially free of DRAQ5.

In an embodiment, 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 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 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 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 200nm and about 350 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 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 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 200nm 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 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 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 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

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.

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 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 embodiments, the pre-platelets and the original platelets as well as the platelet-like particles are all 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 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 embodiments, the human pluripotent stem cells are HSCs produced by the liver, kidney, spleen, or bone marrow. In embodiments, the HSCs are produced by the liver. In embodiments, the HSCs are produced by the kidneys. In embodiments, the HSCs are produced by the spleen. In embodiments, the HSCs are produced from bone marrow. In 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 embodiments, the molecular signal is TPO. In embodiments, the molecular signal is GM-CSF. In embodiments, the molecular signal is IL-3. In embodiments, the molecular signal is IL-6. In embodiments, the molecular signal is IL-11. In embodiments, the molecular signal is IL-6. In embodiments, the molecular signal is SCF. In an embodiment, the molecular signal is IL-6.SCF. In embodiments, the molecular signal is Flt3L. In embodiments, the molecular signal is IL-6. In embodiments, the molecular signal is IL-9.

In embodiments, the molecular signal is a chemokine.

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

In 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-l-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 (iPCS). 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 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 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 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 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 embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of mitochondria. In embodiments, the formulation comprising megakaryocyte-derived extracellular vesicles of the present disclosure is substantially free of exosomes. In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise organelles.

In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure are substantially free of nuclei. In 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 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 load through various mechanisms of target cells for megakaryocyte-derived extracellular vesicle internalization.

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 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 embodiments, megakaryocyte-derived extracellular vesicles home to one or more myelogenous cells in the bone marrow in vivo. In the context of an embodiment of the present invention, 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 embodiments, megakaryocyte-derived extracellular vesicles home to one or more erythropoietic cells in the bone marrow in vivo. In embodiments, the one or more erythropoietic cells are selected from the group consisting of orthoerythroblasts, basophils, multi-chromatic erythroblasts, and orthochromatic erythroblasts. In embodiments, megakaryocyte-derived extracellular vesicles home to one or more of plasma cells, reticulocytes, lymphocytes, monocytes, and megakaryocytes in vivo.

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

In 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 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 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 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 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 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 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 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 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 embodiments, megakaryocyte-derived extracellular vesicles home to HSCs in vivo with about 10-fold higher 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 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 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 embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes 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 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 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 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 embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes 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 embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes 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 embodiments, megakaryocyte-derived extracellular vesicles home to lymphocytes 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 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 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 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 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 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 embodiments, megakaryocyte-derived extracellular vesicles home to regulatory T cells in vivo with about 6-fold higher specificity than to another cell type, or to another organ, or to all other combined cell types. In 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 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 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 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 Fanconi Anemia (FA), including methods of transferring deliverable therapeutic agents.

In some embodiments, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) obtaining megakaryocyte-derived extracellular vesicles; (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 some embodiments, the invention relates to a method of transferring a deliverable therapeutic agent comprising: (a) obtaining megakaryocyte-derived extracellular vesicles; (b) Incubating the megakaryocyte-derived extracellular vesicles with the therapeutic agent to bind the therapeutic agent to 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 one aspect, the present disclosure provides ex 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 Fanconi Anemia (FA) 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 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 Fanconi Anemia (FA) to bind the therapeutic agent to the surface of the megakaryocyte-derived extracellular vesicles and produce 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, 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 Fanconi Anemia (FA) to bind the therapeutic agent to 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 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 embodiments, contacting a deliverable therapeutic agent capable of treating Fanconi Anemia (FA) with a biological cell comprises co-culturing the deliverable therapeutic agent with the biological cell to provide transfer of a load 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 embodiments, the biological cell is one or more of a cancer cell, a tumor cell, a virally 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 embodiments, the biological cell is one or more of a cancer cell, a tumor cell, a virally 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 embodiments, the biological cell is one or more of a cancer cell, a tumor cell, a virally 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 embodiments, the cell culture process is employed to generate allogeneic megakaryocyte-derived extracellular vesicles from primary human peripheral blood cd34+ HSCs. In 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 embodiments, megakaryocyte-derived extracellular vesicles are produced by a method comprising obtaining primary human cord blood cd34+ HSCs. In 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 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 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 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 embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of nucleic acids. In embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of autologous nucleic acids. In embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of RNA. In embodiments, the megakaryocyte-derived extracellular vesicles comprise nucleic acids. In embodiments, megakaryocyte-derived extracellular vesicles of the present disclosure comprise autologous nucleic acids. In 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 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 embodiments, the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA. In 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 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 the CD41 expression and/or presence is measured and the concentration of megakaryocyte-derived extracellular vesicles in the cell culture is analyzed by flow cytometry or nanoparticles. In embodiments, megakaryocyte-derived extracellular vesicles are produced by a series of centrifugation, e.g., at increasing speeds/forces. In embodiments, megakaryocyte-derived extracellular vesicles are produced by the following methods: (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 embodiments, use is made of a pH and pO which are different ₂ Or pCO ₂ And a multi-stage culture process of a mixture of different cytokines to greatly increase megakaryocyte production.

In 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 embodiments, commercial media is used. In embodiments, serum-free medium is used. In embodiments, the pH is changed to increase megakaryocyte production. In embodiments, CO is altered ₂ Percentage to increase megakaryocyte production. In embodiments, the properties of the molecular signal/factor/cytokine are altered to increase megakaryocyte production. In embodiments, the molecular signalThe factor/cytokine mixture comprises one or more of TPO, GM-CSF, IL-3, IL-6, IL-11, SCF, flt3L, IL-9, etc.

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

In embodiments, methods of producing megakaryocyte-derived extracellular vesicles are standardized to achieve large-scale production.

In 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 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 embodiments, methods of producing megakaryocyte-derived extracellular vesicles are developed such that the batch-to-batch/donor-to-donor variability is less than 10%. In embodiments, methods of producing megakaryocyte-derived extracellular vesicles are developed such that the batch-to-batch/donor-to-donor variability is less than 7.5%. In embodiments, methods of producing megakaryocyte-derived extracellular vesicles are developed such that the batch-to-batch/donor-to-donor variability is less than 5%. In embodiments, methods of producing megakaryocyte-derived extracellular vesicles are developed such that the batch-to-batch/donor-to-donor variability is less than 2.5%.

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

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD54. In 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 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 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD54.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD18. In 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 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 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD18.

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

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD11b. In 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 megakaryocyte-derived extracellular vesicles in the population comprise CD11b. In 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD11b.

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

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD41. In embodiments, from about 15% to about 90%, from about 30% to about 80%, or from about 50% to about 70% of megakaryocyte-derived extracellular vesicles in the population comprise CD41. In 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 embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD61. In embodiments, from about 40% to about 100%, from about 60% to about 100%, or from about 85% to about 10% of megakaryocyte-derived extracellular vesicles in the population comprise 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 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 embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD21. In 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 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 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD21.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD51. In 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 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 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD51.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CLEC-2. In 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 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CLEC-2.

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

In 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 embodiments, megakaryocyte-derived extracellular vesicles in the population are substantially free of DRAQ5. In 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 megakaryocyte-derived extracellular vesicles in the population comprise DRAQ5. In 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 embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD63. In embodiments, from about 1% 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 CD63. In 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 a population comprise CD63. In embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD63.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD42b. In 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 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD42b.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD9. In embodiments, from about 40% to about 100%, from about 50% to about 80%, or from about 60% to about 70% of megakaryocyte-derived extracellular vesicles in the population comprise CD9. In 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 embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD31. In embodiments, from about 1% to about 30%, from about 1% to about 25%, from about 1% to about 20%, or from about 1% to about 15% of megakaryocyte-derived extracellular vesicles in the population comprise CD31. In 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 embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD31.

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

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

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise CD32a. In 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 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 a population comprise CD32a. In embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of CD32a.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In 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 megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In 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 megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of GVPI.

In embodiments, substantially all megakaryocyte-derived extracellular vesicles in the population comprise phosphatidylserine. In embodiments, from about 15% to about 90%, from about 30% to about 80%, or from about 50% to about 70% of megakaryocyte-derived extracellular vesicles in the population comprise phosphatidylserine. In 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 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 megakaryocyte-derived extracellular vesicles in the population comprise GVPI. In embodiments, all megakaryocyte-derived extracellular vesicles in the population are free or substantially free of phosphatidylserine.

In embodiments, megakaryocyte-derived extracellular vesicles are produced by the following methods: (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 embodiments, human pluripotent stem cells are extracted from a patient and used to generate megakaryocytes, from which extracellular vesicles derived from megakaryocytes comprising a selected load are generated, and then administered to the patient. In 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 a selected load are produced, and then administered to the patient.

In embodiments, the megakaryocyte-derived extracellular vesicles are allogeneic to the patient. In embodiments, human pluripotent stem cells are extracted from a human subject that is not a patient and used to generate megakaryocytes, from which extracellular vesicles derived from megakaryocytes comprising a selected load are generated, and then administered to the patient. In 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 a selected load are produced, and then administered to the patient.

In embodiments, the megakaryocyte-derived extracellular vesicles are allogenic with the patient. In embodiments, pluripotent stem cells are extracted from a non-human subject and used to generate megakaryocytes, from which extracellular vesicles derived from megakaryocytes comprising a selected load are generated, and then administered to a patient. In 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 extracellular vesicles derived from megakaryocytes comprising a selected load 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 embodiments, the amount of gamma radiation is between about 12kGy and 15 kGy. In embodiments, the amount of gamma radiation is between about 15kGy and 20 kGy. In embodiments, the amount of gamma radiation is between about 20kGy and 25 kGy. In embodiments, the amount of gamma radiation is between about 25kGy and 30 kGy. In embodiments, the amount of gamma radiation is between about 30kGy and 35 kGy. In embodiments, the amount of gamma radiation is between about 35kGy and 40 kGy. In embodiments, the amount of gamma radiation is between about 40kGy and 45 kGy. In embodiments, the amount of gamma radiation is between about 45kGy and 50kGy. In embodiments, the amount of gamma radiation is between about 50kGy and 55 kGy. In embodiments, the amount of gamma radiation is between about 55kGy and 60 kGy.

In embodiments, the method is substantially serum-free. In embodiments, the method is greater than 60% serum-free. In embodiments, the method is greater than 70% serum-free. In embodiments, the method is greater than 80% serum-free. In 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 embodiments, megakaryocyte-derived extracellular vesicles are purified using size exclusion filtration. In an embodiment, the filter has a pore size of about 650 nm. In embodiments, megakaryocyte-derived extracellular vesicles are purified using size exclusion filtration. In embodiments, the filter has a pore size of about 50nm to about 600 nm. In embodiments, the filter has a pore size of at least 50 nm. In an embodiment, the filter has a pore size of about 600 nm.

Load of megakaryocyte-derived extracellular vesicles (cargo)

Megakaryocyte-derived extracellular vesicles may contain a variety of loads, such as mRNA, micrornas, and cytokines. Megakaryocyte-derived extracellular vesicles are capable of transferring their load to alter the function of target cells. They exert effects on target cells through surface receptor signaling, plasma membrane fusion and internalization. By loading biological or therapeutic loads into megakaryocytes or megakaryocyte-derived extracellular vesicles, megakaryocyte-derived extracellular vesicles can be further used as delivery vehicles to achieve targeted therapeutic effects. 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, and are particularly suitable for delivering payloads, such as therapies.

In embodiments, megakaryocyte-derived extracellular vesicles are suitable for loading into the lumen. In embodiments, the load is selected from one or more of RNA, DNA, proteins, carbohydrates, lipids, biomolecules, and small molecules. In embodiments, the load is a biologically produced component. In embodiments, the load is a synthetically produced component. In embodiments, the load is preloaded into megakaryocyte-derived extracellular vesicles. In embodiments, the biological component is overexpressed in megakaryocytes, such that the resulting megakaryocyte-derived extracellular vesicles comprise the biological component. In embodiments, the load is post-loaded into megakaryocyte-derived extracellular vesicles. In embodiments, purified megakaryocyte-derived extracellular vesicles are mixed with a payload to produce loaded megakaryocyte-derived extracellular vesicles. In embodiments, the load is hydrophobic. In embodiments, the load is hydrophilic. In embodiments, the load is incorporated into the lipid bilayer of megakaryocyte-derived extracellular vesicles. In embodiments, the load is located in the lumen of megakaryocyte-derived extracellular vesicles.

In embodiments, the load is associated with megakaryocyte-derived extracellular vesicles in addition to or as an alternative to the load located in the lumen of megakaryocyte-derived extracellular vesicles. In embodiments, the load is associated with the surface and/or exterior of megakaryocyte-derived extracellular vesicles. Non-limiting examples of loads that bind to megakaryocyte-derived extracellular vesicles include loads that are covalently bound to the surface of the vesicle or that bind to the surface by electrostatic interactions. As will be appreciated by those of ordinary skill in the art, the load associated with megakaryocyte-derived extracellular vesicles can be transported even when not loaded into the vesicle lumen.

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

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

In 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 embodiments, the nucleic acid is DNA. In embodiments, the nucleic acid is plasmid DNA.

In embodiments, the loading efficiency for loading a load such as a 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 embodiments, the load is a nucleic acid. In embodiments, the nucleic acid is DNA. In embodiments, the nucleic acid is plasmid DNA. In an embodiment, the load efficiency is calculated using the following equation:

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

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

In embodiments, the load is one or more therapeutic agents. 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 embodiments, the nucleic acid therapeutic agent is selected from one or more of mRNA, miRNA, siRNA and snoRNA.

In embodiments, one or more non-autologous and/or recombinant nucleic acid constructs are incorporated into the vector. In embodiments, the vector is an expression vector comprising an expression control sequence operably linked to a nucleotide sequence. In embodiments, the vector is a plasmid, phagemid, phage derivative, cosmid or viral vector. In embodiments, the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentiviral vector, a sendai virus vector, a herpes simplex virus vector, a cytomegalovirus vector, or a chimeric virus vector.

In embodiments, the therapeutic agent is a small molecule therapeutic or a biologic therapeutic. In embodiments, the therapeutic agent is used in gene therapy. In embodiments, the biotherapeutic agent encodes a functional protein or a recombinant protein. In embodiments, functional or recombinant proteins include wild-type proteins, fusion proteins, cytokines, antigens and peptides, antibodies or antibody fragments. In embodiments, the therapeutic agent is a nucleic acid therapeutic agent. In embodiments, the nucleic acid therapeutic agent expresses a wild-type functional FA gene, and/or comprises a nucleic acid encoding a functional FA-related gene or a protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-related gene or a protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-related gene or a protein product thereof.

In embodiments, the therapeutic agent is any nucleic acid delivery system known in the art that can be used for vaccination. In one embodiment, the nucleic acid delivery system is a vaccine vector, DNA plasmid, or mRNA vaccine. In embodiments, the therapeutic agent is a vaccine and/or an immunogenic antigen. In embodiments, the nucleic acid therapeutic encodes a wild-type gene (i.e., FA-associated 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 the context of an embodiment of the present invention, 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-pseudoisocytidine, 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-aminocytidine, N4-amino-5-hydroxy-pseudoisocytidine, N4-hydroxy-5-azacytidine, N4-hydroxy-pseudoisocytidine, 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 embodiments, the methods of the invention comprise gene editing and/or gene correction. In embodiments, the methods of the invention include 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 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 embodiments, the gene editing and/or gene correction of the present invention is sufficient for in vivo applications. In embodiments, the gene editing and/or gene correction methods of the invention are sufficiently effective without the need for cell selection (e.g., selecting cells that have been edited). In 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 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 embodiments, the methods of the invention include high efficiency gene editing proteins comprising engineered nuclease cleavage or DNA modification domains. In embodiments, the methods comprise a high efficiency gene editing protein comprising an engineered nuclease cleavage or DNA modification domain. In embodiments, the methods of the invention include high fidelity gene editing proteins comprising engineered DNA binding domains. In embodiments, the high fidelity gene editing protein comprises an engineered DNA binding domain. In embodiments, the methods comprise a gene-editing protein comprising an engineered repeat sequence. In embodiments, the methods comprise a gene-editing protein comprising one or more CRISPR-associated family members. In embodiments, the methods comprise altering the DNA sequence of a cell by transfecting the cell with a gene-editing protein or inducing the cell to express the gene-editing protein. In embodiments, the method comprises altering the DNA sequence of a cell present in an in vitro culture. In embodiments, the method comprises altering the DNA sequence of a cell present in vivo.

In 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 embodiments, the method comprises contacting the cell or patient with a glucocorticoid, such as hydrocortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, or betamethasone. In 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 embodiments, the nucleic acid molecule comprises synthetic RNA. In embodiments, the steroid is hydrocortisone. In embodiments, hydrocortisone is present in the medium at a concentration of about 0.1uM to about 10uM or about 1 uM. In 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 embodiments, the antioxidant is ascorbic acid or ascorbic acid-2-phosphate. In 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 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 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 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 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 agent. In embodiments, the biologic therapeutic is a protein. In embodiments, the biologic therapeutic is an interferon, a monoclonal antibody, and/or an interleukin. In 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 biologic therapeutic is a recombinant protein.

In embodiments, the biologic therapeutic is a virus.

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

In embodiments, the therapeutic agent is a small molecule therapeutic agent. In embodiments, the small molecule therapeutic is one or more of a drug, an inhibitor, or a cofactor. In embodiments, the medicament is for use in cancer treatment. In 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 an immunogenic antigen.

Pharmaceutical composition

In one aspect, the present disclosure provides a composition useful for treating Fanconi Anemia (FA), wherein the composition comprises megakaryocyte-derived extracellular vesicles of the present disclosure. In another aspect, the present disclosure provides a composition useful for treating Fanconi Anemia (FA), the 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 a load and/or the load is associated with a surface of the megakaryocyte-derived extracellular vesicle; and the lipid bilayer membrane comprises one or more proteins associated therewith or embedded therein. In embodiments, the load comprises one or more agents useful in treating Fanconi Anemia (FA). In embodiments, the agent is one or more therapeutic agents, including therapeutic agents for treating Fanconi Anemia (FA).

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 embodiments, an 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 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 publication No. US2008/0213377, which is incorporated herein by reference in its entirety. In various aspects, the present disclosure 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 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 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 embodiments, the pharmaceutical composition is formulated for infusion. In 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 embodiments, the patient may also receive supportive medications or treatments, such as hydration, via infusion. In embodiments, the pharmaceutical composition is formulated for intravenous infusion. In embodiments, the infusion is a continuous infusion, a secondary intravenous therapy (IV) and/or an IV bolus. In 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 disclosure 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 disclosure 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 isolated fromThe stem cell maintenance medium is transformed into HSC expansion medium. During this time, HSCs significantly proliferate. These cultures were then placed in megakaryocyte differentiation medium and megakaryocyte-derived extracellular vesicles were collected from the culture supernatant. 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 cd41+ mkevs, cd41+ PFP mkevs, and cd41+ PLT EVs of the present disclosure (fig. 2C) and fold-change in marker expression between mkevs and PFP mkevs of the present disclosure (fig. 2D), and fold-change in marker expression between mkevs and PLT EVs 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 example, experiments were 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: determination of the Gene load of MKEV

These embodiments describe data related to the successful loading of various loads into the MkEV.

In one example, 9.8kb pDNA encoding wild-type human FANCA is Electroporated (EP) into MKEV. Different electroporation conditions allow for reproducible titration of the load. To quantify only successfully internalized and thus protected copies of pDNA within the MkEV, the MkEV was treated with dnase after electroporation to remove any free and EV-related loads. The protected internalized pDNA payload was then extracted and quantified by qPCR. The amount of supported pDNA in nanograms (ng) was calculated from a standard curve run in parallel and the pDNA copy number/MkEV was calculated. As shown in fig. 11A, a repeatable titratable amount of pDNA was loaded into the MkEV, from as few as 10 copies of pDNA to >1000 copies of pDNA, depending on electroporation conditions.

In another example, cas9 is loaded into a MkEV. MkEV was electroporated with Cas9 and either treated with proteinase K to remove any uninhibited load (free load and load associated with the vesicle surface) or subjected to a filtration treatment to remove any free load, followed by western blot analysis to quantify Cas9. Controls included MkEV plus Cas9, no electroporation ± proteinase K ± filtration. After filtration, cas9 was present in electroporated mkevs, but not in control non-electroporated mkevs, indicating successful vesicle association and/or internalization of protein load. Similarly, cas9 was present in electroporated mkevs after proteinase K digestion, but not in control non-electroporated mkevs, indicating successful internalization and protection of the loaded protein load following electroporation (fig. 11B).

In a non-limiting example, to determine gene loading efficiency, 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 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 mv#

pDNA copy # = [ supported pDNA (ng) # 10≡9/molecular weight ]

* Avogaldel number

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.

Example 5: biodistribution data related to MkEV

This example provides data related to MkEV biodistribution data, including delivery of mkevs loaded, mkEV targeting ex vivo, and in vivo MkEV biodistribution data.

Confocal microscopy was performed to determine internalization of the load delivered by primary HSPCs to mkevs. First, bone marrow was harvested from wild-type mice and NH was used ₄ Cl (STEMCELL Technologies) and lineage depletion (STEMCELL Technologies) for erythrocyte lysis. Activation of cells by fluorescenceHSPCs were isolated by sorting (FACS) and defined as lineage depleted cd150+cd48-cells. MkEV is loaded with GFP-tagged Cas9 (Sigma) and guide RNA (Sigma) preformed as RNPs. HSPCs were co-cultured with loaded mkevs for 18 hours. After the incubation period was completed, the unfixed cells were transferred to a microscope slide and imaged using a Zeiss 780 confocal microscope and a 63-fold objective lens. Image capture and processing was performed using Zen Black software.

Using NH ₄ Cl (STEMCELL Technologies) after lysis of erythrocytes, co-culture was performed using primary wild type whole bone marrow cells. MkEV either carries GFP-tagged Cas9 or is tagged with a DiD Vybrant dye (Invitrogen). DiD labeling was performed according to manufacturer's instructions followed by 2 wash steps. The loaded or labeled MkEV was co-cultured with 1e6 whole bone marrow cells for 24 hours. The co-cultures were then analyzed by flow cytometry (LSR Fortessa X-20, BD Bioscience) using the following antibodies: CD3, CD11B, CD19, GR-1, TER119, CD45R/B220, 7AAD, cKit, sca-1. Flow cytometry data analysis was performed using FlowJo (version 10.8.1,BD Bioscience).

Confocal microscopy images of HSPCs (lineage depleted cd150+cd48-murine bone marrow cells) co-cultured with mkevs loaded with GFP-tagged Cas9 Ribonucleoprotein (RNP) were obtained (fig. 8). Cells co-cultured with the loaded mkevs showed GFP-positive cells, indicating that the cells ingested GFP-tagged Cas 9-loaded mkevs. In contrast, control samples comprising cells alone and cells co-cultured with RNP-loaded, as mock MkEV (no electroporation (no EP)), showed no GFP-positivity. These data indicate that RNP-loaded mkevs have been successfully delivered to HSPCs.

As shown in fig. 9A-9C, mkevs preferentially target ex vivo hematopoietic stem and progenitor cells. MkEV was loaded with GFP-labeled Cas9 protein (fig. 9A) or with lipophilic fluorescent dye DiD (fig. 9B). Loaded and DiD-tagged mkevs were then co-cultured with primary whole bone marrow from wild-type mice. After 24 hours of co-culture, the cells were analyzed for the percentage of gfp+ or did+ (i.e., mkev+) cells by flow cytometry. Quantification of the percentage of gfp+ or did+ cells by flow cytometry indicated cellular uptake/binding of MkEV. In addition, the percentage of lineage positive (Lin+), lineage negative (Lin-) and lineage negative/c-kit+/Sca-1+ (LSK) cells was determined simultaneously using fluorescently labeled antibodies against the lineage positive markers, sca-1 and c-Kit cell surface proteins. The percentage of each cell subtype in the heterogeneous whole bone marrow population is shown in fig. 9A. For cells co-cultured with MkEV loaded with GFP-tagged Cas9 (as shown by the bar graph in fig. 9A), while the vast majority (95%) of the cells in culture were lin+ cells (differentiated cells), only up to 23% of these cells were MkEV positive. In contrast, although the proportion of hematopoietic stem and progenitor cells (Lin-cells) was <5%, almost 50% of these cells were MkEV positive at 300EV per cell dose. Finally, LSK cells account for only 0.25% of the cell population, but almost 40% of the cell population is MkEV positive, for the least and most potent hematopoietic stem cells evaluated in these cultures. These data indicate that bone marrow-derived hematopoietic stem and progenitor cells are preferentially targeted ex vivo. Also, as shown in fig. 9B, for whole bone marrow cells co-cultured with DiD-tagged MkEV; only 20% of Lin+ cells were MKEV positive. In contrast, 30% of the more rare Lin-cell populations were MKEV positive. Finally, for the least and most potent hematopoietic stem cells (LSKs) evaluated in these cultures, up to 48% of the cell population were MkEV positive. The percentage of total Lin+ and Lin-cells in whole bone marrow cultures did not significantly change compared to controls under all conditions of MKEV co-culture (FIG. 9C), indicating lack of toxicity.

As shown in fig. 10A-10K, the in vivo biodistribution of the fluorescence-labeled MkEV was detected after in vivo delivery to wild-type mice. The experimental design is shown in fig. 10A (n=3-5 mice/group). The fluorescently labeled MkEV was intravenously injected into wild-type mice via the tail vein, and tissues were harvested 16 hours post injection and analyzed for fluorescence. Fig. 10B shows fluorescence signals detected by IVIS in the femur dissected from mice. N=5 mice/group. Fluorescence in each homogenized tissue was measured by plate reader and normalized to tissue weight (fig. 10C). As shown in fig. 10C, there was a significant in vivo MkEV targeting bone marrow after injection. Figures 10D and 10E show graphs of experimental data of bone marrow cells stained with antibodies against CD45, lineage markers, CD150, CD201, and CD48, analyzed by flow cytometry to determine the percentage of MkEV positive hematopoietic cells (cd45+ cells; figure 10D) and the percentage of very primitive long term hematopoietic stem cells (cd45+/Lin-/cd150+/cd201+/CD 48-cells; figure 10E). MkEV preferentially targets hematopoietic cells within the bone marrow (fig. 10D), and within this compartment, very rare (less than 0.03% of bone marrow cells) long-term hematopoietic stem cells (fig. 10E). 16 hours after MkEV injection, there was no change in peripheral white blood cell count (fig. 10F), hemoglobin (fig. 10G), platelet count (fig. 10H), WBC differential (fig. 10I), or percentage of cd45+ and CD 45-cells (fig. 10J) or percentage of long-term hematopoietic stem cells in bone marrow (fig. 10K), indicating no evidence of toxicity to hematopoietic compartments.

Example 6: therapeutic delivery vehicle using megakaryocyte-derived extracellular vesicles (mkevs) as FAs

In a non-limiting example, mkevs are produced by obtaining human primary cd34+ HSCs derived from peripheral blood. Human primary cd34+ cells were differentiated into megakaryocytes, and mkevs were isolated from megakaryocytes. MkEV is loaded with therapeutic loads. In a non-limiting example, the payload is plasmid DNA encoding a wild-type FANCA protein. Plasmid DNA was loaded into MKEV using electroporation, and the loaded MKEV was intravenously injected into FANCA-/-mice. Injected mkevs home back to bone marrow, deliver their load to hematopoietic stem cells and resume expression of fasca protein. Successful gene correction and restoration of FANCA protein expression and function is demonstrated by western blotting, increased mitomycin C resistance and/or restoration of FANCD function.

In another non-limiting example, the payload is pDNA encoding a wild-type human FANCA protein. Murine lineage depleted cells were harvested from mouse bone marrow and co-cultured with a pDNA-loaded MkEV (drug (DP) -EV). Total RNA was extracted from cells within 48 hours after co-culture, and pDNA-encoded human FANCA mRNA was quantified using qPCR. There was strong expression of human fasca mRNA in those cells exposed to load-loaded mkevs (fig. 12A), whereas cells treated with load-mimicked mkevs (no-load electroporation) showed no human fasca expression. Quantification by densitometry showed that this increase in mRNA expression in cells treated with load-loaded mkevs was 18.5-fold higher than load-simulated mkevs (fig. 12B). When cells were co-cultured with MkEV loaded with one tenth of the dose of pDNA encoding human FANCA, there was a dose-dependent decrease in the amount of human FANCA mRNA in the co-cultured cells (fig. 13A). This was confirmed by densitometry readings, which showed 1.3 and 4.4 fold increases in FANCA mRNA expression in cells treated with low and high doses of DP-EV, respectively, compared to mock-treated cells (FIG. 13B)

In another non-limiting example, mkevs loaded with pDNA encoding wild-type human FANCA are co-cultured with murine bone marrow lineage depleted cells isolated from FANCA-/-mice. After 48 hours of co-culture, human FANCA mRNA (amplicon length 413 bp) was quantified by qPCR. As shown in fig. 14A, cells lacking any endogenous FANCA expression showed strong expression of human FANCA from MkEV-mediated pDNA delivery when co-cultured with DP-EV. In contrast, mock controls (cells co-cultured with MkEV treated in parallel but not loaded with pDNA load) did not show FANCA expression. Optical density readings indicated a 60-fold increase in FANCA mRNA expression in cells treated with DP-EV compared to mock-treated cells (FIG. 14B).

In another non-limiting example, the hematopoietic cells are derived from FA patients. Hematopoietic stem and progenitor cells carrying FA-specific mutations, or primary cd34+ cells from FA-a patients, were transfected ex vivo with a loaded MkEV. Successful gene correction and recovery of FANCA protein expression and function was demonstrated by qPCR, western blotting, increased mitomycin C resistance and/or recovery of FANCD function.

In another non-limiting example, the payload is pDNA encoding a wild-type FANCA protein. Hematopoietic stem and progenitor cells carrying FA-specific mutations, or primary cd34+ cells from FA-a patients, are transfected ex vivo with loaded mkevs and these mkevs-exposed cd34+ cells are intravenously injected into immunocompromised mice, e.g., non-obese diabetic (NOD) immunodeficiency Cg-prkdccid Il2rgtm1Wjl/SzJ mice (NSG). Successful gene correction and restoration of FANCA protein expression and function is demonstrated, for example, by western blotting, increased mitomycin C resistance and/or restoration of FANCD function, increased transplantation, contribution to peripheral blood and/or proliferation advantage.

In another non-limiting example, the payload is a gene editing complex consisting of CRISPR/Cas9 and guide RNA to correct the FANCA mutation. In this example, hematopoietic cells derived from FA-A patients were transfected in vitro with a load-bearing MKEV. Functional gain in proliferation advantage in transfected cells compared to untreated cells or cells treated with mock EV demonstrated successful gene editing and restoration of FANCA expression (fig. 15). This proliferation advantage of cells recovering wild-type FANCA is a well-documented phenomenon (R I o, P. Et al Nat Med 25,1396-1401 (2019), rom n-Rodr I guez, F.J. et al Cell Stem Cell 25,607-621.e7 (2019), nicolett, E. Et al Ann Hematol 99,913-924 (2020), and R I o, P. Et al Blood 130,1535-1542 (2017)).

In another non-limiting example, hematopoietic stem and progenitor cells carrying FA-specific mutations, or FA-a patient-derived cells, are transfected ex vivo with a loaded MkEV. Successful gene editing depends on functional next generation sequencing and recovery of functional FANCA proteins. Cd34+ cells exposed to MkEV were then maintained in cell culture and the indels were re-assessed by next generation sequencing to reveal the proliferation advantage of successfully editing the cells and normal in vitro differentiation pattern.

In another non-limiting example, the payload is a gene editing complex comprising CRISPR/Cas9 and guide RNA to correct the FANCA mutation. Hematopoietic stem and progenitor cells carrying FA-specific mutations, or primary cd34+ cells from FA-a patients, are transfected ex vivo with loaded mkevs and these mkevs-exposed cd34+ cells are intravenously injected into immunocompromised mice, e.g., non-obese diabetic (NOD) immunodeficiency Cg-prkdccid Il2rgtm1Wjl/SzJ mice (NSG). Indels were measured by NGS before and after implantation. Changes in transplantation and contribution to peripheral blood, proliferation advantage and/or resistance to mitomycin C were measured.

In another non-limiting example, the payload is a gene editing complex comprising CRISPR/Cas9 and guide RNA to correct the FANCA mutation. The human bone marrow xenograft mouse model is generated with hematopoietic stem and progenitor cells, hematopoietic stem and progenitor cells carrying FA-specific mutations, or primary cd34+ cells from FA-a patients. Loaded MkEV was injected intravenously into human bone marrow xenograft mice. The injected MkEV home back to the bone marrow, carries its load to hematopoietic stem cells and successfully edits the fasca gene. Successful gene editing and restoration of FANCA protein expression and function was demonstrated by western blotting, increased mitomycin C resistance and/or restoration of FANCD function. Indels were measured by NGS before and after implantation. Changes in transplantation and contribution to peripheral blood were measured.

Equivalent scheme

While the present disclosure has been described in connection with the specific embodiments thereof, it will be understood that the present disclosure is capable of additional modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the present disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure 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 disclosure is not entitled to antedate such publication by virtue of prior disclosure.

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.

Claims

1. A method for modifying a cell, the method comprising:

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

wherein:

the MkEV lumen comprises a load comprising an agent suitable for modifying the cell; and/or the number of the groups of groups,

a load comprising an agent suitable for modifying the cell is associated with a surface of the MkEV; and

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

(b) Modifying the cell to provide a functional Fanconi Anemia (FA) related gene and/or repairing a functional FA related gene mutated therein.

2. The method of claim 1, wherein the lipid bilayer membrane comprises one or more proteins selected from CD18, CD43, CD11b, CD62P, CD41, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a, CD54 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 megakaryocyte-derived extracellular vesicles are derived from human pluripotent stem cells, optionally wherein the human pluripotent stem cells are primary cd34+ hematopoietic stem cells.

18. The method of claim 17, wherein the primary cd34+ hematopoietic stem cells are derived from peripheral blood or umbilical cord blood.

19. The method of claim 18, wherein the peripheral blood is adult peripheral blood (mPB) mobilized by granulocyte colony-stimulating factor.

20. The method of any one of claims 1-19, wherein the human pluripotent stem cell is an Embryonic Stem Cell (ESC).

21. The method of any one of claims 1-20, wherein the human pluripotent stem cells are induced pluripotent stem cells (iPS).

22. The method of any one of claims 1-21, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the absence of added erythropoietin.

23. The method of any one of claims 1-22, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the presence of added thrombopoietin.

24. The method of any one of claims 1-23, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient or carrier.

25. The method of any one of claims 1-24, wherein the load is located in the lumen and the load is associated with a surface of the megakaryocyte-derived extracellular vesicle.

26. The method of any one of claims 1-25, wherein the load and/or the agent suitable for modifying a cell comprises one or more therapeutic agents.

27. The method of claim 26, wherein the therapeutic agent is a small molecule therapeutic or a biologic therapeutic.

28. The method of claim 27, wherein the biologic therapeutic is for gene therapy.

29. The method of claim 28, wherein the therapeutic agent encodes a functional protein or a recombinant protein.

30. The method of claim 29, wherein the functional protein or the recombinant protein comprises wild-type proteins, fusion proteins, cytokines, antigens and peptides, antibodies or antibody fragments.

31. The method of claim 26, wherein the therapeutic agent is a nucleic acid therapeutic agent.

32. The method of claim 31, wherein the nucleic acid therapeutic expresses a wild-type functional FA gene, and/or comprises a nucleic acid encoding a functional FA-related gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-related gene or protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-related gene or protein product thereof.

33. The method of claim 31, 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, plasmid DNA or DNA fragments.

34. The method of claim 33, wherein the one or more non-autologous and/or recombinant nucleic acid constructs are incorporated into a vector.

35. The method of claim 34, wherein the vector is an expression vector comprising an expression control sequence operably linked to a nucleotide sequence.

36. The method of claim 34, wherein the vector is a plasmid, phagemid, phage derivative, cosmid or viral vector.

37. The method of claim 36, wherein the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentivirus vector, a sendai virus vector, a herpes simplex virus vector, a cytomegalovirus vector, or a chimeric virus vector.

38. The method of any one of claims 31-37, wherein the nucleic acid therapeutic encodes a gene-editing protein and/or a related element for gene-editing function.

39. The method of claim 38, 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.

40. The method of claim 39, wherein the CRISPR-associated protein is selected from the group consisting of Cas9, xCas9, cas12a (Cpf 1), cas13a, cas14, casX, casY, class 1 Cas protein, class 2 Cas protein, MAD7, and gRNA complexes thereof.

41. The method of any one of claims 26-40, wherein the therapeutic agent is a vaccine and/or an immunogenic antigen.

42. The method of claim 26, wherein the therapeutic comprises a FA-associated gene or fragment thereof comprising FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7) and FANCW (RFWD 3).

43. The method of claim 26, wherein the therapeutic increases or restores FA-associated gene expression and/or level and/or function of one or more FA-associated proteins.

44. The method of claim 44, wherein the FA-related proteins comprise FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (Rad 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7) and FANCW (RFWD 3).

45. A method for treating Fanconi Anemia (FA), the method comprising:

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

(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 FA; 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.

46. The method of claim 45, wherein the lipid bilayer membrane comprises one or more proteins selected from the group consisting of CD18, CD43, CD11b, CD62P, CD41, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a, CD54 and GPVI, and/or the lipid bilayer membrane comprises phosphatidylserine.

47. The method of claim 46, wherein:

48. The method of claim 46 or 47, 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).

49. The method of any one of claims 46-48, 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).

50. The method of any one of claims 45-49, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 600 nm.

51. The method of any one of claims 45-49, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 30nm to about 100 nm.

52. The method of any one of claims 45-50, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 300 nm.

53. The method of any one of claims 45-50, 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.

54. The method of any one of claims 45-50, 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.

55. The method of any one of claims 45-51, wherein the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA.

56. The method of any one of claims 45-51, wherein the megakaryocyte-derived extracellular vesicles are substantially free of:

(a) Megakaryocytes, and/or

(b) Platelets.

57. The method of any one of claims 45-56, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to hematopoietic stem cells in vivo and/or in vitro.

58. The method of any one of claims 45-57, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vivo and/or in vitro.

59. The method of claim 58, wherein said megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vivo and/or in vitro.

60. The method of claim 59, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vivo and/or in vitro.

61. The method of any one of claims 45-60, 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.

62. The method of claim 45, wherein the incubating is performed in vivo.

63. The method of claim 45, wherein the incubating is performed ex vivo.

64. The method of claim 63, wherein the method further comprises obtaining biological cells from the patient.

65. The method of claim 63 or 64, wherein contacting the deliverable therapeutic agent with the biological cell comprises co-culturing the deliverable therapeutic agent with the biological cell.

66. The method of any one of claims 45-65, wherein the megakaryocyte-derived extracellular vesicles are autologous to the patient.

67. The method of any one of claims 45-65, wherein the megakaryocyte-derived extracellular vesicles are allogeneic to the patient.

68. The method of any one of claims 45-65, wherein the megakaryocyte-derived extracellular vesicles are allogenic with the patient.

69. The method of any one of claims 45-65, wherein the therapeutic agent is a small molecule therapeutic agent.

70. The method of any one of claims 45-65, wherein the therapeutic agent is a biologic therapeutic agent.

71. The method of claim 70, wherein the biologic therapeutic is for gene therapy.

72. The method of claim 70, wherein the therapeutic agent encodes a functional protein or a recombinant protein.

73. The method of claim 72, wherein the functional protein or the recombinant protein comprises wild-type proteins, fusion proteins, cytokines, antigens and peptides, antibodies or antibody fragments.

74. The method of any one of claims 45-65, wherein the therapeutic agent is a nucleic acid therapeutic agent.

75. The method of claim 74, wherein the nucleic acid therapeutic expresses a wild-type functional FA gene that is defective in the patient, and/or comprises a nucleic acid encoding a functional FA-related gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-related gene or protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-related gene or protein product thereof.

76. The method of claim 74, 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, plasmids or DNA fragments.

77. The method of claim 76, wherein the one or more non-autologous and/or recombinant nucleic acid constructs are incorporated into a vector.

78. The method of claim 77, wherein the vector is an expression vector comprising an expression control sequence operably linked to a nucleotide sequence.

79. The method of claim 77, wherein said vector comprises a plasmid, phagemid, phage derivative, cosmid or viral vector.

80. The method of claim 79, wherein the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentiviral vector, a sendai virus vector, a herpes simplex virus vector, or a cytomegalovirus vector or a chimeric virus vector.

81. The method of any one of claims 74-80, wherein the nucleic acid therapeutic encodes a gene-editing protein and/or associated elements for gene-editing function.

82. The method of claim 81, 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.

83. The method of claim 82, 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.

84. The method of any one of claims 45-83, wherein the therapeutic agent is a vaccine and/or an immunogenic antigen.

85. The method of claim 45, wherein the therapeutic increases or restores FA-associated gene expression and/or level and/or function of one or more FA-associated proteins.

86. The method of claim 85, wherein the FA-related protein comprises FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7), and FANCW (RFWD 3).

87. The method of any one of claims 45-86, 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.

88. The method of claim 87, wherein the primary cd34+ hematopoietic stem cells are derived from peripheral blood or umbilical cord blood.

89. The method of claim 88, wherein the peripheral blood is adult peripheral blood (mPB) mobilized by granulocyte colony-stimulating factor.

90. The method of any one of claims 45-89, wherein the human pluripotent stem cell is an Embryonic Stem Cell (ESC).

91. The method of any one of claims 45-89, wherein the human pluripotent stem cell is an induced pluripotent stem cell (iPS).

92. The method of any one of claims 45-91, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the absence of added erythropoietin.

93. The method of any one of claims 45-92, wherein the megakaryocyte-derived extracellular vesicles are isolated from megakaryocytes produced in the presence of added thrombopoietin.

94. The method of any one of claims 45-68, 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.

95. The method of any one of claims 45-94, wherein the incubating comprises one or more of electroporation, transformation, transfection, and microinjection.

96. The method of any one of claims 45-95, wherein the megakaryocyte-derived extracellular vesicles bind to a cell surface receptor on a cell of the patient.

97. The method of any one of claims 45-96, wherein the megakaryocyte-derived extracellular vesicles bind to cell surface receptors on the contacted biological cells of step (c).

98. The method of any one of claims 45-97, wherein the megakaryocyte-derived extracellular vesicles are fused with the extracellular membrane of cells of the patient.

99. The method of any one of claims 45-98, wherein the megakaryocyte-derived extracellular vesicles are fused to the extracellular membrane of the biological cell of step (c).

100. The method of any one of claims 45-99, wherein the megakaryocyte-derived extracellular vesicles are endocytosed by cells of the patient.

101. The method of any one of claims 45-98, wherein the megakaryocyte-derived extracellular vesicles are endocytosed by the biological cells of step (c).

102. A method for treating Fanconi Anemia (FA), the method 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:

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

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 increasing or restoring FA-associated gene expression and/or level and/or function of one or more FA-associated proteins; and, a step of, in the first embodiment,

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 restoring or increasing expression of FA-associated genes in the patient to a normal level in a patient not suffering from FA.

103. The method of claim 102, wherein the lipid bilayer membrane comprises one or more proteins selected from CD18, CD43, CD11b, CD62P, CD41, CD61, CD21, CD51, CLEC-2, LAMP-1 (CD 107 a), CD63, CD42b, CD9, CD31, CD47, CD147, CD32a, CD54 and GPVI, and/or the lipid bilayer membrane comprises phosphatidylserine.

104. The method of claim 103, wherein:

105. The method of claim 103 or 104, 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).

106. The method of any one of claims 103-105, 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).

107. The method of any one of claims 103-106, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 600 nm.

108. The method of any one of claims 103-106, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 30nm to about 100 nm.

109. The method of any one of claims 103-107, wherein the megakaryocyte-derived extracellular vesicles have a diameter substantially in the range of about 100nm to about 300 nm.

110. The method of any one of claims 103-107, 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.

111. The method of any one of claims 103-105, 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.

112. The method of any one of claims 102-111, wherein the megakaryocyte-derived extracellular vesicles are substantially free of autologous DNA.

113. The method of any one of claims 102-111, wherein the megakaryocyte-derived extracellular vesicles are substantially free of:

a. Megakaryocytes, and/or

b. Platelets.

114. The method of any one of claims 102-113, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to hematopoietic stem cells in vivo and/or in vitro.

115. The method of any one of claims 102-113, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to bone marrow in vivo and/or in vitro.

116. The method of claim 115, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to lymphocytes in vivo and/or in vitro.

117. The method of claim 116, wherein the megakaryocyte-derived extracellular vesicles are suitable for homing to regulatory T cells in vivo and/or in vitro.

118. The method of any one of claims 102-117, wherein the megakaryocyte-derived extracellular vesicle is suitable for loading the therapeutic into the lumen and/or loading the therapeutic in association with a surface of the megakaryocyte-derived extracellular vesicle.

119. The method of any one of claims 102-118, wherein the therapeutic agent is a small molecule therapeutic agent.

120. The method of any one of claims 102-118, wherein the therapeutic agent is a biologic therapeutic agent.

121. The method of claim 120, wherein the biologic therapeutic is for gene therapy.

122. The method of claim 121, wherein the therapeutic agent encodes a functional protein or a recombinant protein.

123. The method of claim 122, wherein the functional protein or the recombinant protein comprises wild-type proteins, fusion proteins, cytokines, antigens and peptides, antibodies, or antibody fragments.

124. The method of any one of claims 102-118, wherein the therapeutic agent is a nucleic acid therapeutic agent.

125. The method of claim 124, wherein the nucleic acid therapeutic expresses a wild-type functional FA gene that is defective in the patient, and/or comprises a nucleic acid encoding a functional FA-related gene or protein product thereof, or a nucleic acid encoding a gene-editing protein capable of producing a functional FA-related gene or protein product thereof, or a ribonucleoprotein gene-editing complex capable of producing a functional FA-related gene or protein product thereof.

126. The method of claim 124, 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, plasmids or DNA fragments.

127. The method of claim 126, wherein the one or more non-autologous and/or recombinant nucleic acid constructs are incorporated into a vector.

128. The method of claim 127, wherein the vector is an expression vector comprising an expression control sequence operably linked to a nucleotide sequence.

129. The method of claim 127, wherein the vector is a plasmid, phagemid, phage derivative, cosmid or viral vector.

130. The method of claim 129, wherein the viral vector comprises an adenovirus vector, an adeno-associated virus vector, a poxvirus vector, a retrovirus vector, a lentiviral vector, a sendai virus vector, a herpes simplex virus vector, or a cytomegalovirus vector or a chimeric virus vector.

131. The method of any one of claims 102-118, wherein the nucleic acid therapeutic encodes a gene-editing protein and/or a related element for gene-editing function.

132. The method of claim 131, wherein the gene editing protein is selected from the group consisting of Zinc Fingers (ZFs), transcription activator-like effectors (TALEs), meganucleases, and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated proteins.

133. The method of claim 132, 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.

134. The method of any one of claims 102-133, wherein the therapeutic agent is a vaccine and/or an immunogenic antigen.

135. The method of claim 134, wherein the therapeutic increases or restores FA-associated gene expression and/or level and/or function of one or more FA-associated proteins.

136. The method of claim 135, wherein the FA-related protein comprises FANCA, FANCB, FANCC, FANCD (BRCA 2), FANCD2, FANCE, FANCF, FANCG, FANCI, FANCJ (BRIP 1), FANCL, FANCM, FANCN (PALB 2), FANCO (RAD 51C), FANCP (SLX 4), FANCQ (ERCC 4), FANCR (RAD 51), FANCS (BRCA 1), FANCT (UBE 2T), FANCU (XRCC 2), FANCV (REV 7), and FANCW (RFWD 3).

137. The method of any one of claims 45-136, wherein the treatment reduces, ameliorates, or eliminates a defect in one or more of white blood cell count, neutrophil count, reticulocyte count, platelet count, and red blood cell count, bone marrow, or normal cell production in the patient.

138. The method of any one of claims 45-137, wherein the treatment reduces, ameliorates, or eliminates the likelihood of the patient developing one or more of headache, dizziness, fatigue, shortness of breath, anemia, thrombocytopenia, neutropenia, myelodysplastic syndrome (MDS), kidney-related disorders, and Acute Myeloid Leukemia (AML).

139. The method of any one of claims 45-138, wherein the treatment eliminates the need for blood and/or bone marrow transplantation, androgen therapy, synthetic growth factor therapy, chemotherapy, and/or surgery.

140. The method of any one of claims 45-139, wherein the treatment reduces, improves, or eliminates FA, as detected or detectable with one or more of a chromosome breakage assay, a complete peripheral blood count, a mitomycin C resistance assay, a cell cycle analysis in peripheral blood lymphocytes, and a mutation analysis.