CN111182890A

CN111182890A - Methods and compositions for treating epidermolysis bullosa

Info

Publication number: CN111182890A
Application number: CN201880061329.5A
Authority: CN
Inventors: 埃万盖洛斯·V·巴迪阿瓦斯
Original assignee: University of Miami
Current assignee: University of Miami
Priority date: 2017-09-22
Filing date: 2018-09-21
Publication date: 2020-05-19
Also published as: MX2022015753A; CA3076610A1; EP3684336A4; KR20200088799A; IL273407A; WO2019060719A1; KR102722950B1; JP7525396B2; MX2020002777A; EA202090814A1; EP3684336A1; JP2020534344A; AU2018335788A1; AU2018335788B2

Abstract

The present invention provides methods and compositions for treating epidermolysis bullosa.

Description

Methods and compositions for treating epidermolysis bullosa

RELATED APPLICATIONS

This application claims priority to USSN 15/712,294 filed on 22/9/2017, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to the fields of medicine, cell biology, molecular biology and genetics. In particular, the present invention relates to methods and compositions for treating epidermolysis bullosa.

Background

Epidermolysis Bullosa (EB) is a group of inherited skin disorders that cause blisters on the skin and mucous membranes, with an incidence of 20 per million newborns in the united states. This is a result of anchoring defects between the epidermis and the dermis, resulting in a fragile skin. Its severity ranges from mild to fatal.

Dystrophic Epidermolysis Bullosa (DEB) is a genetic variation affecting the skin and other organs. Children with this disease are called "butterfly children" because their skin is described as vulnerable and fragile like the wings of a butterfly. The skin of DEB patients is very prone to severe blistering. Open wounds on the skin heal slowly or not at all, often extensive scarring, and are particularly susceptible to infection. Many individuals bathe in a mixture of bleach and water to combat these infections. Chronic inflammation leads to errors in DNA of affected skin cells, which in turn leads to Squamous Cell Carcinoma (SCC). Most DEB patients succumb to SCC or to DEB-related complications before the age of 30.

DEB is caused by a mutation in the human COL7a1 gene encoding the protein type VII collagen (collagen VII). Mutations that cause DEBs can be autosomal dominant (DDEB) or autosomal Recessive (RDEB). COL7a1 is located on the short arm of human chromosome 3 in the chromosomal region denoted 3p 21.31. The size of this gene is about 31,000 base pairs, and it is notable that its coding sequence is extremely split into 118 exons. COL7A1 was transcribed as 9,287 base pairs of mRNA. In the skin, type VII collagen is synthesized by keratinocytes and dermal fibroblasts.

Collagen VII is a 300kDa protein that dimerizes to form a semi-circular structure: anchoring fibrils. The anchoring fibrils are believed to form a structural link between fibrous collagen in the epidermal basement membrane and the upper dermis. Collagen VII is also associated with the epithelium of the esophageal lining, and DEB patients may suffer from chronic scarring, webbing (webbing), and esophageal obstruction. Affected individuals are often severely malnourished due to trauma to the oral and esophageal mucosa, and require feeding tubes for nutrition. They also suffer from iron deficiency anemia of unknown origin, which leads to chronic fatigue.

There remains a need to provide methods and compositions for treating EB.

Disclosure of Invention

The present invention provides methods for isolating Microvesicles (MVs), such as extracellular vesicles (MVs), from biological fluids without compromising the structural and/or functional integrity of the microvesicles. The present invention also provides methods of isolating ectosomes, microparticles, microvesicles, nanovesicles, shedding vesicles, apoptotic bodies, or membrane particles from a biological fluid without compromising their structural and/or functional integrity. The invention further provides MVs (e.g., EVs) and methods of using MVs (e.g., EVs) for treating EBs (e.g., RDEBs and/or DDEBs).

In one aspect, there is provided a method of treating epidermolysis bullosa in a subject in need thereof, the method comprising: administering to a subject a pharmaceutical composition comprising isolated microvesicles purified by precipitation from a biological fluid; and alleviating and reducing one or more symptoms of epidermolysis bullosa in the subject.

In certain exemplary embodiments, the epidermolysis bullosa is dystrophic epidermolysis bullosa, such as recessive dystrophic epidermolysis bullosa.

In certain exemplary embodiments, the isolated microvesicles are extracellular microvesicles, which are optionally precipitated from the biological fluid using a precipitating agent selected from the group consisting of calcium ions, magnesium ions, sodium ions, ammonium ions, iron ions, ammonium sulfate, alginate, and polyethylene glycol.

In certain exemplary embodiments, the biological fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate (bladder ejaculate), sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stool, pancreatic juice, lavage fluid, cell-derived fluid, tissue sample-derived fluid, and cell culture fluid. In certain exemplary embodiments, the biological fluid is from a mammalian (e.g., human) cell. In certain exemplary embodiments, the mammalian cell is a mesenchymal cell.

In certain exemplary embodiments, the precipitating agent is polyethylene glycol, optionally having a molecular weight of about 6,000Da, about 8,000Da, about 10,000Da, or about 20,000 Da.

In certain exemplary embodiments, the one or more symptoms of epidermolysis bullosa are selected from any combination of: callus thickening, epidermal blistering (e.g. of the hands, feet, elbows and/or knees), oral mucosal blistering, thickening of the nails and/or toenails, sepsis, malnutrition, dehydration, electrolyte imbalance, obstructive airway complications, defects in collagen VII expression, anemia, esophageal stenosis, growth retardation, finning or fusion of the fingers and/or toes, tooth malformations, microcurrent malformations, and corneal abrasions.

In certain exemplary embodiments, the treatment comprises increasing collagen VII expression in the subject.

In another aspect, there is provided a method of treating epidermolysis bullosa in a subject in need thereof, the method comprising: administering to a subject a pharmaceutical composition comprising an isolated extracellular vesicle; and alleviating and reducing one or more symptoms of epidermolysis bullosa in the subject.

In certain exemplary embodiments, the biological fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluids, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stool, pancreatic juice, lavage fluids, fluids derived from cells, fluids derived from tissue samples, and cell culture fluids. In certain exemplary embodiments, the biological fluid is from a mammalian (e.g., human) cell. In certain exemplary embodiments, the mammalian cell is a mesenchymal cell.

In another aspect, a method of increasing collagen VII levels in a cell is provided, the method comprising contacting a cell with an isolated extracellular vesicle from a mammalian fluid, wherein the cell expresses a epidermolysis bullosa genotype.

In certain exemplary embodiments, the mammalian fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, CSF, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid, female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stool, pancreatic juice, lavage fluid, fluid derived from cells, fluid derived from tissue samples, and cell culture fluid. In certain exemplary embodiments, the mammalian fluid is a conditioned medium. In certain exemplary embodiments, the conditioned medium is derived from mesenchymal stem cells.

In certain exemplary embodiments, the cell comprises a mutation in the COL7a1 gene.

In certain exemplary embodiments, the epidermolysis bullosa genotype is recessive dystrophic epidermolysis bullosa.

In certain exemplary embodiments, one or both of the following is performed: stimulating the proliferation of cells and enhancing the resistance of cells to trypsin digestion.

In certain exemplary embodiments, the isolated extracellular vesicles deliver collagen VII protein and/or COL7a1mRNA to cells.

In another aspect, a method of delivering one or more bioactive agents to a cell is provided, the method comprising contacting the cell with an isolated extracellular vesicle from a mammalian fluid.

In certain exemplary embodiments, the one or more bioactive agents are selected from collagen VII proteins, collagen VII mrnas, STAT3 signaling activators (e.g., interferons, epidermal growth factors, interleukin-5, interleukin-6, MAP kinase, and/or c-src non-receptor tyrosine kinase), and canonical Wnt activators.

In certain exemplary embodiments, STAT3 is phosphorylated.

In certain exemplary embodiments, the one or more bioactive agents are one or more pharmaceutical compounds.

In certain exemplary embodiments, the cell has a recessive dystrophic epidermolysis bullosa genotype.

In another aspect, a method is provided for isolating and/or purifying microvesicles from a cell culture supernatant or biological fluid using a precipitating agent that precipitates the microvesicles from the cell culture supernatant or biological fluid by displacing solvated water.

In another aspect, an isolated microvesicle formulation is provided. In one embodiment, the isolated microvesicle preparation is subsequently purified. In one embodiment, the isolated microvesicle preparation is subsequently purified to produce a nuclear exosome preparation. In one embodiment, the isolated microvesicle formulation is subsequently purified to produce a particulate formulation. In one embodiment, the isolated microvesicle formulation is subsequently purified to produce a nanovesicle formulation. In one embodiment, the isolated microvesicle preparation is subsequently purified to produce a preparation of shedding vesicles. In one embodiment, the isolated microvesicle formulation is subsequently purified to produce a membrane particle formulation. In one embodiment, the isolated microvesicle formulation is subsequently purified to produce an apoptotic body preparation.

In another aspect, an isolated microvesicle formulation that promotes or enhances angiogenesis is provided. In one embodiment, the isolated microvesicle formulation promotes or enhances angiogenesis in a patient.

In another aspect, isolated microvesicle formulations that promote or enhance neuronal regeneration are provided. In one embodiment, the isolated microvesicle formulation promotes or enhances neuronal regeneration in a patient.

In another aspect, an isolated microvesicle formulation that promotes or enhances cell proliferation is provided. In one embodiment, the isolated microvesicle preparation promotes or enhances cell proliferation in a patient.

In another aspect, an isolated microvesicle formulation that promotes or enhances cell migration is provided. In one embodiment, the isolated microvesicle preparation promotes or enhances cell migration in a patient. In one embodiment, the invention provides an isolated microvesicle formulation that promotes or enhances wound healing. In one embodiment, the wound is a full thickness burn. In one embodiment, the wound is a degree II burn.

In another aspect, a separate microcapsule brew that reduces scar formation in a patient is provided.

In another aspect, an isolated microcapsule brew is provided that reduces wrinkle formation in the skin of a patient.

In another aspect, an isolated microvesicle formulation for use in diagnosing the presence and/or progression of a disease in a patient is provided. In one embodiment, the disease is metastatic melanoma. In an alternative embodiment, the disease is an inflammatory/autoimmune disorder, such as rheumatoid arthritis. In one embodiment, the disease is graft versus host disease.

In another aspect, isolated microvesicle formulations that can promote functional regeneration and tissue complex tissue structures are provided. In one embodiment, an isolated microvesicle formulation that can regenerate hematopoietic tissue in a patient suffering from aplastic anemia is provided. In one embodiment, an isolated microvesicle formulation is provided that can regenerate at least one tissue selected from the group consisting of: epithelial tissue, stromal tissue, neural tissue, vascular tissue, and adnexal structures. In one embodiment, the invention provides an isolated microvesicle formulation that can regenerate tissue and/or cells from all three germ layers.

In another aspect, an isolated microcapsule formulation for modulating the immune system of a patient is provided.

In another aspect, an isolated microvesicle formulation that enhances the survival of tissue or cells transplanted into a patient is provided. In one embodiment, the patient is treated with the isolated microvesicle formulation prior to receiving the transplanted tissue or cells. In another embodiment, the patient is treated with the isolated microvesicle formulation after receiving the transplanted tissue or cells. In another embodiment, the tissue or cells are treated with an isolated microencapsulating agent. In one embodiment, the tissue or cells are treated with an isolated microvesicle infusion prior to transplantation.

In another aspect, an isolated microvesicle formulation comprising at least one molecule selected from the group consisting of RNA, DNA, and protein from a host cell is provided. In one embodiment, the host cell is engineered to express at least one molecule selected from the group consisting of RNA, DNA, and protein. In one embodiment, an isolated microvesicle formulation comprising at least one molecule selected from the group consisting of RNA, DNA and protein from a host cell is used as a therapeutic agent.

Drawings

The accompanying drawings, which are incorporated herein and form a part of the specification, illustrate various embodiments of the present invention and, together with the description, further serve to explain the principles of the invention and to enable a person skilled in the pertinent art to make and use the invention. In the drawings, like reference numbers can indicate identical or functionally similar elements. A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

figure 1 shows a schematic overview of the protocol for separating microvesicles by ultracentrifugation.

Figure 2 shows an embodiment of the microvesicle separation method of the present invention.

Figure 3 shows another embodiment of the microvesicle separation method of the present invention.

Figure 4 shows an embodiment of the device of the invention that promotes clarification of the biological fluid and collection of precipitated microvesicles by filtration.

Fig. 5A to 5D show, at the magnifications shown in the figures, electron micrographs of microvesicles derived from a medium conditioned with human bone marrow-derived mesenchymal stem cells, isolated by the ultracentrifugation method described in example 1 (fig. 5A & 5B) and according to the method of the invention (fig. 5C & 5D).

Fig. 6A to 6D show, at the magnifications shown in the figures, electron micrographs of microvesicles derived from a culture medium conditioned with porcine bone marrow-derived mesenchymal stem cells, isolated by the ultracentrifugation method described in example 1 (fig. 6A & 6B) and according to the method of the present invention (fig. 6C & 6D).

Fig. 7A to 7D show, at the magnifications shown in the figures, electron micrographs of microvesicles derived from a culture medium conditioned with murine bone marrow-derived mesenchymal stem cells, isolated by the ultracentrifugation method described in example 1 (fig. 7A & 7B) and according to the method of the invention (fig. 7C & 7D).

Fig. 8A to 8C show electron micrographs of microvesicles isolated from human plasma according to the method of the present invention. Fig. 8A to 8C show microvesicles at increased magnification, as shown on a scale bar in the figure.

Fig. 9A to 9C show electron micrographs of microvesicles isolated from porcine plasma according to the method of the present invention. Fig. 9A to 9C show microvesicles at increased magnification, as not shown to scale in the figures.

Fig. 10A to 10C show electron micrographs of microvesicles isolated from human urine according to the method of the present invention. Fig. 10A to 10C show microvesicles at increased magnification, as shown on a scale bar in the figures.

Figure 11 shows a western blot reporting the expression of HSP70, CD63, STAT3 and phosphorylated STAT3 in lysates of human bone marrow-derived mesenchymal stem cells, microvesicles isolated from media conditioned with human bone marrow-derived stem cells, prepared by ultracentrifugation (hMSC MV ultracentrifugation) or the method of the invention as described in example 3 (hMSC PEG pellet). Microvesicles derived from human plasma and human urine prepared by the method of the present invention as described in example 3 were also analyzed. Respectively (human plasma PEG precipitation) and (human urine PEG precipitation).

Fig. 12A to 12C show the effect of microvesicles isolated from a medium conditioned with human bone marrow-derived mesenchymal stem cells on the proliferation of normal human dermal fibroblasts (fig. 12A), dermal fibroblasts obtained from diabetic foot ulcers (fig. 12B), and dermal fibroblasts obtained from pressure foot ulcers (fig. 12C). The effect of microvesicles isolated by ultracentrifugation (MV U/C) and microvesicles isolated by the method of the invention (MV PEG) were compared. Fibroblasts treated with PBS or microvesicle depleted medium were included as controls. Proliferation was determined using the MTT assay.

Fig. 13A to 13G show the effect of microvesicles isolated from a medium conditioned with human bone marrow-derived mesenchymal stem cells on human dermal fibroblast migration, determined by the ability of fibroblasts to migrate into the lacerated area. The plot labeled "pretreatment" shows a representative area of the cell culture plate where cells were removed prior to addition of the test treatment. The effect of fibroblast migration was tested using microvesicles isolated according to the method of the invention (PEG precipitation) and microvesicles isolated by ultracentrifugation (ultracentrifugation) at the indicated concentrations. Fibroblasts treated with PBS or microvesicle depleted medium were included as controls.

Fig. 14A to 14G show the effect of microvesicles isolated from a medium conditioned with human bone marrow-derived mesenchymal stem cells on migration of human dermal fibroblasts obtained from diabetic foot ulcers, as determined by the ability of fibroblasts to migrate into the lacerated area. The plot labeled "pretreatment" shows a representative area of the cell culture plate where cells were removed prior to addition of the test treatment. The effect of fibroblast migration was tested using microvesicles isolated according to the method of the invention (PEG precipitation) and microvesicles isolated by ultracentrifugation (ultracentrifugation) at the indicated concentrations. Fibroblasts treated with PBS or microvesicle depleted medium were included as controls.

Fig. 15A to 15D show that the microvesicles of the present invention are absorbed into human dermal fibroblasts. Nuclei resolved (resolve) using Hoechst33342 dye are shown in the plot labeled "Hoechst 33342". Cells resolved using the Vybrant dye are shown in the figure labeled "Vybrant-Dio". Microvesicles resolved using PKH dyes are shown in the graph labeled "PKH labeled MV". A graph covering the images obtained from all three dyes is seen in the graph labeled "Composite".

Fig. 16A to 16D show that the microvesicles of the present invention are absorbed into human dermal fibroblasts. Nuclei resolved using Hoechst33342 dye are shown in the graph labeled "Hoechst 33342". Cells resolved using the Vybrant dye are shown in the figure labeled "Vybrant-Dio". Microvesicles resolved using PKH dyes are shown in the graph labeled "PKH labeled MV". A graph covering the images obtained from all three dyes is seen in the graph labeled "composite".

Figure 17 shows a western blot of lysates of human dermal fibroblasts treated with the following method: microvesicles (human plasma MV PEG pellets) isolated from plasma obtained from patients suffering from rheumatoid arthritis according to the process of the invention; microvesicles (human hMSC MV PEG pellet) isolated from a culture medium conditioned with bone marrow-derived mesenchymal stem cells according to the method of the present invention; microvesicles isolated from media conditioned with bone marrow-derived mesenchymal stem cells via ultracentrifugation (human hMSC MV ultracentrifugation); PBS control; and depleted media control (MV depleted hMSC conditioned media).

Figure 18 shows the presence of a region comprising exon 15 of BRAF containing the T1799A mutation in: SK-MEL28 cells from RNA amplified using primer 1 (lane 3); SK-MEL28 cells from RNA amplified using primer 2 (lane 4); microvesicles isolated from the medium conditioned with SK-MEL28 cells according to the method of the invention, derived from RNA amplified using primer 1 (lane 5); microvesicles isolated from media conditioned with SK-MEL28 cells according to the method of the invention, derived from RNA amplified using primer 2 (lane 6); SK-MEL28 cells from DNA amplified using primer 1 (lane 7); SK-MEL28 cells from DNA amplified using primer 2 (lane 8); microvesicles isolated from the medium conditioned with SK-MEL28 cells according to the method of the invention, derived from DNA amplified using primer 1 (lane 9); and microvesicles isolated from the medium conditioned with SK-MEL28 cells according to the method of the present invention, from DNA amplified using primer 2 (lane 10).

FIG. 19 shows the presence of V600E BRAF in lysates of SK-MEL28 cells and in lysates of microvesicles isolated from media conditioned with SK-MEL28 cells according to the methods of the invention.

Fig. 20A to 20D show that microvesicles isolated from a culture medium conditioned with bone marrow-derived stem cells obtained from a mouse expressing Green Fluorescent Protein (GFP) are taken up into human dermal fibroblasts according to the method of the present invention. Nuclei resolved using Hoechst33342 dye are shown in the graph labeled "Hoechst 33342". Cells resolved using the Vybrant dye are shown in the figure labeled "Vybrant-Dio". GFP-labeled microvesicles are shown in the figure labeled "GFP". A graph covering the images obtained from all three dyes is seen in the graph labeled "composite".

Fig. 21A to 21D show that microvesicles isolated from a culture medium conditioned with bone marrow-derived stem cells obtained from a mouse expressing GFP are taken up into human dermal fibroblasts according to the method of the present invention. Nuclei resolved using Hoechst33342 dye are shown in the graph labeled "Hoechst 33342". Cells resolved using the Vybrant dye are shown in the figure labeled "Vybrant-Dio". GFP-labeled microvesicles are shown in the figure labeled "GFP". A graph covering the images obtained from all three dyes is seen in the graph labeled "composite".

Fig. 22A-22D show histological sections of full thickness wounds from: figure 22A-untreated animals; figure 22B-microvesicles isolated from a culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method of the present invention; FIG. 22C-saline; and figure 22D-microvesicles isolated from autologous bone marrow-derived mesenchymal stem cells by ultracentrifugation 5 days after injury.

Figures 23A-23D show pictures of a degree II burn in animals treated with: figure 23A-microvesicles isolated by ultracentrifugation from media conditioned with autologous bone marrow-derived mesenchymal stem cells; figure 23B-microvesicles isolated from a culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method of the present invention; and figure 23C-untreated animals 7 days after injury. Figure 23D-shows a full thickness wound in animals treated 7 days after injury with microvesicles isolated by ultracentrifugation from media conditioned with autologous bone marrow-derived mesenchymal stem cells. Arrows indicate abscess formation (40X) in full thickness wounds treated with microvesicles isolated by ultracentrifugation on day 7. This was not observed in full thickness wounds treated with microvesicles prepared according to the method of the present invention.

Figure 24 shows histological sections of degree II wounds from microvesicle treated animals isolated from culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method of the present invention 28 days after injury.

Figure 25 shows histological sections of II degree wounds from saline treated animals 28 days after injury.

Figure 26 shows histological sections of full thickness wounds from microvesicle treated animals isolated from culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method of the present invention 28 days after injury.

Figures 27A-27C show histological sections of full thickness wounds from microvesicle treated animals isolated from culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method of the present invention 28 days after injury. Fig. 27A shows new nerve growth (arrows) and angiogenesis (circles). Fig. 27B shows new nerve growth (arrows). Fig. 27C shows new blood vessel growth (arrows).

Figure 28 shows histological sections of full thickness wounds in animals treated 7 days after injury with microvesicles obtained from media conditioned with autologous bone marrow-derived mesenchymal stem cells.

Fig. 29A to 29B show the presence or absence of chimerism in irradiated animals following administration of GFP-labeled bone marrow.

Fig. 30A-30C show the effect of MSC treatment on hair growth after gamma irradiation (fig. 30A and 30B), and the absence of chimerism in irradiated animals after administration of GFP-labeled bone marrow (fig. 30C).

Fig. 31A to 31F show the effect of bone marrow-derived microvesicles obtained using the method of the present invention on angiogenesis using an in vitro assay for angiogenesis. The top three panels are representative images taken using epifluorescence microscopy of cultures of HUVEC cells treated with bone marrow-derived microvesicles ("bone marrow aspirate MV") obtained using the method of the present invention. The three lower panels are representative images of cultures of HUVEC cells treated with a vehicle control ("vehicle control") taken using an epifluorescence microscope.

Fig. 32A to 32C show the effect of bone marrow-derived microvesicles obtained using the method of the present invention on cell growth or proliferation using an in vitro assay of cell growth. Fig. 32A shows representative images taken three days after treatment using an epitactic microscope of cultures of normal adult fibroblasts treated with bone marrow-derived microvesicles ("bone marrow MV") or PBS ("PBS") obtained using the method of the present invention. Figure 32B shows the average cell number in cultures of normal adult fibroblasts treated three days after treatment with bone marrow-derived microvesicles obtained using the method of the invention ("bone marrow MV") or PBS ("PBS"). Fig. 32C graphically depicts cell number.

Fig. 33A to 33B show the results of chronic wound treatment with bone marrow stem cells (including BM-MSCs). Figure 33A-prior to treatment and prior to wound debridement. Necrotic achilles tendon was seen. FIG. 33B-bone marrow cells healed after administration (i.e., topical administration).

Fig. 34A-34C show dermal remodeling in a wound treated with bone marrow stem cells. (A) Fig. 34A-pre-treatment biopsy of fibrotic scar wound. A post-treatment biopsy producing a large number of reticulin fibers (fig. 34B) and elastic fibers (fig. 34C) is shown.

Fig. 35A-35C show deep II degree burns. Patients were administered BM-MSCs twice at 11 days intervals. Figure 35A-deep II degree burn day 0 (before treatment). The circled area represents the deepest part of the burn. Figure 35B-hair follicle enhancement 11 days after the first (i.e. topical) administration of BM-MSCs. Enhanced blebs were recorded in the circled area of a. Figure 35C-hair growth in the circled area of figure 35A 34 days after the second administration of BM-MSCs.

Fig. 36A-36C show healing of burn patients treated with two topically applied MSCs given ten days apart. Figure 36A-prior to treatment. Figure 36B-10 days after treatment (i.e., topical administration) with the first dose of MSC. Figure 36 CA-7 days after treatment with the second dose of BM-MSCs (i.e., 17 days after figure 36A).

Figures 37A-37B show no evidence of scar formation in burn patients evaluated one year after treatment with BM-MSC. The upper diagram: left ventral forearm (lower panel shows area outlined in yellow). The patient's skin showed signs of normal elasticity, while there were no signs of scar formation in the original burn area.

Fig. 38A-38B show full thickness wounds (day 5) generated on yorkshires. Figure 38A-untreated control. Fig. 38B-wounds treated with BM-MSC EV according to certain embodiments of the invention. The closure of the wound was significantly higher after BM-MSC EV treatment. The arrows indicate regions of increased dermal remodeling according to certain embodiments of the present invention.

Fig. 39A-39C show full thickness wounds (day 28) generated on yorkshires treated with BM-MSC EV according to certain exemplary embodiments. Fig. 39A-arrows highlight nerve growth, while stars show blood vessel growth. Figure 39B-higher magnification shows blood vessel growth (arrows). Figure 39C-higher magnification shows nerve growth (arrows).

Fig. 40A-40B show a degree II burn wound in pigs 5 days after treatment with intralesional injection of porcine BM-MSC EV, according to certain exemplary embodiments. Left: EVs prepared by ultracentrifugation methods known in the art are used to treat burn wounds. The wound bulges and is severely inflamed, forming a sterile pustule (indicative of an induced inflammatory response, not an infection) and healing is reduced. And (3) right: EVs prepared using the exemplary methods described herein are used to treat burn wounds. Wound healing is accelerated and inflammation is reduced compared to traditional EV prepared by ultracentrifugation.

FIGS. 41A-41B graphically depict enrichment of COL7A1mRNA in BM-MSC EV (bars in the middle of each group). EV treatment increased expression of COL7A1 in RDEB fibroblasts, the left panel shows COL7A1 expression detected with primer pair 1, the right panel shows COL7A1 expression detected with primer pair 2, gene expression normalized by the common EV housekeeping gene β -actin expression.

FIG. 42 graphically depicts a chemoselective ligation assay (using "click iT" reaction chemistry) that revealed that RDEB fibroblasts produced new collagen VII, which was integrated into newly synthesized proteins, after co-treatment with BM-MSC EV (10 μ g/mL) and the L-methionine analog L-Homopropargylglycine (HPG) (modified amino acids).

Figures 43A-43B graphically depict BM-MSC EV significantly promoting both RDEB proliferation (figure 43A) and resistance to trypsin digestion (figure 43B), both of which standard in vitro assays assessing function gain support the wound-healing potential of RDEB dermal fibroblasts.

Fig. 44A to 44C show validation of in vitro cell lines derived from infants diagnosed with RDEB (halopeau-Siemens type). RDEB fibroblasts expressed significantly less COL7a1 than fibroblasts from unaffected subjects (NHF). FIG. 44A-

primer pairs

1 and 2 were designed near the 3 'end of the cDNA, corresponding to the 5' end of the mRNA. FIG. 44B-COL 7A1 gene expression in Normal Human Fibroblasts (NHF) and RDEB fibroblasts. Figure 44C-RDEB cells secrete low levels of collagen VII protein relative to normal (control) human fibroblasts.

Fig. 45A-45B show vesicle exchange between BM-MSCs and RDEB fibroblasts. RDEBF (stained with lipid dye Dil (red)) and BM-MSC (stained with lipid dye DiO (green)) were co-cultured and uptake of extracellular vesicles (yellow) started within one day. Scale bar, 10 μm.

Fig. 46A to 46D show co-separation (co-isolate) of collagen VII protein with BM-MSC Extracellular Vesicles (EV). FIG. 46A-Transmission Electron micrograph of extracellular vesicles isolated from BM-MSC serum free Conditioned Medium (CM). FIG. 46B-NanoSigt image of BM-MSC EV diluted 1: 500. FIG. 46C-histogram of size versus concentration (diluted 1: 500). The inset shows that EV comprises a CD63 exosome marker. FIG. 46 collagen VII proteins in D-BM-MSC CM and correlation with purified BM-MSC EV.

FIGS. 47A-47B show enrichment of COL7A1mRNA in BM-MSC EV (bars in the middle of each group). EV treatment increased expression of COL7A1 in RDEB fibroblasts, the left panel shows COL7A1 expression detected with primer pair 1, the right panel shows COL7A1 expression detected with primer pair 2, gene expression normalized by the common EV housekeeping gene β -actin expression.

Fig. 48A to 48C show that RDEB fibroblasts treated with BM-MSC EV contained more collagen VII protein in the medium 3 days after washing. Fig. 48A-process schematic. FIG. 48B-Western blot of collagen VII in RDEB medium. Figure 48C-figure 48B densitometry quantification (above baseline collagen VII detection).

Figures 49A to 49C depict chemoselective ligation assays (using "click iT" reaction chemistry) (figures 49A and 49B) which revealed that after co-treatment with BM-MSC EV (10 μ g/mL) and the L-methionine analogue L-homopropargyl glycine (HPG) (modified amino acids), RDEB fibroblasts produced new collagen VII, which was integrated into the newly synthesized protein (figure 49C).

Fig. 50A-50B show that BM-MSC EV increases in vitro surrogate assays related to RDEB fibroblast wound healing (proliferation and trypsin resistance). FIG. 50A-proliferation (MTT) assay. FIG. 50B-Trypsin resistance assay.

Fig. 51A-51E show BM-MSCs delivered to burn patients in saline in clinical trials. BM-MSCs secreted large amounts of EV (positive for CD63) in saline within a few hours (showing 4 hours). Upper left panel, saline buffered background NanoSight; upper right panel, NanoSight EV in saline (diluted 1: 500); the following figures: histogram of 1: 500 saline dilutions delivered in burn clinical trial, bar graph quantification. Western blot inset shows CD63 (exosome marker) secreted by BM-MSCs at 4 hours.

Figure 52 depicts a model in which the secreted proteome (secretome) of the BM-MSC comprises EV-related and non-EV-related proteins that deliver multiple wound healing promoting functions to RDEB fibroblasts comprising collagen VII protein, collagen VII mRNA, STAT3 signaling activator, and canonical Wnt activator, according to certain exemplary embodiments of the invention.

Detailed Description

For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the invention.

Method for isolating microvesicles of the invention

The term "microvesicles" as used herein refers to vesicles comprising lipid bilayers, which are formed by the plasma membrane of a cell, and are non-uniform in size, ranging from about 2nm to about 5000 nm. Cells that form microvesicles are referred to herein as "host cells". Microvesicles are a non-uniform population of vesicles and include, but are not limited to, Extracellular Vesicles (EVs), extranuclear bodies, microparticles, microvesicles, nanovesicles, shedding vesicles, membrane particles, and the like.

The microvesicles display membrane proteins from their host cells on their membrane surface, and may also comprise molecules from within the microvesicles of the host cells, such as, for example, mRNA, miRNA, tRNA, RNA, DNA, lipids, proteins or infectious particles. These molecules may be produced by, or introduced into, recombinant molecules of the host cell. Microvesicles play a key role in cell-cell communication and can act locally and remotely in vivo, inducing cellular changes by introducing molecules transported on and/or in the microvesicles into target cells by fusion with the target cells. For example, microvesicles are associated with anti-tumor reversal, cancer, tumor immunosuppression, metastasis, tumor-matrix interactions, angiogenesis and tissue regeneration. Microvesicles can also be used for the diagnosis of diseases, as they have been shown to carry several biomarkers of diseases, including e.g. heart disease, HIV and leukemia.

In one embodiment, microvesicles are isolated from a microvesicle-containing biological fluid in a process comprising the steps of:

a) obtaining a biological fluid containing microvesicles,

b) the biological fluid is clarified to remove cellular debris,

c) the microvesicles are precipitated by adding a precipitating agent to the clarified biological fluid,

d) collecting the precipitated microvesicles and washing the material to remove the precipitant, an

e) The washed microvesicles are suspended in a solution for storage or subsequent use.

In one embodiment, the biological fluid is clarified by centrifugation. In an alternative embodiment, the biological fluid is clarified by filtration.

In one embodiment, the precipitated microvesicles are collected by centrifugation. In an alternative embodiment, the precipitated microvesicles are collected by filtration.

a) obtaining a biological fluid containing microvesicles,

b) the biological fluid is clarified to remove cellular debris,

d) the precipitated microvesicles are collected and the material is washed to remove the precipitant,

e) suspending the washed microvesicles in a solution, and

f) the microvesicles are processed to analyze nucleic acid, carbohydrate, lipid, small molecule and/or protein content.

In one embodiment, the invention provides reagents and kits for isolating microvesicles from a biological fluid according to the methods of the invention.

The biological fluid may be: peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchoalveolar lavage fluid, semen (including prostatic fluid), cowper's fluid or pre-ejaculatory fluid (pre-ejaculatory fluid), female ejaculate, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, watery stool, pancreatic juice, lavage fluid from nasal cavities, bronchopulmonary aspirates, or other lavage fluids.

The biological fluid may also be derived from the blastocoel, umbilical cord blood or maternal circulation, which may be of fetal or maternal origin. The biological fluid may also be derived from a tissue sample or biopsy.

The biological fluid may be derived from plant cells of a plant cell culture. The biological fluid may be derived from a yeast cell or a culture of yeast cells.

In one embodiment, the biological fluid is a cell culture medium. In one embodiment, the cell culture medium is conditioned with tissues and/or cells prior to isolation of microvesicles according to the methods of the present invention.

The term "conditioned" or "conditioned medium" refers to a medium in which a population of cells or tissues or a combination thereof is grown and acted upon by the population of cells or tissues or a combination thereof. In one such use, a population of cells or tissues, or a combination thereof, is removed from the culture medium while retaining the factors produced by the cells. In one embodiment, the factor produced is a microvesicle. The medium may be conditioned via any suitable method selected by one of ordinary skill in the art. For example, the culture medium may be cultured according to the method described in EP1780267A 2.

In one embodiment, microvesicles are isolated from cells or tissues that have been pre-treated prior to isolation of the microvesicles. The pretreatment can include, for example, culturing in a specified medium, a medium containing at least one additive, growth factor, serum-free medium, or a combination thereof. Alternatively, the pretreatment may comprise contacting the cell or tissue with an additive (e.g., interleukins, VEGF, inducers of transcription factors, hormones, neurotransmitters, pharmaceutical compounds, micrornas), a transforming agent (e.g., liposomes, viruses, transfection agents, etc.). Alternatively, the pretreatment may comprise exposing the cells or tissue to an altered physical condition (e.g., hypoxia, cold shock, heat shock, etc.).

In one embodiment, the microvesicles are isolated from a medium conditioned using cells or tissues, which has been pretreated prior to isolating the microvesicles. The pretreatment can include, for example, culturing in a specified medium, a medium containing at least one additive, growth factor, serum-free medium, or a combination thereof. Alternatively, the pretreatment may comprise contacting the cell or tissue with an additive (e.g., interleukins, VEGF, inducers of transcription factors, hormones, neurotransmitters, pharmaceutical compounds, micrornas), a transforming agent (e.g., liposomes, viruses, transfection agents, etc.). Alternatively, the pretreatment may comprise exposing the cells or tissue to an altered physical condition (e.g., hypoxia, cold shock, heat shock, etc.).

In one embodiment, the biological fluid is an extract from a plant. In an alternative embodiment, the biological fluid is a cell culture medium from a plant cell culture. In an alternative embodiment, the biological fluid is a yeast extract. In an alternative embodiment, the biological fluid is cell culture medium from a yeast cell culture.

Although the methods of the present invention may be performed at any temperature, one of ordinary skill in the art will readily appreciate that certain biological fluids may degrade and that such degradation may be reduced if the sample is maintained at a temperature below the degradation temperature of the biological fluid. In one embodiment, the process of the invention is carried out at 4 ℃. In an alternative embodiment, at least one step of the process of the invention is carried out at 4 ℃. In certain embodiments, the biological fluid may be diluted prior to performing the methods of the present invention. If the viscosity of the sample is too great to obtain acceptable microvesicle yields, the viscous biological fluid may need to be diluted to reduce the viscosity of the sample. The dilution may be a 1: 2 dilution. Alternatively, the dilution may be a 1: 3 dilution. Alternatively, the dilution may be a 1: 4 dilution. Alternatively, the dilution may be a 1: 5 dilution. Alternatively, the dilution may be a 1: 6 dilution. Alternatively, the dilution may be a 1: 7 dilution. Alternatively, the dilution may be a 1: 8 dilution. Alternatively, the dilution may be a 1: 9 dilution. Alternatively, the dilution may be a 1: 10 dilution. Alternatively, the dilution may be a 1: 20 dilution. Alternatively, the dilution may be a 1: 30 dilution. Alternatively, the dilution may be a 1: 40 dilution. Alternatively, the dilution may be a 1: 50 dilution. Alternatively, the dilution may be a 1: 60 dilution. Alternatively, the dilution may be a 1: 70 dilution. Alternatively, the dilution may be a 1: 80 dilution. Alternatively, the dilution may be a 1: 90 dilution. Alternatively, the dilution may be a 1: 100 dilution.

The biological fluid may be diluted with any diluent provided that the diluent does not affect the functional and/or structural integrity of the microvesicle. One of ordinary skill in the art can readily select a suitable diluent. The diluent may be, for example, phosphate buffered saline, cell culture media, and the like.

In one embodiment, the biological fluid is clarified by applying centrifugal force to remove cellular debris. The centrifugal force applied to the biological fluid is sufficient to remove any cells, lysed cells, tissue debris from the biological fluid, but the magnitude, duration, or both of the applied centrifugal force is insufficient to remove microvesicles. The biological fluid may require dilution to facilitate clarification.

The duration and magnitude of the centrifugal force used to clarify the biological fluid may vary according to a number of factors that are readily understood by one of ordinary skill in the art, including, for example, the biological fluid, the pH of the biological fluid, the desired purity of the isolated microvesicles, the desired size of the isolated microvesicles, the desired molecular weight of the microvesicles, and the like. In one embodiment, a centrifugal force of 2000 × g is applied to the biological fluid for 30 minutes.

The clarified biological fluid is contacted with a precipitating agent to precipitate the microvesicles. In one embodiment, the precipitating agent may be any agent that surrounds the microvesicles and displaces the solvated water. Such precipitating agents may be selected from the group consisting of polyethylene glycol, dextran, and polysaccharides.

In an alternative embodiment, the precipitating agent may cause aggregation of the microvesicles.

In an alternative embodiment, the precipitating agent is selected from the group consisting of calcium ions, magnesium ions, sodium ions, ammonium ions, iron ions, organic solvents (e.g., ammonium sulfate), and flocculants (e.g., alginates).

Contacting the clarified biological fluid with a precipitating agent for a period of time sufficient to precipitate the microvesicles. The period of time sufficient to precipitate the microvesicles may vary depending on a number of factors that are readily understood by one of ordinary skill in the art, including, for example, the biological fluid, the pH of the biological fluid, the desired purity of the isolated microvesicles, the desired size of the isolated microvesicles, the desired molecular weight of the microvesicles, and the like. In one embodiment, the period of time sufficient to allow the microvesicles to precipitate is 6 hours.

In one embodiment, the clarified biological fluid is contacted with a precipitating agent for a period of time sufficient to precipitate the microvesicles at 4 ℃.

The concentration of the precipitating agent used to precipitate the microvesicles from the biological fluid may vary according to a number of factors that are readily understood by one of ordinary skill in the art, including, for example, the biological fluid, the pH of the biological fluid, the desired purity of the isolated microvesicles, the desired size of the isolated microvesicles, the desired molecular weight of the microvesicles, and the like.

In one embodiment, the precipitating agent is polyethylene glycol. The polyethylene glycol used in the process of the invention may have a molecular weight of from about 200Da to about 10,000 Da. In one embodiment, the polyethylene glycol used in the method of the invention may have a molecular weight greater than 10,000 Da. In certain embodiments, the polyethylene glycol used in the methods of the invention has a molecular weight of 10,000Da or 20,000 Da. The choice of molecular weight may be influenced by a number of factors including, for example, the viscosity of the biological fluid, the desired purity of the microvesicles, the desired size of the microvesicles, the biological fluid used, etc. In one embodiment, the molecular weight of the polyethylene glycol used in the process of the present invention may be from about 200Da to about 8,000Da, or any one of, or any range or molecular weight in between, about 200Da, 300Da, 400Da, 600Da, 1000Da, 1450Da, 1500Da, 2000Da, 3000Da, 3350Da, 4000Da, 6000Da, 8000Da, 10000Da, 20000Da, or 35000 Da.

In one embodiment, the polyethylene glycol used in the method of the invention has a molecular weight of about 6000 Da.

In one embodiment, the polyethylene glycol used in the method of the present invention has an average molecular weight of about 8000 Da.

In one embodiment, the polyethylene glycol used in the method of the invention has an average molecular weight of about 10000 Da.

In one embodiment, the polyethylene glycol used in the method of the invention has an average molecular weight of about 20000 Da.

The concentration of polyethylene glycol used in the methods of the invention may be from about 0.5% w/v to about 100% w/v. The concentration of polyethylene glycol used in the methods of the invention may be affected by a variety of factors including, for example, the viscosity of the biological fluid, the desired purity of the microvesicles, the desired size of the microvesicles, the biological fluid used, and the like.

In certain embodiments, polyethylene glycol is used at a concentration of about 5% to 25% w/v in the concentrations of the present invention. In certain embodiments, the concentration is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, or a range between any two of these values.

In one embodiment, the concentration of polyethylene glycol used in the method of the invention is about 8.5% w/v.

In one embodiment, the concentration of polyethylene glycol used in the method of the invention is about 6% w/v.

In one embodiment, polyethylene glycol having an average molecular weight of 6000Da is used at a concentration of 8.5% w/v. In one embodiment, the polyethylene glycol is diluted in 0.4M sodium chloride.

In one embodiment, the concentration of polyethylene glycol used in the method of the invention is inversely proportional to the average molecular weight of the polyethylene glycol. For example, in one embodiment, polyethylene glycol having an average molecular weight of 4000Da is used at a concentration of 20% w/v. In an alternative embodiment, polyethylene glycol having an average molecular weight of 8000Da is used at a concentration of 10% w/v. In an alternative embodiment, a polyethylene glycol with an average molecular weight of 20000Da is used at a concentration of 4% w/v.

In one embodiment, the precipitated microvesicles are collected by applying a centrifugal force. The centrifugal force is sufficient and the duration of application is sufficient to cause the microvesicles to form particles, but not sufficient to damage the microvesicles.

The duration and magnitude of the centrifugal force used to precipitate the microvesicles from the biological fluid may vary according to a number of factors that are readily understood by one of ordinary skill in the art, including, for example, the biological fluid, the pH of the biological fluid, the desired purity of the isolated microvesicles, the desired size of the isolated microvesicles, the desired molecular weight of the microvesicles, and the like. In one embodiment, the precipitated microvesicles are collected by applying a centrifugal force of 10000 × g for 60 minutes.

The precipitated microvesicles may be washed with any liquid as long as the liquid does not affect the functional and/or structural integrity of the microvesicles. One of ordinary skill in the art can readily select a suitable liquid. The liquid may be, for example, phosphate buffered saline, cell culture media, and the like.

In one embodiment, the washing step removes the precipitating agent. In one embodiment, the microvesicles are washed by centrifugation using a filtration device having a cut-off molecular weight of 100 kDa.

The isolated microvesicles may be suspended with any liquid as long as the liquid does not affect the functional and/or structural integrity of the microvesicles. One of ordinary skill in the art can readily select a suitable liquid. The liquid may be, for example, phosphate buffered saline, cell culture media, and the like.

In one embodiment, the isolated microvesicles may be further processed. Further processing may be to isolate microvesicles of a particular size. Alternatively, the further processing may be to isolate microvesicles of a particular size range. Alternatively, the further processing may be to isolate microvesicles of a particular molecular weight. Alternatively, the further processing may be to isolate microvesicles of a particular molecular weight range. Alternatively, the further processing may be to isolate microvesicles displaying or containing a particular molecule.

In one embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle preparation having a size of about 2nm to about 1000nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle preparation having a size of about 2nm to about 500nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle preparation having a size of about 2nm to about 400nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle formulation having a size ranging from about 2nm to about 300nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle preparation having a size of about 2nm to about 200nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle preparation having a size of about 2nm to about 100nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle preparation having a size of about 2nm to about 50nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle formulation having a size ranging from about 2nm to about 20nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention are further processed to isolate a microvesicle formulation having a size ranging from about 2nm to about 10nm, as determined by electron microscopy.

In one embodiment, the subsequent purification is performed using a method selected from the group consisting of immunoaffinity, HPLC, tangential flow filtration, phase separation/partitioning, and microfluidics.

In one embodiment, the isolated microvesicles are further processed to analyze molecules displayed on the microvesicles or contained within the microvesicles. The molecule analyzed is selected from the group consisting of nucleic acids, carbohydrates, lipids, small molecules, ions, metabolites, proteins, and combinations thereof.

Biological fluid comprising a cell culture medium conditioned with cultured cells:in one embodiment, the microvesicles are obtained from a culture medium conditioned with cultured cells. Any cultured cell or population of cells can be used in the methods of the invention. The cell may be a stem cell, a primary cell, a cell line, a tissue or organ explant, or any combination thereof. The cells may be of allogeneic, autologous or xenogeneic origin.

In one embodiment, the cells are cells derived from bone marrow aspirate. In one embodiment, the cells derived from bone marrow aspirate are bone marrow derived mesenchymal stem cells. In one embodiment, the cells derived from bone marrow aspirate are mononuclear cells. In one embodiment, the cells derived from bone marrow aspirate are a mixture of mononuclear cells and bone marrow-derived mesenchymal stem cells.

In one embodiment, bone marrow-derived mesenchymal stem cells are isolated from bone marrow aspirates by culturing the bone marrow aspirates in plastic tissue culture flasks for a period of up to about 4 days, and then washed to remove non-adherent cells.

In one embodiment, mononuclear cells are separated from the bone marrow aspirate by low density centrifugation using a ficoll gradient and collected at the interface.

In one embodiment, prior to isolation of microvesicles according to the method of the invention, the cells are subjected to a suitable temperature and gas mixture (typically 37 ℃, 5% CO for mammalian cells) in a cell culture incubator₂) Culturing, growing or maintaining. The culture conditions for each cell type vary widely and can be readily determined by one of ordinary skill in the art.

In one embodiment, one or more than one culture condition is altered. In one embodiment, such changes result in different phenotypes.

In one embodiment, when the cells require serum in their culture medium to start the microvesicle isolation process, the cell culture medium is supplemented with microvesicle-free serum and then added to the cells to be conditioned. Microvesicles were collected from the conditioned cell culture medium. The serum can be depleted by any suitable method (e.g., such as ultracentrifugation, filtration, precipitation, etc.). The choice of medium, serum concentration, and culture conditions is influenced by a variety of factors as will be readily understood by one of ordinary skill in the art, including, for example, the type of cells being cultured, the desired microvesicle purity, the desired phenotype of the cultured cells, and the like. In one embodiment, the cell culture medium conditioned for use in the microvesicle isolation process is the same type of cell culture medium in which the cells were grown prior to the microvesicle isolation process.

In one embodiment, to begin the microvesicle isolation process, the cell culture medium is removed and serum-free medium is added to the cells to be conditioned. Microvesicles were then collected from the conditioned serum-free medium. The choice of medium and culture conditions are influenced by a variety of factors as will be readily understood by one of ordinary skill in the art, including, for example, the type of cells being cultured, the desired purity of the microvesicles, the desired phenotype of the cultured cells, and the like. In one embodiment, the serum-free medium is supplemented with at least one additional factor that promotes or enhances cell survival in the serum-free medium. Such factors may, for example, provide nutritional support to the cell, inhibit or prevent apoptosis of the cell.

Culturing the cells in the culture medium for a period of time sufficient to allow the cells to secrete the microvesicles into the culture medium. The period of time sufficient to allow the cells to secrete microvesicles into the culture medium is influenced by a variety of factors, which are readily understood by one of ordinary skill in the art, including, for example, the cell type in culture, the desired purity of the microvesicles, the desired phenotype of the cultured cells, the desired yield of microvesicles, and the like.

The microvesicles are then removed from the culture medium by the method of the invention.

In one embodiment, prior to the microvesicle isolation process, the cells are treated with at least one agent selected from the group consisting of: anti-inflammatory compounds, anti-apoptotic compounds, fibrosis inhibitors, compounds capable of enhancing angiogenesis, immunosuppressive compounds, compounds that promote cell survival, chemotherapeutic agents, compounds capable of enhancing cell migration, neurogenic compounds, and growth factors. In one embodiment, when culturing the cells in the medium in which the microvesicles are collected, the cells are treated with at least one agent selected from the group consisting of: anti-inflammatory compounds, anti-apoptotic compounds, fibrosis inhibitors, compounds capable of enhancing angiogenesis, immunosuppressive compounds, compounds that promote cell survival, and growth factors.

In one embodiment, the anti-inflammatory compound may be selected from the compounds disclosed in U.S. patent No.6,509,369.

In one embodiment, the anti-apoptotic compound may be selected from the compounds disclosed in U.S. patent No.6,793,945.

In one embodiment, the fibrosis inhibitor may be selected from the compounds disclosed in U.S. patent No.6,331,298.

In one embodiment, the compound capable of enhancing angiogenesis may be selected from the compounds disclosed in U.S. patent application 2004/0220393 or U.S. patent application 2004/0209901.

In one embodiment, the immunosuppressive compound may be selected from the compounds disclosed in U.S. patent application 2004/0171623.

In one embodiment, the compound that promotes cell survival may be selected from the compounds disclosed in U.S. patent application 2010/0104542.

in one embodiment, the growth factor may be at least one molecule selected from the group consisting of members of the TGF- β family including TGF- β 1,2 and 3, bone morphogenetic proteins (bone morphogenic proteins, BMP-2, -3, -4, -5, -6, -7, -11, -12 and-13), fibroblast growth factors-1 and-2, platelet derived growth factors-AA, -AB and-BB, platelet rich plasma, insulin growth factors (insulin growth factors, IGF-I, II), growth differentiation factors (growth differentiation factors, GDF-5, -6, -8, -10, -15), vascular endothelial cell derived growth factors (vaso endonexal-derived growth factors, VEGF), growth factors (pleiotrophin), and like, other pharmaceutical compounds may include, for example, hypoxia inducible factors 1-alpha, GLP-derived peptides (peptide-derived from fibronectin 2, such as the phosphopeptides of the mouse laminin-binding domain of fibronectin 2, and/laminin-binding domain of the fibronectin 2, as well as the laminin-2, fibronectin-derived laminin-binding domain of the fibronectin 2, as well as the laminin-derived laminin-binding domain of the laminin, as disclosed in U.S. Patientia patent application publication No. 2, and/3.

In one embodiment, microvesicles are isolated from a biological fluid comprising a cell culture medium conditioned with a culture of bone marrow-derived mesenchymal stem cells, comprising the steps of:

a) diluting the cells by 1: 4 to obtain a mesenchymal stem cell population and an inoculation bottle,

b) culturing the cells in a medium until the cells are 80% to 90% confluent,

c) the medium is removed and clarified to remove cell debris,

d) the microvesicles are precipitated by adding a precipitating agent to the clarified culture medium,

e) collecting the precipitated microvesicles and washing the material to remove the precipitant, an

f) The washed microvesicles are suspended in a solution for storage or subsequent use.

In one embodiment, microvesicles are isolated from a biological fluid comprising a cell culture medium conditioned with a culture of bone marrow-derived mononuclear cells, comprising the steps of:

a) obtaining a mononuclear cell population from bone marrow and an inoculation bottle by diluting cells at a ratio of 1: 4,

b) culturing the cells in a medium until the cells are 80% to 90% confluent,

c) the medium is removed and clarified to remove cell debris,

In one embodiment, 95% humidified air and 5% CO at 37 ℃₂in (1), bone marrow-derived mesenchymal stem cells are cultured in a medium comprising α -MEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin/glutamine.

In one embodiment, at 37 ℃ in 95% humid air and 5% CO₂in (1), bone marrow-derived mononuclear cells were cultured in a medium comprising α -MEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin/glutamine.

In one embodiment, the medium is clarified by centrifugation.

In one embodiment, the precipitating agent is polyethylene glycol having an average molecular weight of 6000. In one embodiment, polyethylene glycol is used at a concentration of about 8.5 w/v%. In one embodiment, the polyethylene glycol is diluted in a sodium chloride solution with a final concentration of 0.4M.

In one embodiment, the precipitated microvesicles are collected by centrifugation.

In one embodiment, the isolated microvesicles are washed via centrifugal filtration using phosphate buffered saline using a membrane having a cut-off molecular weight of 100 kDa.

Biological fluid containing plasma: in one embodiment, the microvesicles are obtained from plasma. Plasma may be obtained from healthy individuals, or alternatively from individuals with a particular disease phenotype.

In one embodiment, microvesicles are isolated from a biological fluid comprising plasma, comprising the steps of:

a) plasma was obtained and diluted with cell culture medium,

b) the microvesicles are precipitated by adding a precipitating agent to the diluted plasma,

c) collecting the precipitated microvesicles and washing the material to remove the precipitant, an

d) The washed microvesicles are suspended in a solution for storage or subsequent use.

in one embodiment, the plasma is diluted 1: 10 with medium.

Biological fluid containing bone marrow aspirate: in one embodiment, the microvesicles are obtained from a bone marrow aspirate. In one embodiment, the microvesicles are obtained from a cellular fraction of a bone marrow aspirate. In one embodiment, the microvesicles are obtained from a cell-free fraction of a bone marrow aspirate.

In one embodiment, the microvesicles are obtained from cells cultured in bone marrow aspirate. In one embodiment, cells cultured from bone marrow aspirate are used to condition the cell culture medium from which microvesicles are isolated.

In one embodiment, microvesicles are isolated from a biological fluid comprising bone marrow aspirate, comprising the steps of:

a) obtaining a bone marrow aspirate and separating the bone marrow aspirate into a cell-free fraction and a cellular fraction,

b) the cell-free fraction is diluted and,

c) the diluted cell-free fraction is clarified to remove cell debris,

d) precipitating microvesicles in the cell-free fraction by adding a precipitating agent to the diluted cell-free fraction,

In one embodiment, the cell-free fraction is diluted 1: 10 with culture medium.

in one embodiment, the medium is α -MEM.

In one embodiment, the diluted cell-free fraction is clarified by centrifugation.

In one embodiment, the cell fraction is further processed to isolate and collect cells. In one embodiment, the cell fraction is further processed to isolate and collect bone marrow-derived mesenchymal stem cells. In one embodiment, the cell fraction is further processed to isolate and collect bone marrow-derived mononuclear cells. In one embodiment, the cell fraction is used to condition the medium from which microvesicles can subsequently be obtained.

In one embodiment, microvesicles are isolated from a cellular fraction. The cell fraction may be incubated for a period of time before isolating the microvesicles. Alternatively, microvesicles may be isolated from the cell fraction immediately after the cell fraction is collected.

In one embodiment, the cell fraction is also treated with at least one agent selected from the group consisting of: anti-inflammatory compounds, anti-apoptotic compounds, fibrosis inhibitors, compounds capable of enhancing angiogenesis, immunosuppressive compounds, compounds that promote cell survival, chemotherapeutic agents, compounds capable of enhancing cell migration, neurogenic compounds, and growth factors.

in one embodiment, the growth factor may be at least one molecule selected from the group consisting of TGF- β family members including TGF- β 1,2, and 3, bone morphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and-13), fibroblast growth factor-1 and-2, platelet derived growth factors-AA, -AB, and-BB, platelet rich plasma, insulin growth factors (IGF-1, II), growth differentiation factors (GDF-5, -6, -8, -10, -15), vascular endothelial cell derived growth factors (VEGF), pleiotropic growth factors, endothelin, and the like other pharmaceutical compounds may include, for example, nicotinamide, hypoxia inducible factor 1- α, glucagon-like peptide-1 (GLP-1), GLP-1 and GLP-2 mimetibodies, and II, Excrein-4, nodal, nogal, NGF, retinoic acid, parathyroid hormone, Protophan C, tropoelastin, thrombin derived peptides, fibronectin, anti-laminin, fibronectin-2 mimetibodies, as disclosed in the embodiments of the NodeB family of the invention and as well as a component of the anti-adhesion protein, as disclosed in the NonA, e.g. in U.S. Pub. patent application, and/or S. Pub. No. Pub. 7, and/or S. 7.

Biological fluid containing urine: in one embodiment, the microvesicles are obtained from urine. Urine may be obtained from healthy individuals, or alternatively from individuals with a particular disease phenotype.

In one embodiment, microvesicles are isolated from a biological fluid comprising urine, comprising the steps of:

a) a urine sample is obtained and,

b) the urine is clarified to remove cellular debris,

c) the microvesicles are precipitated by adding a precipitating agent to the clarified urine,

In one embodiment, the urine is clarified by centrifugation.

In an alternative embodiment of the invention, the biological fluid is clarified by filtration. In an alternative embodiment, the precipitated microvesicles are collected by filtration. In an alternative embodiment, the biological fluid is clarified and the precipitated microvesicles are collected by filtration. In certain embodiments, filtration of the biological fluid and/or precipitated microvesicles requires the application of an external force. The external force may be gravity (normal gravity or centrifugal force). Alternatively, the external force may be a suction force.

In one embodiment, this embodiment provides a device that facilitates clarification of biological fluids by filtration. In one embodiment, the present invention provides a device that facilitates collection of precipitated microvesicles by filtration. In one embodiment, the invention provides a device that helps to clarify biological fluids and collect precipitated microvesicles by filtration. In one embodiment, the device also washes microvesicles.

In one embodiment, the device is the device shown in fig. 4. In this embodiment, a biological fluid is added to the internal chamber. The inner chamber has a first filter having a pore size that allows the microvesicles to pass through while retaining any particles having a size larger than the microvesicles in the inner chamber. In one embodiment, the filter of the inner chamber has a pore size of 1 μm. In this embodiment, when the biological fluid passes from the internal chamber through the filter, particles larger than 1 μm remain in the internal chamber and all other particles accumulate in the region between the bottom of the internal chamber and the second filter.

The pore size of the second filter does not allow the microvesicles to pass through. In one embodiment, the second filter of the inner chamber has a pore size of 0.01 μm. In this embodiment, as the biological fluid passes through the second filter, the microvesicles are retained in the region between the bottom of the internal chamber and the second filter, and all remaining particles and fluid accumulate in the bottom of the device.

One of ordinary skill in the art can readily appreciate that the device can have more than two filters, e.g., with different pore sizes to select microvesicles of a desired size.

In one embodiment, a precipitating agent is added to the biological fluid in the internal chamber. In one embodiment, the precipitating agent is added to the filtrate after having passed through the first filter. The filter membrane utilized in the device of the present invention may be made of any suitable material, provided that the filter membrane does not react with or bind to components within the biological fluid. For example, the filter membrane may be made of a low binding material such as, for example, polyethersulfone, nylon 6, polytetrafluoroethylene, polypropylene, zeta-modified glass microfibers, nitrocellulose, cellulose acetate, polyvinylidene fluoride, regenerated cellulose.

Microvesicles of the invention

In one embodiment, the microvesicles of the present invention have a size of about 2nm to about 5000nm as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 1000nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 500nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 400nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 300nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 200nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 100nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 50nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 20nm, as determined by electron microscopy. In an alternative embodiment, the microvesicles of the present invention have a size of about 2nm to about 10nm, as determined by electron microscopy.

In one embodiment, the microvesicles of the present invention have a molecular weight of at least 100 kDa.

Microvesicles isolated according to the method of the invention can be used in therapy. Alternatively, microvesicles isolated according to the method of the present invention can be used for diagnostic tests. Alternatively, the microvesicles of the present invention may be used to modify or engineer cells or tissues. Where the microvesicles of the present invention are used to alter or engineer a cell or tissue, the microvesicles may be loaded, labeled with RNA, DNA, lipids, carbohydrates, proteins, drugs, small molecules, metabolites, or a combination thereof, which will alter or engineer the cell or tissue. Alternatively, microvesicles may be isolated from cells or tissues that express and/or contain RNA, DNA, lipids, carbohydrates, proteins, drugs, small molecules, metabolites, or combinations thereof.

Use of microvesicles of the invention in diagnostic tests

Microvesicles of the invention can be used in diagnostic tests that can detect biomarkers that identify a particular phenotype, such as, for example, a disorder or disease, or a stage or progression of a disease. Biomarkers or markers from cell-derived specific microvesicles can be used to determine treatment regimens for diseases, disorders, disease stages and stages of disorders, and can also be used to determine treatment efficacy. Markers from specific microvesicles of cellular origin can also be used to identify disorders of unknown origin.

The term "biomarker" as used herein refers to an indicator of a biological state. It is an objectively measured and evaluated feature that can be used as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions. One or more biomarkers of the microvesicles may be assessed to characterize the phenotype. The biomarker may be a metabolite, nucleic acid, peptide, protein, lipid, antigen, carbohydrate or proteoglycan, such as DNA or RNA. The RNA can be mRNA, miRNA, snorRNA, snRNA, rRNA, tRNA, siRNA, hnRNA, or shRNA.

The phenotype of a subject may be characterized by obtaining a biological sample from the subject and analyzing one or more microvesicles from the sample. For example, characterizing the phenotype of a subject or individual can include detecting a disease or disorder (including early detection before symptoms), determining the prognosis, diagnosis, or theranosis of a disease or disorder, or determining the stage or progression of a disease or disorder. Characterizing a phenotype may also include determining an appropriate treatment or therapeutic efficacy for a particular disease, disorder, disease stage, and disorder stage, predicting disease progression, particularly disease recurrence (disease recurrence), metastatic spread, or disease recurrence (disease relapse), and likelihood analysis. A phenotype can also be a clinically distinct type or subtype of a disorder or disease, such as a cancer or tumor. The phenotypic determination may also be a determination of a physiological condition, or an assessment of organ distress or organ rejection, such as an assessment after transplantation. The products and methods described herein allow for the evaluation of subjects on an individual basis, which may provide the benefit of more effective and economical treatment decisions.

The phenotype may be any of those listed in U.S. patent 7,897,356. The phenotype may be a tumor, neoplasm, or cancer. Cancers detected or assessed by the products or methods described herein include, but are not limited to, breast cancer, ovarian cancer, lung cancer, colon cancer, hyperplastic polyps, adenomas, colorectal cancer, high grade dysplasia, low grade dysplasia, prostatic hyperplasia, prostate cancer, melanoma, pancreatic cancer, brain cancer (e.g., glioblastoma), hematological malignancies, hepatocellular carcinoma, cervical cancer, endometrial cancer, head and neck cancer, esophageal cancer, gastrointestinal stromal tumors (GIST), Renal Cell Carcinoma (RCC), or gastric cancer. The colorectal cancer may be CRC Dukes B or Dukes C-D. The hematologic malignancy can be B cell chronic lymphocytic leukemia, B cell lymphoma-DLBCL-germinal center-like, B cell lymphoma-DLBCL activated B cell-like, and Burkitt's lymphoma. The phenotype may also be a precancerous condition, such as barrett's esophagus.

The phenotype may also be an inflammatory disease, an immune disease, or an autoimmune disease. For example, the disease may be Inflammatory Bowel Disease (IBD), Crohn's Disease (CD), Ulcerative Colitis (UC), pelvic inflammatory disease, vasculitis, psoriasis, diabetes, autoimmune hepatitis, multiple sclerosis, myasthenia gravis, type I diabetes, rheumatoid arthritis, psoriasis, Systemic Lupus Erythematosus (SLE), hashimoto's thyroiditis, grave's disease, ankylosing spondylitis, sjogren's disease, CREST syndrome, scleroderma, rheumatic disease, organ rejection, graft-versus-host disease, primary sclerosing cholangitis or sepsis. In certain exemplary embodiments, the disease is EB, e.g., RDEB and/or DDEB, borderline EB, simple EB, and/or acquired form of EB.

The phenotype may also be a cardiovascular disease, such as atherosclerosis, congestive heart failure, vulnerable plaque, stroke, or ischemia. The cardiovascular disease or condition may be hypertension, stenosis, vessel occlusion or a thrombotic event.

The phenotype can also be a neurological Disease, such as Multiple Sclerosis (MS), Parkinson's Disease (PD), Alzheimer's Disease (AD), schizophrenia, bipolar disorder, depression, autism, prion Disease, pick's Disease, dementia, Huntington's Disease (HD), down syndrome, cerebrovascular Disease, lamotrigine encephalitis, viral meningitis, nervous system systemic lupus erythematosus (NPSLE), amyotrophic lateral Sclerosis, creutzfeldt-jakob Disease, gerstmann-straussler-scheinker Disease, transmissible spongiform encephalopathy, ischemia reperfusion injury (e.g., stroke), brain trauma, microbial infection, or chronic fatigue syndrome. The phenotype may also be a condition such as fibromyalgia, chronic neuropathic pain, or peripheral neuropathic pain.

The phenotype may also be an infectious disease, such as a bacterial, viral or yeast infection. For example, the disease or condition may be whipple's disease, prion disease, cirrhosis, methicillin-resistant staphylococcus aureus, HIV, hepatitis, syphilis, meningitis, malaria, tuberculosis, or influenza. Viral proteins, such as HIV or HCV-like particles, can be evaluated in exosomes to characterize the viral condition.

The phenotype can also be a perinatal or pregnancy related disorder (e.g., preeclampsia or preterm labor), a metabolic disease or disorder, such as a metabolic disease or disorder associated with iron metabolism. The metabolic disease or disorder may also be diabetes, inflammation or a perinatal disorder.

The phenotype may be detected via any suitable assay, such as, for example, western blot, ELISA, PCR, and the like. The assay methods can be combined to perform multiple analyses for more than one phenotype. Examples of assay methods applicable to microvesicles of the present invention are disclosed in PCT applications WO2009092386A3 and WO2012108842a 1.

Where the biomarker is RNA, RNA can be isolated from the microvesicles of the present invention by the method disclosed in U.S. patent No. 8,021,847.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for a disease, as disclosed in us patent 7,897,356.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for cancer according to the method disclosed in us patent 8,211,653.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for cancer according to the method disclosed in us patent 8,216,784.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for prostate cancer according to the method disclosed in us patent 8,278,059. In one embodiment, the microvesicles of the present invention are used in a diagnostic test for the prognosis of cancer survival according to the method disclosed in us patent 8,343,725.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for the prognosis of cancer survival according to the method disclosed in us patent 8,349,568.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for acute lymphocytic leukemia according to the method disclosed in us patent 8,349,560.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for acute lymphocytic leukemia according to the method disclosed in us patent 8,349,561.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for hepatitis c virus. In one embodiment, hepatitis c viral RNA is extracted from microvesicles of the present invention to test for the presence of hepatitis c virus in a patient according to the method described in us patent 7,807,438.

In one embodiment, the microvesicles of the present invention are used in a diagnostic test for determining the response of a patient to a cancer treatment according to the method disclosed in us patent 8,349,574.

In one embodiment, the microvesicles of the invention are used in a diagnostic test for the diagnosis of a malignant tumor, according to the method disclosed in US patent application US20120058492a 1.

In one embodiment, the microvesicles of the invention are used in a diagnostic test for diagnosing cancer or poor pregnancy outcome, according to the method disclosed in US patent application US20120238467a 1.

In one embodiment, the microvesicles of the invention are used in a diagnostic test for HIV in urine, according to the method disclosed in US patent application US20120214151a 1. In one embodiment, the microvesicles of the invention are used in a diagnostic test of a cardiovascular event according to the method disclosed in US patent application US20120309041a 1.

In one embodiment the microvesicles of the invention are used in a diagnostic test of a cardiovascular event according to the method disclosed in PCT application WO2012110099a 1.

In one embodiment the microvesicles of the invention are used in a diagnostic test of a cardiovascular event according to the method disclosed in PCT application WO2012126531a 1.

In one embodiment the microvesicles of the invention are used in a diagnostic test of a cardiovascular event according to the method disclosed in PCT application WO2013110253 A3.

In one embodiment, the microvesicles of the invention are used in a diagnostic test for melanoma according to the method disclosed in PCT application WO2012135844a 2.

In one embodiment, microvesicles of the invention are used in a diagnostic test for metastatic melanoma by testing for the presence of the biomarker BRAF in microvesicles isolated according to the method of the invention. The presence of BRAF can be determined by western blotting or alternatively by PCR. In one embodiment, the metastatic melanoma test is capable of detecting both wild-type and malignant BRAF. In one embodiment, the metastatic melanoma test is capable of detecting splice variants of malignant BRAF.

In one embodiment, microvesicles used in a diagnostic test for metastatic melanoma are isolated using a method comprising the steps outlined in figure 3.

In one embodiment, the microvesicles are obtained from a patient in whom it is desired to be diagnosed that metastatic melanoma is present. In one embodiment, the microvesicles are obtained from the plasma of a patient.

In one embodiment, the presence of metastatic melanoma is determined by PCR using one of the following two primer sets:

sequence 1:

forward direction: AGACCTCACAGTAAAAATAGGTGA (SEQ ID NO: 1)

And (3) reversing: CTGATGGGACCCACTCCATC (SEQ ID NO: 2)

Amplicon length: 70

Sequence 2:

forward direction: GAAGACCTCACAGTAAAAATAGGTG: (SEQ ID NO: 3)

And (3) reversing: CTGATGGGACCCACTCCATC (SEQ ID NO: 4)

Amplicon length: 82

In another embodiment, the presence of metastatic melanoma is determined by western blotting using a mouse anti-BRAFV 600E antibody (new east Biosciences, Malvern, PA).

Use of microvesicles of the invention in therapy

The microvesicles of the present invention can be used as a therapy for treating diseases.

In one embodiment, the microvesicles of the invention are used as a vaccine according to the method described in US patent application US20030198642a 1.

In one embodiment, the microvesicles of the invention are used to modulate or inhibit an immune response in a patient according to the method described in US patent application US20060116321a 1.

In one embodiment, the microvesicles of the invention are used to modulate or inhibit an immune response in a patient according to the method described in PCT patent application WO06007529 A3.

In one embodiment, the microvesicles of the invention are used to modulate or inhibit an immune response in a patient according to the method described in PCT patent application WO2007103572 A3.

In one embodiment, the microvesicles of the invention are used to modulate or inhibit an immune response in a patient according to the method described in us patent 8,288,172.

In one embodiment, the microvesicles of the invention are used as a treatment for cancer according to the method described in PCT patent application WO2011000551a 1. In one embodiment, the microvesicles of the invention are used as a treatment of cancer or an inflammatory disease according to the method described in US patent application US20120315324a 1.

In one embodiment, the microvesicles of the present invention are used as a treatment for vascular injury according to the method described in us patent 8,343,485.

In one embodiment, the microvesicles of the present invention are used to deliver a molecule to a cell. Delivery of the molecules can be used to treat or prevent disease. In one embodiment, the delivery is performed according to the method described in PCT application WO04014954a 1. In an alternative embodiment, the delivery is performed according to the method described in PCT application WO2007126386a 1. In an alternative embodiment, the delivery is performed according to the method described in PCT application WO2009115561a 1. In an alternative embodiment, the delivery is performed according to the method described in PCT application WO2010119256a 1.

In one embodiment, the microvesicles of the present invention are used to promote or enhance wound healing. In one embodiment, the wound is a full thickness burn. In one embodiment, the wound is a second degree burn.

In one embodiment, the microvesicles of the present invention are used to promote or enhance angiogenesis in a patient.

In one embodiment, the microvesicles of the invention are used to promote or enhance neuronal regeneration in a patient.

In one embodiment, the microvesicles of the present invention are used to reduce scar formation in a patient.

In one embodiment, the microvesicles of the present invention are used to reduce wrinkle formation in the skin of a patient.

In one embodiment, the microvesicles of the invention are used to coordinate complex tissue regeneration in a patient.

In one embodiment, the present invention provides isolated preparations of microvesicles that can promote functional regeneration and organization of complex tissue structures. In one embodiment, the invention provides isolated preparations of microvesicles that can regenerate hematopoietic tissues in a patient suffering from aplastic anemia. In one embodiment, the present invention provides an isolated preparation of microvesicles that can regenerate at least one tissue in a patient having diseased, injured, or deleted skin selected from the group consisting of: epithelial tissue, stromal tissue, neural tissue, vascular tissue, and adnexal structures. In one embodiment, the invention provides an isolated preparation of microvesicles that can regenerate tissues and/or cells from all three germ layers.

In one embodiment, the invention provides an isolated preparation of microvesicles that is used to modulate the immune system of a patient.

In one embodiment, the present invention provides isolated preparations of microvesicles for use in alleviating one or more symptoms of EB (e.g., RDEB and/or DDEB, borderline EB, simple EB, and/or acquired form of EB) in a patient.

In another embodiment, the invention provides isolated preparations of microvesicles for use in increasing collagen VII expression in a patient with EB (e.g., RDEB and/or DDEB, borderline EB, simple EB and/or acquired form of EB).

In one embodiment, the present invention provides an isolated preparation of microvesicles that enhances the survival of a tissue or cell transplanted into a patient. In one embodiment, the patient is treated with the isolated preparation of microvesicles prior to receiving the transplanted tissue or cells. In an alternative embodiment, the patient is treated with the isolated preparation of microvesicles after receiving the transplanted tissue or cells. In an alternative embodiment, the tissue or cells are treated with an isolated preparation of microvesicles. In one embodiment, the tissue or cells are treated with an isolated preparation of microvesicles prior to transplantation.

In one embodiment, the present invention provides an isolated preparation of microvesicles comprising at least one molecule from a host cell selected from the group consisting of: RNA, DNA, lipids, carbohydrates, metabolites, proteins, and combinations thereof. In one embodiment, the host cell is engineered to express at least one molecule selected from the group consisting of: RNA, DNA, lipids, carbohydrates, metabolites, proteins, and combinations thereof. In one embodiment, an isolated preparation of microvesicles comprising at least one molecule from a host cell selected from the group consisting of: RNA, DNA, lipids, carbohydrates, metabolites, proteins, and combinations thereof.

Use of microvesicles of the invention in therapy

For therapeutic use, MV is preferably combined with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" means buffers, carriers and excipients suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. A carrier should be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

Thus, the EV compositions of the invention may comprise any suitable excipient, such as, but not limited to, at least one of diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. Pharmaceutically acceptable excipients are preferred. Some non-limiting examples of such sterile solutions and methods of preparation are well known in the art, such as, but not limited to, those described in Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Edition, mack publishing Co. (Easton, Pa.) 1990. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and/or stability of the EV composition can be routinely selected, as is well known in the art or as described herein.

Pharmaceutically acceptable excipients and additives that may be used in the compositions of the present invention include, but are not limited to, proteins that may be present individually or in combination, comprising from 1% to 99.99% by weight or volume, individually or in combination; a peptide; an amino acid; a lipid; and carbohydrates (e.g., sugars, including mono-, di-, tri-, tetra-, and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers). Some exemplary protein excipients include serum albumin, such as Human Serum Albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody molecule components that may also exert buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.

Carbohydrate excipients suitable for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), inositol, and the like. Some preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

The EV composition may also include a buffering agent or pH adjuster; generally, the buffer is a salt prepared from an organic acid or base. Representative buffers include organic acid salts, such as salts of citric acid, acetic acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, or phthalic acid; tris, tromethamine hydrochloride (tromethamine hydrochloride) or phosphate buffer.

additionally, the EV compositions of the invention may comprise polymeric excipients/additives such as polyvinylpyrrolidone, ficoll (polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- β -cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates, such as "tween 20" and "tween 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol) and chelating agents (e.g., EDTA).

These and further known pharmaceutical excipients and/or additives suitable for use in the antibody molecule composition according to the invention are known in the art, e.g. as listed in "Remington: the Science & Practice of pharmacy, "19 th ed., Williams & Williams, (1995), and those in The" Physician's Desk Reference, "52 nd ed., Medical Economics, Montvale, N.J. (1998). Some preferred carrier or excipient materials are carbohydrates (e.g., sugars and sugar alcohols) and buffers (e.g., citrate) or polymeric agents.

The present invention provides a stable composition comprising MV in a pharmaceutically acceptable formulation. The preservative formulation comprises at least one known preservative, or optionally at least one selected from the group consisting of: phenol, m-cresol, p-cresol, o-cresol, chlorocresol; benzyl alcohol; a phenylmercuric nitrite salt; phenoxyethanol; formaldehyde; chlorobutanol; magnesium chloride (e.g., hexahydrate); alkyl parabens (alkylparabens) (methyl paraben, ethyl paraben, propyl paraben, butyl paraben, and the like); benzalkonium chloride; benzethonium chloride; sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. As known in the art, any suitable concentration or mixture may be used, such as 0.001% to 5%, or any range or value therein, such as, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.6, 4, 4.6, or any range therein. Some non-limiting examples include no preservative; 0.1% to 2% m-cresol (e.g., 0.2%, 0.3%, 0.4%, 0.5%, 0.9%, or 1.0%); 0.1% to 3% benzyl alcohol (e.g., 0.5%, 0.9%, 1.1%, 1.5%, 1.9%, 2.0%, or 2.5%); 0.001% to 0.5% thimerosal (e.g. 0.005% or 0.01%); 0.001% to 2.0% phenol (e.g., 0.05%, 0.25%, 0.28%, 0.5%, 0.9%, or 1.0%); 0.0005% to 1.0% of an alkyl paraben (e.g., 0.00075%, 0.0009%, 0.001%, 0.002%, 0.005%, 0.0075%, 0.009%, 0.01%, 0.02%, 0.05%, 0.075%, 0.09%, 0.1%, 0.2%, 0.3%, 0.5%, 0.75%, 0.9%, or 1.0%), and the like.

Pharmaceutical compositions comprising MV as disclosed herein may be presented in dosage unit form and may be prepared by any suitable method. The pharmaceutical composition should be formulated to be compatible with its intended route of administration. Some examples of routes of administration are Intravenous (IV) administration, intradermal administration, inhalation administration, transdermal administration, topical administration, transmucosal administration, and rectal administration. One preferred route of administration of MV is topical administration. Useful formulations may be prepared by methods known in the pharmaceutical art. See, for example, Remington's Pharmaceutical Sciences (1990) supra. Formulation components suitable for parenteral administration include sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycols, glycerol, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as EDTA; buffers such as acetate, citrate or phosphate; and agents for adjusting tonicity, such as sodium chloride or dextrose.

The carrier should be stable under the conditions of manufacture and storage and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

The pharmaceutical formulation is preferably sterile. Sterilization may be accomplished by any suitable method, such as filtration through sterile filtration membranes. When the composition is lyophilized, filter sterilization may be performed before or after lyophilization and reconstitution.

The compositions of the present invention may be in a variety of forms. These include, for example, liquids; semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions); dispersions or suspensions, and liposomes. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions. Preferred modes of administration are parenteral (e.g., intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular). In a preferred embodiment, the preparation is administered by intravenous infusion or injection. In another preferred embodiment, the preparation is administered by intramuscular or subcutaneous injection.

The phrases "parenteral administration" and "parenterally administered" as used herein mean modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, subcutaneous, intraarterial, intrathecal, intracapsular, intraorbital, intravitreal, intracardiac, intradermal, intraperitoneal, transtracheal, inhalation, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection (intraspinal injection) and infusion.

The present invention provides a kit comprising packaging material and at least one vial comprising a solution of MV with specified buffers and/or preservatives, optionally in an aqueous diluent. The aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preservatives include those selected from: phenol; m-cresol; p-cresol; o-cresol; chlorocresol; benzyl alcohol; alkyl parabens (methyl paraben, ethyl paraben, propyl paraben, butyl paraben, and the like); benzalkonium chloride, benzethonium chloride, sodium dehydroacetate, and thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is a concentration sufficient to produce an antimicrobial effect. Such concentrations depend on the preservative selected and are readily determined by the skilled artisan.

Further excipients, such as isotonic agents, buffers, antioxidants, preservative enhancers may optionally and preferably be added to the diluent. Isotonic agents, such as glycerol, are generally used at known concentrations. Physiologically tolerable buffers may be added to provide better pH control. The formulation may cover a wide range of pH, for example from about pH 4.0 to about pH 10.0, from about pH 5.0 to about pH 9.0, or from about pH 6.0 to about pH 8.0.

The following further additives may optionally be added to the formulation or composition to reduce aggregation: for example, pharmaceutically acceptable solubilizers such as Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymer) and PEG (polyethylene glycol); or a non-ionic surfactant, such as polysorbate 20 or 80 or poloxamer 184 or 188,

Polyols, other block copolymers; and chelating agents such as EDTA and EGTA. These additives are particularly useful if the formulation is to be administered using a pump or a plastic container. The presence of the pharmaceutically acceptable surfactant reduces the tendency of the protein to aggregate.

A variety of delivery systems are available for administering MVs to a subject. In certain exemplary embodiments, the application of MV is topical, optionally with the addition of dressings, bandages, medical tape, pads, gauze, and the like. Suitable dressings to facilitate surface delivery are well known in the art and are commercially available. In other embodiments, the MV is administered by pulmonary delivery, e.g., by intranasal administration or by oral inhalation administration. Pulmonary delivery can be achieved by a syringe or inhaler device (e.g., nebulizer, pressurized metered dose inhaler, multi-dose liquid inhaler, thermal aerosol device, dry powder inhaler, etc.). Suitable methods for pulmonary delivery are well known in the art and are commercially available.

Any of the above formulations can be stored in liquid or frozen form and optionally subjected to a preservation process.

In certain exemplary embodiments of the invention, the EVs described herein are used to deliver one or more bioactive agents to a target cell. The term "bioactive agent" is intended to include, but is not limited to, proteins (e.g., non-membrane bound proteins), peptides (e.g., non-membrane bound peptides), transcription factors, nucleic acids, and the like, that are expressed in cells and/or in the cytosol and that are added during purification and/or preparation of the EVs described herein; and/or pharmaceutical compounds, proteins (e.g., non-membrane bound proteins), peptides (e.g., non-membrane bound peptides), transcription factors, nucleic acids, etc., of the EVs described herein during exposure to one or more of the purification and/or preparation steps described herein. In certain embodiments, the bioactive agent is collagen VII protein, collagen VII mRNA, STAT3 signaling activator (e.g., interferon, epidermal growth factor, interleukin-5, interleukin-6, MAP kinase, c-src non-receptor tyrosine kinase, or other molecule that phosphorylates and/or otherwise activates STAT 3) and/or canonical Wnt activator (see, e.g., McBride et al, (2017) Transgenic expression of antigenic Wnt inhibitor, kallistatin. is associated with secreted circulating CD19+ B lymphocytes in the periphytol. In other embodiments, the bioactive agent is one or more pharmaceutical compounds known in the art.

It will be apparent to those skilled in the art that other suitable modifications and adaptations to the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, they will be more clearly understood by reference to the following examples, which are included merely for purposes of illustration and are not intended to be limiting. All patents, patent applications, and references described herein are incorporated by reference in their entirety for all purposes.

Examples

Example 1: isolation of microvesicles from cell culture media by ultracentrifugation

This example illustrates a typical method by which microvesicles are isolated from a cell culture medium or any biological fluid. An overview of the process for isolating microvesicles from cell culture media is shown in fig. 1. In summary, cells are cultured in medium supplemented with microvesicle-free serum (serum that can be depleted of microvesicles by ultracentrifugation, filtration, sedimentation, etc.). After culturing the cells for a period of time, the medium was removed and transferred to a conical tube and centrifuged at 400 Xg for 10 minutes at 4 ℃ to pellet the cells. Next, the supernatant was transferred to a new conical tube and centrifuged at 2000 × g for 30 minutes at 4 ℃ to further remove cells and cell debris. This may be followed by another centrifugation step (e.g. 10000 × g for 30 minutes to further deplete cell debris and/or remove larger microvesicles). The resulting supernatant was transferred to an ultracentrifuge tube, weighed to ensure equal weight, and ultracentrifuged at 70000+ × g for 70 minutes at 4 ℃ to pellet microvesicles.

The supernatant was then discarded and the pellet resuspended in ice-cold PBS. The solution was ultracentrifuged at 70000+ × g for 70 minutes at 4 ℃ to precipitate microvesicles. The microvesicle-rich pellet is resuspended in a small volume (about 50 to 100 μ l) of a suitable buffer (e.g. PBS).

Example 2: isolation of microvesicles from cell culture media by the method of the invention

This example illustrates how microvesicles can be isolated from a cell culture medium by the method of the invention. An overview of the process for isolating microvesicles from the culture medium of cultured cells is shown in fig. 2 and 3. In summary, cells are cultured in medium supplemented with microvesicle-free serum (serum that can be depleted of microvesicles by ultracentrifugation, filtration, sedimentation, etc.). After culturing the cells for a period of time, the medium was removed and transferred to a conical tube and centrifuged at 400 Xg for 10 minutes at 4 ℃ to pellet the cells. Next, the supernatant was transferred to a new conical tube and centrifuged at 2000 × g for 30 minutes at 4 ℃ to further remove cells and cell debris. This may be followed by another centrifugation step (e.g. 10000 × g for 30 minutes to further deplete cell debris and remove larger particles).

Microvesicles were then precipitated at 4 ℃ using 8.5% w/v PEG 6000 and 0.4M NaCl. The mixture was spun at 10000 Xg for 30 minutes at 4 ℃. The supernatant is removed and the pellet is resuspended in a suitable buffer (e.g., PBS). It can be used for immediate downstream reactions or further purification. Further purification processes may include the use of centrifugal filters (e.g., MWCO of 100kDa), immunoaffinity, HPLC, tangential flow filtration, phase separation/partitioning, microfluidics, and the like.

Example 3: isolation of microvesicles from a culture medium conditioned with bone marrow-derived stem cells by the method of the present invention

Normal donor human bone marrow was obtained from AllCells LLC (Emeryville, CA, http:// www.allcells.com.) MSC was isolated by standard plastic adhesion method MSC was isolated according to the manufacturer's protocol (GE Healthcare Life sciences, Pittsburgh, Pa.) bone marrow mononuclear cells were isolated by low density centrifugation using Ficoll-Paque Premium (density: 1.077 g/mi.) mononuclear cells were collected at the interface, washed 3 times in Phosphate Buffered Saline (PBS) supplemented with 2% FBS (Atlanta biologics, Atlanta, GA), and resuspended in MSC media consisting of α -minimal essential medium (α -minimal essential medium, a-MEM) (Mediash tec., Inc, VA), 20% FBS, 1% penicillin/streptomycin (Lonza, Alllene, NJ) and 1% glutamine (Lonza).

Initial cultures of MSCs or mononuclear cells at 2X 10⁵To 3X 10⁵Individual cell/cm²Seeded in tissue culture treated dishes (BD Biosciences, San Jose, Calif.) and placed at 37 ℃ in 95% humidified air and 5% CO₂In a cell culture incubator. After 48 to 72 hours, non-adherent cells were removed, the culture flask was washed once with PBS, and fresh medium was added to the flask. Cells were grown until 80% confluence was reached and then by trypsin-EDTA (Life technologies, Carlsbad, Calif.)And (5) carrying out passage. The cells were divided into 5-layered multi-flasks (BDbiosciences) at a ratio of 1: 4. Alternatively, cryopreserved MSCs were thawed at 37 ℃ and immediately supplemented with 20% non-microvesicle fetal bovine serum and 1% penicillin/streptomycin/glutamine a-MEM at 37 ℃ in 95% humidified air and 5% CO₂Culturing in the medium. It is expanded similarly to the above.

cells were grown in multiple flasks until reaching 80% to 90% confluence, the flasks were washed twice with PBS and added with α -mem supplemented with 1% penicillin/streptomycin/glutamine, after 24 hours, the conditioned medium was transferred to a 50mL conical centrifuge tube (Thermo Fisher Scientific inc., Weston, FL) and immediately centrifuged at 400 × g for 10 minutes at 4 ℃ to pellet any non-adherent cells, the supernatant was transferred to a new 50mL conical centrifuge tube and centrifuged at 2000 × g for 30 minutes at 4 ℃ to further remove cells and cell debris, the supernatant was collected and placed in 250mL sterile polypropylene disposable containers (Corning, NY), the supernatant was added with 8.5 w/v% RNase-free and protease-free polyethylene glycol (Sigma Aldrich, Saint, MO) of average molecular weight 6000 and sodium chloride (final concentration of 0.5000 w/v%) and the solution was placed in a cold room at 4 ℃ and transferred to a 5000 × 5000 Millipore buffer PBS to a phosphate buffer solution (Sigma Aldrich buffer) and the protein concentration was determined by centrifugation in a buffer centrifuge tube (buffer) and the bottom buffer cell buffer of the buffer enriched RNA buffer for a second time (buffer) was transferred to a buffer for centrifugation device (buffer for centrifugation at 5000 × 5000 mm +1 g, buffer.

Example 4: isolation of microvesicles from plasma by the method of the invention

approximately 6 to 8ml of blood (human and porcine) was collected by venipuncture and placed in a BD Vacutainer plastic EDTAlavender tube (BD Biosciences, San Jose, Calif.) the venipuncture tube was centrifuged at 400 Xg for 30 minutes at room temperature to remove plasma (approximately 3 to 4ml) and placed in a new 50ml conical centrifuge tube (Thermo Fisher Scientific Inc., Weston, FL.) sterile α -minimum essential medium (α -MEM) was added at a ratio of 1: 10 (plasma to medium) (Mediatech Inc., assManas, Va.).

To the solution were added 8.5 w/v% RNase and protease free polyethylene glycol (SigmaAldrich, Saint Louis, Mo.) with an average molecular weight of 6000 and sodium chloride (final concentration 0.4M). The solution was placed in a cold room at 4 ℃ overnight with shaking. The solution was centrifuged at 10000 Xg for 30 minutes at 4 ℃. The supernatant was decanted and the microvesicle-rich pellet was resuspended in Phosphate Buffered Saline (PBS). The solution enriched in microvesicles was transferred to an Amicon ultra-15 centrifugal filter unit (nominal molecular weight limit of 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000 Xg for 30 min. The filter unit was washed with phosphate buffered saline and centrifuged again at 5000 Xg for 30 min. The concentrated sample (about 200. mu.l to 400. mu.l) was recovered from the bottom of the filter device. Protein concentration was determined by micro BSA protein assay kit (Pierce, Rockford, IL) and the microvesicle enriched solution was stored or processed at-70 degrees for downstream uses (e.g. protein, RNA and DNA extraction).

Example 5: isolation of microvesicles from Bone Marrow Aspirate (Bone Marrow aspiration) by the method of the present invention

Porcine bone marrow was isolated from the iliac crest. The skin area was carefully cleaned with 7.5% povidone-iodine and 70% isopropyl alcohol. An 11-gauge (gauge)3mm trocar (Ranafac, Avon, MA) was inserted into the iliac crest. The aspiration syringe is filled with 5000 to 1000 units of heparin to prevent coagulation of the marrow sample. About 20 to 25ml of marrow was aspirated and the solution was transferred to a 50ml conical centrifuge tube. Alternatively, normal donor human bone marrow (approximately 50ml) was obtained from AllCells LLC (Emeryville, Calif., URL: AllCells. com).

the supernatant (cell free fraction) was collected by centrifugation at 400 Xg for 30 minutes at room temperature in a 50ml conical centrifuge tube (approximately 10 to 12ml per 50ml) and placed in a new 50ml conical centrifuge tube (Thermo Fisher Scientific Inc., Weston, FL.) sterile α -minimal essential medium (α -MEM) was added at a ratio of 1: 10 (bone marrow supernatant to medium) (Mediatech Inc., Manassas, Va.) the solution was transferred to a new 50ml conical tube and centrifuged at 2000 Xg for 30 minutes at 4 ℃ the supernatant was transferred to a new 50ml conical tube and 8.5 w/v% RNase and protease free polyethylene glycol of average molecular weight 6000 (Sigma Aldrich, Saint Louis, MO) and sodium chloride (final concentration 0.4M) were added to the solution.

The solution was placed in a cold room at 4 ℃ overnight with shaking. The solution was centrifuged at 10000 Xg for 30 minutes at 4 ℃. The supernatant was decanted and the microvesicle-rich pellet was resuspended in Phosphate Buffered Saline (PBS). The solution enriched in microvesicles was transferred to an Amicon ultra-15 centrifugal filter unit (nominal molecular weight limit of 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000 Xg for 30 min. The filter unit was washed with phosphate buffered saline and centrifuged again at 5000 Xg for 30 min. The concentrated sample (about 200. mu.l to 400. mu.l) was recovered from the bottom of the filter device. Protein concentration was determined by the microBSA protein assay kit (Pierce, Rockford, IL) and the microvesicle enriched solution was stored or processed at-70 degrees for downstream uses (e.g. protein, RNA and DNA extraction).

Cell fractions were collected and processed for mesenchymal stem isolation or for complete bone marrow isolation.

Example 6: isolation of microvesicles from urine by the method of the invention

Approximately 500ml of clean retained human urine was isolated and placed in a 50ml conical tube (Thermo FisherScientific inc., Weston, FL).

A50 ml conical tube was centrifuged at 400 Xg for 30 min at 4 ℃. The supernatant was removed and placed in a new 50ml conical centrifuge tube (Thermo Fisher Scientific inc., Weston, FL). The solution was transferred to a new 50ml conical tube and centrifuged at 2000 Xg for 30 min at 4 ℃. The supernatant was transferred to a new 50ml conical tube and 8.5 w/v% RNase and protease free polyethylene glycol (Sigma Aldrich, Saint Louis, MO) with an average molecular weight of 6000 and sodium chloride (final concentration of 0.4M) were added to the solution.

The solution was placed in a cold room at 4 ℃ overnight with shaking. The solution was centrifuged at 10000 Xg for 30 minutes at 4 ℃. The supernatant was decanted and the microvesicle-rich pellet was resuspended in Phosphate Buffered Saline (PBS). The solution enriched in microvesicles was transferred to an Amicon ultra-15 centrifugal filter unit (nominal molecular weight limit of 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000 Xg for 30 min. The filter unit was washed with phosphate buffered saline and centrifuged again at 5000 Xg for 30 min. The concentrated sample (about 200. mu.l to 400ul) was recovered from the bottom of the filter device. Protein concentration was determined by the microBSA protein assay kit (Pierce, Rockford, IL) and the microvesicle enriched solution was stored or processed at-70 degrees for downstream uses (e.g. protein, RNA and DNA extraction).

Example 7: isolation of microvesicles from a culture medium derived from bone marrow cells cultured for a long period of time by the method of the present invention

Bone marrow was obtained from the aspirate (see example 1) and red blood cells were lysed using 0.8% ammonium chloride solution (Stem Cell Technologies, Vancouver, BC) containing 0.1mM EDTA. Nucleated cells were pelleted at 400 Xg for 5 minutes under fetal bovine serum (Atlanta Biologics, Atlanta, GA) buffer. Nucleated cells were washed in McCoy's 5a medium (Mediatech inc., Manassas, VA) by precipitation at 400 × g for 5 minutes. The cells were cultured at 1X 10⁶The individual cells/ml density is resuspended in culture medium and plated at 25, 75 or 225 cm²In a bottle (Corning, Corning, NY).

The medium consisted of McCoy's 5a medium, 1% sodium bicarbonate (Life technologies, Carlsbad, CA), 0 to 4% MEM non-essential amino acids (Life technologies), 0 to 8% MEM essential amino acids (Life technologies), 1% L-glutamine (Lonza, Allendale, NJ), 0.1. mu.M hydrocortisone (Life technologies), 1% penicillin/streptomycin (Lonza), 12% to 5% fetal bovine serum (Atlanta Biologics), and 12% to 5% horse serum (Stem Cell Technology). The culture was incubated at 33 ℃ and 5% CO₂And (4) incubating. Feeding was performed by adding half of the original volume of medium every week without removing any medium during the first nine weeks of culture. If the culture is overgrownAfter nine weeks, the volume of medium was reduced to the original volume and half of the original volume of fresh medium was added weekly.

After about nine weeks of culture, the original medium was removed and stored. Cells were washed twice with Phosphate Buffered Saline (PBS) and incubated for 24 hours in a medium consisting of McCoy's 5a medium, 1% sodium bicarbonate, 0 to 4% MEM non-essential amino acids, 0 to 8% MEM essential amino acids (Life technologies), 1% L-glutamine (Lonza, Allendale, NJ), and 1% penicillin/streptomycin (Lonza).

After 24 hours, the supernatant was transferred to a 50mL conical centrifuge tube (Thermo Fisher scientific inc., Weston, FL) and immediately centrifuged at 400 × g for 10 minutes at 4 ℃ to pellet any non-adherent cells. The stored raw media is added back to the cells. The supernatant was transferred to a new 50mL conical centrifuge tube and centrifuged at 2000 × g for 30 minutes at 4 ℃ to further remove cells and cell debris.

The supernatant was collected and placed in a 250ml sterile polypropylene disposable container (Corning, NY). 8.5 w/v% RNase and protease free polyethylene glycol (Sigma Aldrich, Saint Louis, Mo.) with an average molecular weight of 6000 and sodium chloride (final concentration 0.4M) were added to the supernatant. The solution was placed in a cold room at 4 ℃ overnight with shaking. The solution was transferred to a 50mL conical centrifuge tube and centrifuged at 10000 Xg for 30 minutes at 4 ℃. The supernatant was decanted and the microvesicle-rich pellet was resuspended in Phosphate Buffered Saline (PBS). The solution enriched in microvesicles was transferred to an amiconultratra-15 centrifugal filter unit (nominal molecular weight limit of 100kDa) (Millipore, Billerica, MA) and centrifuged at 5000 × g for 30 min. The filter unit was washed with phosphate buffered saline and centrifuged again at 5000 Xg for 30 min. The concentrated sample (about 200. mu.l) was recovered from the bottom of the filter device. Protein concentration was determined by micro BSA protein assay kit (Pierce, Rockford, IL) and the microvesicle enriched solution was stored or processed at-70 degrees for downstream uses (e.g. protein, RNA and DNA extraction).

Example 8: analysis of microvesicles of the invention

Microvesicle samples were analyzed by electron microscopy. For Transmission Electron Microscopy (TEM), each sample of microvesicles was loaded on a Formvar-coated 150 mesh copper grid (Electron Microscopy Sciences, Fort Washington, PA) for 20 minutes. The mesh was drained and floated on 2% glutaraldehyde droplets for 5 minutes, then washed in double distilled water (DDOH), followed by dyeing on 4% aqueous uranyl acetate droplets and multiple washes in DDOH. The grid was examined in a Philips CM10 electron microscope at 80 kV.

Figure 5 shows electron micrographs of microvesicles derived from human bone marrow-derived mesenchymal stem cells isolated by the ultracentrifugation method described in example 1 (panels a & B) and according to the method of the invention as described in example 3 (panels C & D). Figure 6 shows electron micrographs of microvesicles derived from porcine bone marrow-derived mesenchymal stem cells isolated by the ultracentrifugation method described in example 1 (panels a & B) and according to the method of the invention as described in example 3 (panels C & D). Figure 7 shows electron micrographs of microvesicles derived from murine mesenchymal stem cells isolated by the ultracentrifugation method described in example 1 (panels a & B) and according to the method of the invention as described in example 3 (panels C & D).

Figures 5 to 7 illustrate the difference between microvesicles isolated by the method of the present invention compared to ultracentrifugation. The microvesicles separated according to the method of the invention are smoother at their borders, not wavy (uncooked) and appear more "intact".

Figure 8 shows an electron micrograph of microvesicles isolated from human plasma according to the method of the invention. The heterogeneity of shape and size obtained with PEG separation indicates that all types of microvesicles are separated. Similar heterogeneity was observed in microvesicles of porcine plasma (fig. 9) and human urine (fig. 10) isolated according to the method of the present invention.

To analyze protein expression in microvesicle samples, cells and microvesicles were lysed in RIPA buffer (CellSignaling Technology, Danvers, MA) and protein concentration was estimated by the micbsa assay kit (Pierce, Rockford, IL). Approximately 20 micrograms of lysate were loaded in each lane and the membrane probed overnight with rabbit anti-63 antibody (SBI Biosciences, Mountain View, CA), rabbit anti-hsp 70(SBI Biosciences), rabbit STAT3(Cellsignaling technology), and/or rabbit phospho-STAT 3(Cell signaling technology) (1: 1000).

The presence of exosome markers (HSP 70 and CD63) confirms that the methods of the invention are capable of isolating exosomes. In addition, exosomes also contain the transcription factor STAT3 and the activated phosphorylated form of phospho-STAT 3. See fig. 11.

Example 9: effect of microvesicles of the present invention on proliferation and migration of fibroblasts

To investigate the ability of the microvesicles of the present invention to promote or enhance wound healing, the ability of the microvesicles to stimulate proliferation of dermal fibroblasts was tested. Normal human adult dermal fibroblasts were obtained from Life Technology (Carlsbad, CA). According to the IRB approved protocol (IND # BB IND 13201), fibroblasts from chronic wound patients (pressure foot ulcers) and diabetic foot ulcers (diabetic foot ulcers) were collected from wounds lasting 2 years in situations where no cure was indicated, despite standard care and advanced wound care treatment. Normal and chronic wound fibroblasts were added at 5X 10 per well³The cells were plated on 24-well tissue culture plates (BD Biosciences, San Jose, CA). MTT cell proliferation assays were performed on day 0 and day 3. Microvesicles were added on day 0. After 3 days, both PEG-isolated and ultracentrifuged microvesicles were approximately equal in increasing growth of both normal and chronic wound fibroblasts. Phosphate Buffered Saline (PBS) and microvesicle depleted conditioned MSC media showed little growth. See fig. 12.

In a co-culture experiment, normal adult fibroblasts and fibroblasts from diabetic foot ulcers were seeded in twenty-four well plates. Each well was seeded to 100% confluence (approximately 1X 10 per well)⁵Individual cells). To prevent the effect of cell proliferation, the medium was replaced 2 hours before scratching with fresh serum-free medium containing 10. mu.g/ml mitomycin. The confluent monolayers were then scored with a 1ml sterile pipette tip to leave a score of 0.4mm to 0.5mm in width. The medium (along with any detached cells) is then immediately removed. The removed medium was replaced with fresh medium (10% FBS) containing microvesicles (PEG or ultracentrifugation derived), PBS or microvesicle depleted MSC conditioned medium. The scratched area was monitored by collecting digitized images immediately after scratching and 3 days after treatment. The digitized images were captured using an inverted IX81Olympus microscope (Olympus America, Center Valley, PA, URL: olympusameria. com) and an ORCA-AG Hamamatsu digital camera (Hamamatsu Photonics K.K., Hamamatsu City, ShizuokaPref., Japan, URL: Hamamatsu. com). Three days after treatment, microvesicles isolated according to the method of the invention showed the greatest migration (causing the wound to substantially close) followed by microvesicles derived from ultracentrifugation. Control (PBS) and microvesicle depleted MSC conditioned media (depleted) showed little migration. See fig. 13.

Figure 14 shows the effect of microvesicles on cell migration of fibroblasts derived from diabetic foot ulcers. Similar to the results in fig. 13, microvesicles isolated according to the method of the present invention induced the greatest migration, followed by microvesicles isolated using the ultracentrifugation method described in example 1. Control (PBS) and microvesicle depleted MSC conditioned media (depleted) showed little migration.

Example 10: uptake of microvesicles of the invention into cells

Human MSC microvesicles isolated from conditioned medium according to the method of the invention were labeled with the phospholipid cell linker dye PKH-26 (red) according to the manufacturer's instructions (Sigma-Aldrich, st. Normal dermal fibroblasts were labeled with Vybrant-Dio (Life technology) according to the manufacturer's instructions. Normal dermal fibroblasts were plated on fibronectin (Sigma-Aldrich) coated 4-well Nunc Lab-Tek II chamber slides (thermo fisher Scientific inc., Weston, FL) (5 × 10 cells per well). Cells were stained with the nuclear dye Hoechst33342 (Life technology) according to the manufacturer's instructions. Dio-labeled fibroblasts were treated with PKH-26-labeled microvesicles for 24 hours. Images were captured with an inverted 1X81Olympus microscope and an ORCA-AG Hamamatsu digital camera. Normal dermal fibroblasts (stained with the green lipid membrane dye Dio) showed uptake of PKH-26-labeled human MSC MV isolated by PEG precipitation at the perinuclear sites. See fig. 15 and 16. In fig. 16, microvesicles are seen in the perinuclear locations.

Example 11: use of microvesicles of the invention as a diagnosis of rheumatoid arthritis

Normal dermal fibroblasts were cultured at 1X 10⁵Density of individual cells/well was plated in 6-well tissue culture plates (BDBiosciences). Fibroblasts were serum starved overnight and treated with: PBS (control); 10 microgram of any microvesicle (human plasma MVPEG precipitate) isolated from plasma obtained from patients suffering from rheumatoid arthritis according to the method of the present invention; microvesicles (human hMSC MV PEG pellet) isolated from a culture medium conditioned with bone marrow-derived mesenchymal stem cells according to the method of the present invention; microvesicles isolated by ultracentrifugation from media conditioned with bone marrow-derived mesenchymal stem cells (human hMSC MV ultracentrifugation); PBS control; and spent media control (MY-depleted hMSC conditioned media). The amount of STAT3 phosphorylation observed in fibroblasts was greater in microvesicles isolated according to the method of the present invention. See fig. 17.

Example 12: use of microvesicles of the invention as a diagnosis of metastatic melanoma

BRAF is a human gene that makes a protein called B-Raf. More than 30 mutations of the BRAF gene have been identified that are associated with human cancers. We have designed each primer to amplify BRAF in a mutant form associated with metastatic melanoma. This mutation is the T1799A mutation in exon 15 in BRAF. This resulted in the substitution of valine (V) with glutamic acid (E) at codon 600 (now designated V600E). The presence of this mutation requires treatment by the BRAF inhibitor Vemurafenib (Vemurafenib). SK-Mel28 cell line obtained from ATCC (Washington DC, Maryland) is known to have a T1799A mutation in exon 15 in BRAF. Microvesicles isolated according to the method of the invention are obtained from a culture medium conditioned by incubation for 3 days in EMEM (ATCC) + 10% serum (Atlanta Biologics, Atlanta, Georgia).

The isolated microvesicles were subjected to DNA and RNA isolation using AUPrep DNA/RNA kit from Qiagen (Hilden, Germany). Using iScript^TMReverse transcription Supermix (BioRad, Hercules, Calif.) reverse transcribes approximately 50ng of RNA from SK-MEL28 cells and microvesicles. 2ml aliquots were used according to the manufacturer's instructions

PCR SuperMix (Life technology) PCR. In addition, 80ng of DNA from SK-MEL28 cells and microvesicles was used for use according to the manufacturer's instructions

PCR for SuperMix. The PCR products were electrophoresed on a 3% agarose gel and visualized by the gel-doc system from Bio-Rad. The results are shown in FIG. 18.

The primers used were:

sequence 1:

forward direction: AGACCTCACAGTAAAAATAGGTGA (SEQ ID NO: 1)

And (3) reversing: CTGATGGGACCCACTCCATC (SEQ ID NO: 2)

Amplicon length: 70

Sequence 2:

forward direction: GAAGACCTCACAGTAAAAATAGGTG (SEQ ID NO: 3)

And (3) reversing: CTGATGGGACCCACTCCATC (SEQ ID NO: 4)

Amplicon length: 82

Additionally, microvesicle samples were lysed in RIPA buffer and protein concentration was estimated by the micbsa assay kit. Approximately 50. mu.g was loaded in each lane and membranes probed overnight with a mouse anti-BRAF V600E antibody (New Eastbiosciences, Malverm, Pa) (1: 1000). A secondary goat anti-mouse (Pierce) was applied at a dilution of 1: 10000 for 1 hour. Western blot showed BRAFV600E was detected in SKMEL28 cells and MV lysates.

Example 13: isolation of microvesicles from culture conditioned medium using GFP-labeled bone marrow-derived mesenchymal stem cells by the method of the present invention

Homozygous transgenic mice expressing enhanced Green Fluorescent Protein (GFP) under the direction of the human ubiquitin C promoter (C57BL/6-Tg (UBC-GFP)30Scha/J) were obtained from Jackson laboratories (Bar Harbor, Maine). These mice are known to express GFP in all tissues.

GFP-mice (about 3 to 4 weeks old) were passed over CO₂the hind limbs were harvested and the skin, muscle and all connective tissue removed.the bones were then placed in an ice-cold sterile IX PBS dish and washed several times in PBS the ends of each bone were cut with scissors this was repeated several times to ensure removal of all bone marrow by passing a10 cc syringe containing warmed medium (α -MEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin/glutamine) through the shaft, which was placed in a 150mm plate this was repeated several times to ensure removal of all bone marrow.

The initial culture was incubated at 2X 10⁵To 3X 10⁵Individual cell/cm²Seeded in tissue culture treated dishes (BDbiosciences, San Jose, Calif.) and placed at 37 ℃ in 95% humidified air and 5% CO₂In a cell culture incubator. After 72 to 96 hours, non-adherent cells were removed, the culture flask was washed once with PBS, and fresh medium was added to the flask. Cells were grown until 80% confluence was reached and then passaged through trypsin-EDTA (Life Technologies, Carlsbad, CA). The cells were divided in a 1: 4 ratio.

alternatively, cryopreserved GFP mouse MSCs were thawed at 37 ℃ and immediately placed in α -MEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycin/glutamine at 37 ℃ in 95% humidified air and 5% CO₂Culturing in the medium. It is expanded similarly to the above.

Cells were grown in flasks until 100% confluence was reached (about 1 week). The supernatant was transferred to a 50mL conical centrifuge tube (Thermo Fisher Scientific Inc., Weston, FL) and immediately centrifuged at 400 Xg for 10 minutes at 4 ℃ to pellet any non-adherent cells. The supernatant was transferred to a new 50mL conical centrifuge tube and centrifuged at 2000 × g for 30 minutes at 4 ℃ to further remove cells and cell debris. The supernatant was collected and placed in 250ml sterile polypropylene disposable containers (corning, NY). 8.5 w/v% RNase and protease free polyethylene glycol (Sigma Aldrich, Saint Louis, Mo.) with an average molecular weight of 6000 and sodium chloride (final concentration 0.4M) were added to the supernatant. The solution was placed in a cold room at 4 ℃ overnight with shaking. The solution was transferred to a 50mL conical centrifuge tube and centrifuged at 10000 Xg for 30 minutes at 4 ℃. The supernatant was decanted and the microvesicle-rich pellet was resuspended in Phosphate Buffered Saline (PBS). The solution enriched in microvesicles was transferred to an Amicon ultra-15 centrifugal filter unit (nominal molecular weight limit of 100kDa) (Millipore, Billerica, Mass.) and centrifuged at 5000 Xg for 30 min. The filter unit was washed with phosphate buffered saline and centrifuged again at 5000 Xg for 30 min. The concentrated sample (about 200. mu.l to 400. mu.l) was recovered from the bottom of the filter device. Protein concentration was determined by micro BSA protein assay kit (Pierce, Rockford, IL) and the microvesicle enriched solution was stored or processed at-70 degrees for downstream uses (e.g. protein, RNA and DNA extraction).

To determine cellular uptake of microvesicles, normal human dermal fibroblasts were labeled with Vybrant-Dio (Life technology) according to the manufacturer's instructions. Normal dermal fibroblasts were plated on fibronectin (Sigma-Aldrich) coated 4-well Nunc Lab-Tek II chamber slides (Thermo Fisher Scientific Inc.) with 5 × 10 cells per well. Cells were stained with the nuclear dye Hoechst33342 (Life technology) according to the manufacturer's instructions. Dil-labeled fibroblasts were treated with microvesicles isolated from mouse MSCs expressing GFP for 24 hours. Images were captured with an inverted 1X81Olympus microscope and an ORCA-AG Hamamatsu digital camera. See fig. 20 and 21. Importantly, these images show that microvesicles containing GFP are taken up by cells.

Example 14: use of microvesicles of the invention as a treatment for promoting or enhancing wound healing

A full thickness wound was created on the pig's back using a10 mm punch biopsy instrument. Microvesicles were isolated from the culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method described in example 1 ("conventional ultracentrifugation method") or by the method described in example 3. At the time of injury and on

days

1 and 2, 30 micrograms of microvesicles were applied to the wound by local injection. Controls were treated with saline or allowed to heal by exposure to air. After 5 days, animals were euthanized and the wounds were examined.

Fig. 22 shows the histology of the wound 5 days after injury. On day 5, the microvesicle-treated wounds isolated with the method according to the invention (i.e. according to the method described in example 3) appeared smaller compared to saline controls, air-exposed controls and wounds treated with microvesicles prepared by ultracentrifugation. Wounds treated with microvesicles prepared by ultracentrifugation showed an enhanced inflammatory response compared to wounds treated with microvesicles prepared according to the method of the invention and two controls.

In another study, a second degree burn wound was created on the back of the pig using brass rods heated to 100 ℃. Microvesicles were isolated from the culture medium conditioned with autologous bone marrow-derived mesenchymal stem cells according to the method described in example 1 ("conventional ultracentrifugation method") or by the method described in example 3. At the time of injury and on

days

1 and 2, 30 micrograms of microvesicles were applied to the wound by local injection. Controls were treated with saline or allowed to heal by exposure to air.

In the experimental procedure (up to 28 days after burn injury), wounds treated with microvesicles prepared by ultracentrifugation were more significantly inflamed than wounds treated with microvesicles prepared according to the method of the present invention (i.e., according to the method described in example 3). See fig. 23. Similarly, wounds treated with microvesicles prepared by ultracentrifugation were more significantly inflamed than saline controls and air-exposed controls. Burn wounds treated with microvesicles prepared according to the method of the invention do not appear to be more significantly inflamed compared to controls.

Figure 23 illustrates the difference in inflammation between wounds treated 7 days after injury with microvesicles prepared by ultracentrifugation, microvesicles prepared according to the method of the present invention, and a control exposed to air. Under microscope, abscess formation was seen in both full thickness wounds and burn wounds treated with microvesicles prepared by ultracentrifugation. Without being bound by scientific theory, it is believed that the inflammation noted with microvesicles prepared by ultracentrifugation is due to damage to the microvesicles, which can easily stimulate the inflammatory cascade. The microvesicles of the present invention may also impart additional benefits by comprising additional particles.

Figure 24 shows second degree swine burn wounds treated 28 days after burn with microvesicles isolated by the method of the present invention. There is significant remodeling of collagen, as well as the appearance of the matrix (ground substance). These findings indicate a skin remodeling with collagen type III formation. There is also a skin epidermis inducing effect that produces a thickened epidermis that appears well anchored to the dermis. These findings were not observed in scar formation and were more consistent with skin regeneration. The epidermis formed over the scar is easily re-damaged due to poor anchoring to the dermis of the scar.

Figure 25 shows second degree swine burn wounds treated with saline 28 days after burn. There was minimal skin regeneration while the epidermis was smooth. The lack of height of significant cristae formation suggests inadequate cortical anchoring. These findings are more indicative of the risk of scar formation and continued injury.

Figure 26 shows a full thickness pig wound treated with microvesicles isolated according to the method of the invention 28 days after injury. Nerve ingrowth (as exemplified by arrows) into the remodeled dermis is likely caused by stimulation of the applied microvesicles. Nerve growth is accompanied by an angiogenic response (circled area). Nerves appear to be developing structures, not due to simple axonal sprouting (axon sprouting). This is a unique finding and has never been reported and is not observed in control wounds or wounds treated with microvesicles prepared by ultracentrifugation. These observations are highly indicative of complex tissue regeneration, and the ability to produce mature elements from all germ layers, including the epidermis, stroma, vasculature, and neural tissue. These methods, then, appear to be broadly applicable to the treatment of a variety of conditions, including traumatic, inflammatory, neoplastic and degenerative disorders of ectodermal, endodermal and mesodermal origin tissues.

Figure 27 shows full thickness pig wounds treated 28 days after injury with microvesicles isolated by the method of the invention. This figure illustrates the observation depicted in figure 26 at a greater magnification. In a), nerve growth appears to follow a pathway related to the angiogenic response. This finding is interesting because it is well known that nerve growth will follow angiogenesis during embryonic development. Again, these findings indicate tissue regeneration. B) Nerves are shown with higher efficacy. C) Angiogenesis in the vicinity of nerve growth is better exemplified.

Bone formation was observed in all treatment groups (control and microvesicle treated) in the porcine full-thickness wound model. See fig. 28. Animals received a total of 1.44mg of microvesicles (half prepared according to the method of the invention and half prepared by ultracentrifugation). It then appears to have a systemic effect of stimulating bone formation in all wounds. Bone formation tends to occur more often in more inflammatory wounds, suggesting a synergistic effect of local inflammatory mediators and systemic effects of microvesicles.

Example 15: use of microvesicles of the invention as a treatment for reproducing bone marrow and regenerating complex structures

C57/CJ6(GFP) mice were lethally irradiated with two cycles of 400cGy gamma irradiation to ablate their host bone marrow progenitors. After irradiation, the ablative fraction (relative fractional) erbium: YAG laser treatment of mice in an area of about 2 cm. Following laser treatment, plastic chambers were adhered to the skin and syngeneic GFP was added to the chambers<+>Bone marrow-derived cells obtained from transgenic mice. GFP (green fluorescent protein)⁺Bone marrow cells include freshly harvested total bone marrow cells, lineage negative selected bone marrow cells, mesenchymal stem cells, and bone marrow fully cultured cells (as described herein). Chimerism can be achieved in only a few animals; 4 to 6 weeks after administration of cells by circulating GFP⁺The cells are tested. (see FIG. 29). Surprisingly, many animals survived without evidence of donor bone marrow engraftment. Overall (in all groups given cells) 30% of the animals receiving the cells survived. Animals receiving lineage negative selected cells (45%) and fresh bone marrow cells (30%) survived the highest in the different groups. The mortality rate of control irradiated animals that did not receive cells was 100%. Cytokines failed to similarly rescue similar lethally irradiated animals and did not show functional donor bone marrow engraftment in these surviving animals. Microvesicles secreted by the delivered cells may be responsible for the restoration of the host bone marrow, leading to the survival of these animals. We have demonstrated that fresh bone marrow (which includes lineage negative cells) and mesenchymal stem cells produce large numbers of microvesicles that can achieve this effect. In another study, two cycles of 400cGy gamma irradiation were used for C57/CJ6(GFP)^-) Mice were lethally irradiated to inhibit their hair growth and partially ablate their bone marrow. After irradiation, the backs of the mice were shaved and then treated with ablative fractions erbium: YAG laser treatment of mice in an area of about 2 cm. Following laser treatment, plastic chambers were adhered to the skin and syngeneic GFP was added to the chambers⁺Bone marrow-derived cells obtained from transgenic mice. GFP (green fluorescent protein)⁺Bone marrow cells include freshly harvested bone marrow cells, lineage negative selected bone marrow cells, mesenchymal stem cells, and bone marrow fully cultured cells (as described herein). Chimerism was not achieved in any animal; 4 to 6 weeks after administration of cells by circulating GFP⁺The cells are tested. See fig. 30. Animals receiving laser treatment alone had no to very little coarse hair growth. FIG. 30 (A). In animals given bone marrow cells, hair growth was significant and persistent. FIG. 30(A)&B) In that respect These findings were with GFP⁺Lineage negative selected cells and Total neogenesisFresh GFP⁺The most significant of the bone marrow cell treated mice. Hair growth was detected within 2 weeks and continued for months. Skin biopsies were performed in areas of new hair growth, but no GFP was detected⁺A cell. Functional engraftment of bone marrow cells was also not detected in any animals by FACS analysis. FIG. 30 (C). As with the example in fig. 29, the cytokine has not been demonstrated to have this effect in restoring hair growth. Microvesicles secreted by the delivered cells may be responsible for stimulating hair growth.

Example 16: use of microvesicles of the invention for promoting or stimulating angiogenesis and for promoting or stimulating fibroblast proliferation

Isolation of bone marrow aspirate microvesicles:approximately 25ml of fresh whole bone marrow was obtained from AllCells, Inc. (Alameda, CA.) bone marrow was carefully placed in a new 50ml conical centrifuge tube and centrifuged at 400 × g for 30 minutes at room temperature the supernatant (approximately 15ml) was carefully removed and placed in a new 50ml conical centrifuge tube (Thermo Fisher Scientific Inc., Weston, FL) and centrifuged at 2000 × g for 30 minutes at 4 ℃. the supernatant was again removed carefully and placed in a new 50ml conical centrifuge tube to which sterile α -minimum essential medium (α -MEM) was added at a ratio of 1: 10 (bone marrow supernatant to medium) (Mediatech Inc., Manassas, VA) and 8.5 w/v% of rnnless and protease free polyethylene glycol (Sigma Aldrich, Saint Louis, MO) of average molecular weight 6000 and sodium chloride (final concentration of 0.4M.) was added to the solution and the solution was decanted in a millicell buffer under 5000 × 5000 ℃ buffer room buffer phosphate buffer for approximately 5000 × 5000 μ g and centrifuged at 5000 × 5000 μ M to obtain a supernatant (phosphate enriched cell) and the supernatant was again centrifuged in a supernatant in a sample suspension at 5000 × 5000 μ phosphate buffer cell buffer and centrifuged in a cell suspension (phosphate buffer) and centrifuged at approximately 100 μ filter with a centrifuge cell suspension and centrifuged at 400 × 5000 μ cell buffer for approximately 100 μ buffer.

And (3) angiogenesis measurement:measurement Using an endothelial tube formation assay (Invitrogen Life Technologies, GrandIsland, NY)And measuring angiogenesis. Cryopreserved primary Human Umbilical Vein Endothelial Cells (HUVEC) (Invitrogen Life Technologies) were grown for 6 days in 75-cm tissue culture flasks in medium 200PRF supplemented with 2% low serum growth supplement (Invitrogen Life Technologies). The cells were then plated at 3X 10<4>In 24-well tissue culture plates containing medium without supplements. HUVEC cells were subsequently treated with bone marrow microvesicles (approximately 100. mu.g). PBS was used as vehicle control. The treated cells were incubated at 37 ℃ and 5% CO₂Incubate for 6 hours. Calcein AM fluorescent dye at a concentration of 2 μ g/ml was used for visualization of tube formation. Fluorescence images were captured with an inverted IX81Olympus microscope (Olympus America, Center Valley, Pa.). Bone marrow MV showed significant tube-forming ability compared to vehicle (PBS) control (see figure 31).

And (3) growth determination:normal adult fibroblasts were plated on 24-well plates (10000 cells/well) with growth medium (5% FBS, 1% glutamine, 1% penicillin/streptomycin) for assay. After overnight incubation, three wells were randomly selected and cells were stained with NucBlue Live ready probes reagent (Invitrogen Life technologies) (day 0). Fluorescence images were captured using the EVOS FL automated cell imaging system (Invitrogen Life technologies). Fibroblasts were refeeded with fresh medium containing bone marrow-derived microvesicles (about 100 μ g) or PBS (vehicle control), and after three days (day 3), cells were stained and imaged. The number of fibroblasts treated with bone marrow-derived microvesicles increased approximately three-fold (compared to day 0) and its rate was significantly higher than the vehicle control (fig. 32, panels a and 32, panel B).

Example 17: EV-mediated delivery of bioactive substances to target cells

According to certain exemplary embodiments of the invention, the EVs described herein can be used to deliver one or more bioactive agents (e.g., collagen VII proteins or peptides, collagen VII mRNA, STAT3 signaling activators, canonical Wnt activators, etc.) to a target cell. This example demonstrates the delivery of EV to RDEB fibroblasts lacking COL7a1 expression compared to wild-type fibroblasts. EV stimulates collagen VII expression in RDEB fibroblasts. EV also stimulates expression of markers associated with wound healing in RDEB fibroblasts.

Figure 44 shows validation of in vitro cell lines derived from infants diagnosed with RDEB (halopeau-Siemens type). Vesicle exchange was observed between BM-MSCs and RDEB fibroblasts (fig. 45). Collagen VII was co-isolated with BM-MSCEV (fig. 46), and COL7a1mRNA was enriched in MB-MSC EV (fig. 47).

RDEB fibroblasts were treated with BM-MSC EV on day 1, washed on day 3 and showed increased collagen VII expression on day 6 (fig. 48). Chemoselective ligation assay (using "click iT" reaction chemistry) revealed that new collagen VII was produced from RDEB fibroblasts after co-treatment with BM-MSCEV (figure 49). BM-MSC EV was shown to improve in vitro surrogate assays (e.g., proliferation and trypsin resistance) associated with RDEB fibroblast wound healing (figure 50).

Delivery of BM-MSCs to burn patients in saline was shown to secrete large amounts of EV (CD63 positive) in saline within hours (4 hours shown) in clinical trials (fig. 51).

A model of BM-MSC mediated wound healing is shown in figure 52.

Example 18: treatment of extracellular vesicles derived from mesenchymal stem cells of bone marrow in recessive dystrophic epidermolysis bullosa

Local and intravenous injection of allogeneic bone marrow-derived mesenchymal stem cells (BM-MSCs) has been shown to promote wound healing in Recessive Dystrophic Epidermolysis Bullosa (RDEB). We have described that Extracellular Vesicles (EV) derived from BM-MSCs (BM-MSCEV) are largely responsible for the healing effect, which is attributed to BM-MSCs. We also found that EV can transfer collagen VII (Col VII) to RDEB cells. We propose a first clinical trial in which BM-MSC EV surfaces from healthy allogeneic donors were applied to wounds in RDEB patients, providing maximum safety and comfort to the patients while enhancing wound healing. Treatment with EV has many advantages over cell therapy, including lower risk of genetic instability and malignant transformation. We will obtain FDA approval of an experimental New Drug (IND) and manufacture clinical grade BM-MSC EV for an open-label (open-label) dose escalation phase I study of surface application allogeneic BM-MSC derived EV in 30 RDEB patients.

Specific objects

Purpose 1-obtaining phase I dose escalation trial for surface application of BM-MSC EV in RDEB patients and preparation IND approval from FDA for optimal clinical grade BM-MSC-EV

We will apply specific criteria for donor BM-MSC EV screening, selection and functional characterization for our phase I clinical trial. This includes a defined list of EV manufacturing and product characteristics of IND, as they are particularly relevant to RDEB. We will use the important assays found in our stem cell-based clinical trials to evaluate the donors of BM-MSC EV based on the functional performance of the recipient RDEB cells. In our trial, we will select the best BM-MSC donor and make BM-MSC EV for treatment of RDEB patients.

Purpose 2-development of a superficial allogenic BM-MSC Source EV open Landmark in wound treatment in 30 RDEB patients Tab dose escalation clinical trial

Administration will be based on a successful clinical trial of PI-based surface application of BM-MSCs to burn patients. There will be 3 groups of consecutive ascending doses, 10 patients completing each group. The dosing regimen will be the first dose on treatment day 0, with three additional doses given monthly (four treatments in total over three months). The primary results will evaluate the safety and tolerability of surface applied BM-MSC EVs; secondary outcomes will be assessed for wound healing, pain, itch, and cosmetic effects (including evidence of pigment, scar assessment, and tissue regeneration). The Integrium Contract research organization (Integrium Contract research) will assist in clinical trials.

Research strategy

Purpose 1-obtaining phase I clinical presentation for BM-MSC derived Extracellular Vesicle (EV) surface for treatment of wounds in RDEB patients Use of bed test IND approval from FDA.

The direct application of bone marrow derived stem and progenitor cells to burns and stubborn chronic non-healing wounds can lead to wound closure and skin remodeling. Patients with chronic wounds (lasting more than one year) are treated with bone marrow stem and progenitor cells. MSCs account for approximately 30% of the cells administered to the patient. Evidence of healing was observed in all treated patients, with many patients achieving complete closure of their wounds. Some subjects have remained healed for more than 7 years (eventually disappearing with follow-up). It is of central note in clinical findings that no scar formation in the skin reconstruction and healing wounds was noted both clinically and histologically. Clinically, the wound bed (wind bed) was elevated with little atrophy/recess on the healed wound closure (fig. 33). Histological evidence supports skin reconstruction of treated wounds.

Microscopic findings include increased collagen formation and matrix deposition. Among the most surprising observations was the recovery of structures such as reticulin and elastic fibers (fig. 34). These fibers are characteristically lost in the healing of even simple acute and chronic wounds. Collectively, these findings support the ability to induce healing in non-healing wounds using surface applied bone marrow derived stem cells, restore tissue volume insufficiency, stimulate tissue regeneration and greatly reduce scar formation without adverse events.

Topical application of allogeneic BM-MSCs to burn wounds showed evidence of rapid epithelialization, reduced scarring, pigment restoration, and hair follicle regeneration. No evidence of a relevant serious adverse event or rejection was observed. Follicular regeneration consistent with tissue regeneration (fig. 35) was noted after BM-MSC surfaces were applied to burn wounds. However, no regeneration of hair follicles was observed in the untreated areas of the burn. Severe re-pigmentation indicative of tissue regeneration was also observed (fig. 36).

The rapid re-pigmentation noted in the patient was anecdotal to any other treatment, as these wounds typically experienced an extended (usually permanent) period of ichthyophthiriasis after burns. Recovery of elasticity in burned skin is also a particular finding (fig. 37). Of particular interest with respect to these results is that they occur after only a short period of surface application of BM-MSCs. In particular, it is unlikely that cells will survive and/or engraft for a long period of time by this method of administration. This strongly suggests that the delivered cells are able to rapidly convey complex information, resulting in robust regenerative and healing effects. Naked cytokines (nucleic acids) and transcription factors are not able to survive long term in burn wound environments, and it is not feasible that a single factor can produce such a complex response.

Without being bound by scientific theory, it is hypothesized that membrane-bound EV is capable of producing such a clinical response. In examining only the saline vehicle in which cells were delivered to the patient (after removal of the cells), we found that there was more than 1.6 × 10¹¹EV particles/mL, confirming that we are delivering large amounts of EV to patients. EV is intact in the sample administered to the patient and has characteristic EV markers. Recently, we have published that EV stimulates the Proliferation and migration of Normal and Chronic Wound Fibroblasts and enhances Angiogenesis by activating STAT 3-mediated target genes (Shabbir A, Cox A, Rodriguez-Menocal L, Salgadom, Van Badiavas E.Mesenchyl Stem Cell Exosomes introduction and hybridization of Normal and Chronic Wund Fibrobils, and human angiogenisis Invitro. Stem Cells Dev 2015; 24: 1635-47). Our preclinical studies also strongly supported BM-MSC EV stimulation of wound healing and tissue regeneration (fig. 38 and 39). In our preclinical studies, evidence of tissue regeneration (e.g., nerve growth) has not been achieved by other methods. In particular, our preclinical studies suggest that other methods of EV isolation may result in vesicle damage, which leads to the development of undesirable inflammatory responses. Our new approach does not damage EV and has been shown to induce rapid healing without an inflammatory response (figure 40). Preliminary data indicate that these vesicles, in addition to delivering pro-healing factors, can transport Col VII protein and functional Col7a10mRNA to RDEB fibroblasts (fig. 41 and 42).

In a reaction with chemoselective ligation (Click)

ThermoFisher) for capturing Col VII eggsIn the white assay, we found that RDEB fibroblasts were actually induced by BM-MSC EV (dose 10. mu.g/mL) to produce a new Col VII protein (FIG. 42). BM-MSC EV significantly promoted both RDEB proliferation and resistance to trypsin digestion (fig. 43). These are standard assays for assessing functional acquisition and wound healing potential of RDEB dermal fibroblasts. These data provide evidence that BM-MSC EV has benefits for RDEB in addition to its potential to improve wound healing. To obtain IND approval for clinical trials, we will manufacture within a GMP contract manufacturing facility where PI was successfully used in previous trials. In addition to meeting general manufacturing requirements, we will also address issues particularly relevant to RDEB therapy. We will establish criteria for screening and selection of donors to optimize the potential regeneration-promoting activity of EVs. The properties of the BM-MSC EV product will be defined. These parameters include protein concentration, EV size distribution (e.g. using NanoSight NS300), surface marker characterization, removal using reagents during manufacture, and stability testing of the product. Using mass spectrometry and RNA sequencing, we will define the protein and RNA cargo content (cargo content) of several BM-MSCEV donors and correlate the cargo with functional assay performance. BM-MSC EV functional activity will be defined for recipient RDEB cells, including in vitro studies to establish the efficacy of wound healing and phenotypic reversal, including RDEB fibroblast proliferation and trypsin resistance assays. In addition, endothelial angiogenesis assays will be examined in vitro.

Purpose 2-development of a superficial allogenic BM-MSC Source EV open Landmark in wound treatment in 30 RDEB patients Ticket dose escalation clinical trial

The method comprises the following steps:

this clinical trial will be an open-label pilot study with three escalating treatment dose groups (10 patients per dose level). The investigator will identify 5 to 50cm²To treat the target lesion. EV in saline was applied under a thin silicone sheet dressing as a primary layer while overlaying a secondary standard of care wound dressing. Control wounds will be treated with saline under the silicone sheet. Treatment frequency was baseline, 4 weeks, 8 weeks and 12 weeks. Dosage levels will be derived from PI inLevel of application in burn test. Digital images of the treatment and control wounds will be acquired. Will use

The device measures both treatment and control wounds.

in view of our previous experience, we expect to see more than 50% of healing stimuli in view of clinical and laboratory variations, we assume a merging standard deviation of 20. here, for clinical studies examining this range of differences, we propose a statistical efficacy (not to be confused with probability) of 0.8 (break RH, cam TA, Gaboury i.inadequate statistical power of new clinical trials of which the Journal of clinical trials 2006; 176: 263-6; Ichihara K, boyd.a.applied against clinical trial of statistical procedure using in derived correlation results of more than a threshold of probability of significant outcome: 0. this is considered to be a more significant difference than the statistical probability of 0.1 of the mean of the two cases of more than the threshold of 0. this is considered to be a more significant difference than the probability of 0, which we would have calculated as a more significant difference of the two cases of more than the threshold of the probability of the difference of 0.1 (this is considered to be a more significant difference of the probability of the two cases of more significant outcomes of the two cases of the more than the correct clinical trials of the two cases of the difference (this 1. the two cases of the statistical efficacy) of the two cases of the probability of the difference of the two cases of the probability of the two cases of the difference of the two cases of the difference of the different cases of the probability of the two cases of the different outcomes of the two cases of the different outcomes of the more than the probability of the different outcomes of the probability of the two cases of the difference of the two cases of the more than the two cases of the difference of the two cases.

Qualification standard:

key inclusion criteria include: male or female patients who were 12 years or older at screening and were informed consent from guardians (if under 18 years of age); confirmed RDEB diagnosis defined by clinical presentation and histological confirmation; having 5 to 50cm on the arms and/or legs²At least 1 active wound (active wind); women with fertility potential must test negative for urine or serum pregnancy at screening and use an acceptable form of fertility control (oral contraceptive/implantable contraceptive/injectable contraceptive/transdermal contraceptive, intrauterine device or other form of fertility control). Key exclusion criteria would include: clinical evidence of infection; for any reason, any kind of immunosuppressive therapy is performed in parallel.

Measurement of main results:

the following primary outcome measures will be evaluated.

1) All adverse events, particularly those suspected to be treatment-related, were screened and recorded.

2) Characterization and analysis of all reported adverse events.

3) Participants who discontinued due to voluntary withdrawal from treatment or intolerance to treatment were evaluated.

Notably, in this application, we propose to use EV derived from allogeneic BM-MSC in multiple doses to improve wound healing, and can introduce Col VII in RDEB wounds. Clinical registries of the european union and the united states listed far more than 1,000 clinical trials conducted worldwide using BM-MSCs and, therefore, they produced paracrine substances (including EVs) with about half of all studies using allogeneic sources. To date, no serious adverse events have been reported. In our trial with allogeneic BM-MSCs in burn patients we did not detect any evidence of adverse events, nor immune responses to delivered substances we know to contain large amounts of BM-MSC EV. In our study, the analysis of the immune response included an FDA-approved sensitive ELISA assay that could detect minimal subclinical evidence of immune response in a mixed lymphocyte reaction. Despite the multiple doses given in our experiments, we did not detect any immune response in these assays. This is not surprising, however, since BM-MSCs are known to have Immunomodulatory properties (Bartholomew A, Polcher D, Szilagyi E, Douglas GW, Kenyon N. Mesenital cells in the induction of transduction in 2009; Siegel G, Schafer R, Dazzi F. the transduction in 2009, 87: S45-9; Sundin M, Barrett AJ, Ringden O et al, CT transduction in biochemical transduction in MSC to immune effector in MSCvector J2009; 32: transduction in 755-64) which have been demonstrated to be produced by Brucem in K et al, culture in IV, molecular culture in IV, PCR in vitro culture in MS, PCR mediated by fermentation in IV, molecular culture in IV, PCR, culture in IV, culture in MS, culture in IV, culture in MS, culture in IV, culture in MS, culture in culture, culture in, culture medium, culture in, culture medium, culture, liu L, Yang J et al, external eliminated From Human um center Cell media MiR-181c attaching Burn-induced external infection. EBioMedicine 2016; 8: 72-82). We believe that it is reasonable to prudently advance using EV-based therapy for RDEB, given that many trials (including our own) have been providing EV-based delivery of allogeneic mesenchymal stem cells for many years without evidence of immune response or rejection. Of course, we will monitor the patient's immune response or any evidence of rejection very closely using measurements that are well established in our trial. As we have recently discovered, BM-MSC EV can deliver Col VII and induce RDEB cells to make Col VII, thus raising the question of the potential for patients to generate antibodies against Col VII. This has been a concern in many clinical trials on RDEB patients, but even in trials in which antibodies to ColVII were detected, none of the trials showed an adverse response (Petrof, infra). In preclinical trials with direct administration of the Col VII protein, no worsening of the disease, no increase in blistering, and no binding of circulating antibodies to Col VII were observed, even when circulating antibodies to Col VII were detected (Riazifar, infra; Palazzi X, Marchal T, channel L, Spadaafra A, Magnol JP, Meneguzi G. induced dynamic infection Bullosa extracted logs: A specific tissue amino acids model for therapeutic gene therapy. J. Invitro Dermatol 2000; 115: 135-7; South AP, Uitto J. type VII Collagen replay therapy in modified expression therapy polyethylene Bullosa-Much, Hospit of interest J. Derman et al; Waterzia J. 10. invasion of tissue graft J. 10. 1. 9. 1. 9. 1. 9. origin of "tissue, et. origin of" 1. origin "tissue, et al. 9. origin" tissue, et al. origin "tissue. BM-MSC testing in RDEB patients failed to demonstrate disease progression, increased blistering, or evidence of induction of autoimmunity even when the presence of Col VII could be determined and/or anti-collagen antibodies could be detected (El-Darouti M, Fawzy M, Amin I et al, Treatment of developmental epidemic bullosis with bone mineral non-malignant fibrous cells: a random conditioned controlled particulate ceramic 2016; 29: 96-100; Riazifal M, Point EJ, Lotvale J, Zhuao W.Stem Cell Exceller vehicles: Extended Message Regeneration. Annu Rermacol Toxicol 2017; 125-54; Strystenf G, Martron G, Martian molecular cells Exceller vehicles: Extension molecular culture of molecular culture 2319; Svic strain of molecular culture of human 2319; Svic strain of molecular culture of animal research 2319). The induction of Col VII expression in RDEB patients by both chemistry (Woodley, infra) and Bone marrow stem transplantation (Wagner JE, Ishida-Yamamoto A, McGrath JA et al, Bone marrow transfer for receiveable dynamic Bone graft polymerization bullosa. the New England and J ournal of media 2010; 363: 629-39) did not raise any concern regarding the induction of anti-Col VII antibodies in treated patients. This is not surprising, as more than half of RDEB patients typically express the major antigenic portion of Col VII responsible for antibody production (woody DT, Cogan J, Hou Y et al, genomic antibodies functional type VII collagen in reactive specific antigens membranes patent. J Clinon Invest 2017; joints DA, Hunt SW, 3rd, Prisayan PS, Briggaman RA, Gamma. Immunodourant antigens of type VII collagen of short, Pair peptides with the fibrous peptide of the type III homology region of the genomic genes (J Invest 1), type Derman patent publication No. 1995-5, Laplacode type II, P. III family III reagent of colloidal genes, P. J. experiment J. investigation No. 10. J. specific antigens No. 10. 5. P. III. origin of colloidal antigens patent. J. P. J. III. J. P. III. origin of colloidal genes, P. J. D. U.D. U.D.D.A. U.D.D.A.D.A.A.D.D.A.A.A. 2. expression of colloidal peptides A.A.A.A.A.A.A.A.A.A.A. origin of colloidal peptides A. origin, B.A. A.A.A.A. A.A.A. A.A. A. A.A. B.A. B.A.A.A. B.A. shows a similar genes of colloidal peptides A.A.A.A.A.A.A.A. B.A.A.A.A.A.A.A.A.A.A.A.A.A. shows a similar antigens responsible.

The finding that many RDEB patients experience focal reverse mosaicism (with a persistent region of skin containing intact ColVII) also provides strong evidence that introduction of a VII Col into RDEB patients may not induce a pathogenic response. Even so, if the production of the Col VII antibody is to produce a clinically relevant effect, it will be more like the form of acquired epidermolysis bullosa; diseases that are more manageable than RDEB. It should also be noted that we propose the use of EV, rather than stem cells. Stem cells have engraftment capabilities and are not recyclable. EVs are not viable (viable), cannot be durable, and cannot be replicated. Thus, if we detect any evidence of a poor outcome or suspect that there is a poor outcome (including an increase in anti-ColVII antibody titers), the treatment will cease and be reversible.

Secondary outcome measurement:

the following secondary outcome measures will be evaluated:

1) reduction of blisters/erosion based on Body Surface Area Index (BSAI) changes.

2) Reduction or closure of the target wound size. Wound size reduction is an assessment used to determine the likely efficacy of EV treatment. Target wound will use

(Aranz Medical) were measured, said

(Aranzmedical) is an FDA-approved medical device wound imaging, 3D measurement and recording system that provides accurate, precise, and repeatable wound assessment using non-invasive laser techniques.

3) Assessment of individual signs by the physician-the scale evaluates blistering and erosion, oozing/skinning/weeping, itching, erythema and pain on the periphery of the non-blistering skin. The body area will include the head/neck, upper limbs, torso and lower limbs.

4) Epidermolysis bullosa disease Activity and scar formation Index (EBDASI).

5) VAS pain scoring questionnaires and analgesic (pain diagnosis) were used.

6) The ItchyQuant Scale score (a validated scale for assessing itch) (Haydek CG, Love E, Mollanzar NK et al, Validation and bundling of the ItchyQuant: a Self-Report ItchSeverity Scale.J. invest.Dermatol.2017; 137: 57-61.).

7) Dermatological Quality of Life Index for Children (Children's Dermatology Life Quality Index, CDLQI). The initial test site was the University of Miami (University of Miami) (with world-famous doctor Lawrence Schachner, doctor of paediatric dermatologist). The next location will be determined.

While various aspects of the present invention have been illustrated above by reference to examples and certain preferred embodiments, it is to be understood that the scope of the invention is not limited by the foregoing description, but is defined by the following claims appropriately interpreted in accordance with the principles of patent law.

Claims

1. A method of treating epidermolysis bullosa in a subject in need thereof, the method comprising:

administering to the subject a pharmaceutical composition comprising the isolated microvesicles purified by precipitation from a biological fluid; and

One or more symptoms of epidermolysis bullosa are alleviated and alleviated in the subject.

2. The method of claim 1, wherein the isolated microvesicles are extracellular vesicles.

3. The method of claim 2, wherein the extracellular vesicles are derived from a precipitating agent selected from the group consisting of calcium ions, magnesium ions, sodium ions, ammonium ions, iron ions, ammonium sulfate, alginate, and polyethylene glycol. The biological fluid settles.

4. The method of claim 1, wherein the biological fluid is derived from mammalian cells.

5. The method of claim 4, wherein the mammalian cells are human cells.

6. The method of claim 3, wherein the precipitating agent is polyethylene glycol.

7. The method of claim 6, wherein the polyethylene glycol has an average molecular weight of about 6,000 Da, about 8,000 Da, about 10,000 Da, or about 20,000 Da.

8. The method of claim 1, wherein the one or more symptoms of epidermolysis bullosa are selected from any combination of thickening of the corpus callosum, epidermal blistering, oral mucosal blistering, nails and /or thickened toenails, sepsis, malnutrition, dehydration, electrolyte imbalance, obstructive airway complications, defective collagen VII expression, anemia, esophageal stricture, growth retardation, webbing or fusion of fingers and/or toes , dental deformities, micromouth deformities, and corneal abrasions.

9. The method of claim 8, wherein the epidermal blister is epidermal blistering of hands, feet, elbows and/or knees.

10. The method of claim 1, wherein treating comprises increasing collagen VII expression in the subject.

11. A method of treating epidermolysis bullosa in a subject in need thereof, the method comprising:

administering to the subject a pharmaceutical composition comprising the isolated extracellular vesicles; and

12. The method of claim 11, wherein the isolated extracellular vesicles are precipitated using the group consisting of calcium ions, magnesium ions, sodium ions, ammonium ions, iron ions, ammonium sulfate, alginate, and polyethylene glycols The agent is precipitated from the biological fluid.

13. The method of claim 12, wherein the biological fluid is derived from mammalian cells.

14. The method of claim 13, wherein the mammalian cells are human cells.

15. The method of claim 12, wherein the precipitating agent is polyethylene glycol.

16. The method of claim 15, wherein the polyethylene glycol has an average molecular weight of about 6,000 Da, about 8,000 Da, about 10,000 Da, or about 20,000 Da.

17. The method of claim 11, wherein the one or more symptoms of epidermolysis bullosa are selected from any combination of thickening of the corpus callosum, epidermal blistering, oral mucosal blistering, nails and /or thickened toenails, sepsis, malnutrition, dehydration, electrolyte imbalance, obstructive airway complications, defective collagen VII expression, anemia, esophageal stricture, growth retardation, webbing or fusion of fingers and/or toes , dental deformities, micromouth deformities, and corneal abrasions.

18. The method of claim 17, wherein the epidermal blister is epidermal blistering of hands, feet, elbows and/or knees.

19. The method of claim 11, wherein treating comprises increasing collagen VII expression in the subject.

20. A method of increasing collagen VII levels in a cell, the method comprising contacting the cell with an isolated extracellular vesicle from a mammalian fluid, wherein the cell expresses the epidermolysis bullosa genotype.

21. The method of claim 20, wherein the cell comprises a mutation in the COL7A1 gene.

22. The method of claim 20, wherein the isolated extracellular vesicles deliver collagen VII protein and/or COL7A1 mRNA to the cell.

23. A method of delivering one or more bioactive agents to a cell, the method comprising contacting the cell with an isolated extracellular vesicle from a mammalian fluid.

24. The method of claim 23, wherein the cell comprises a mutation in the COL7A1 gene.

25. The method of claim 23, wherein the one or more bioactive agents are selected from the group consisting of collagen VII protein, collagen VII mRNA, STAT3 signaling activators, and canonical Wnt activators.

26. The method of claim 25, wherein the STAT3 signaling activator is selected from the group consisting of interferon, epidermal growth factor, interleukin-5, interleukin-6, MAP kinase, and c-src non-receptor tyrosine kinase.

27. The method of claim 25, wherein STAT3 is phosphorylated.

28. The method of claim 23, wherein the one or more bioactive agents are one or more pharmaceutical compounds.

29. The method of claim 1, wherein the biological fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen Wax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph fluid, chyme, chyle, Bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stools, pancreatic juice, lavages, fluids derived from cells, fluids derived from tissue samples, and cell cultures .

30. The method of claim 4, wherein the mammalian cell is a mesenchymal cell.

31. The method of claim 12, wherein the biological fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, CSF, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk , Bronchoalveolar lavage, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, tissue Interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stools, pancreatic juice, lavage fluids, fluids derived from cells, fluids derived from tissue samples, and cell culture fluids.

32. The method of claim 13, wherein the mammalian cell is a mesenchymal cell.

33. The method of claim 20, wherein the mammalian fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, CSF, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, Breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyle, chyle, bile, Interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stools, pancreatic juice, lavage fluids, fluids derived from cells, fluids derived from tissue samples, and cell culture fluids.

34. The method of claim 33, wherein the mammalian fluid is a conditioned medium.

35. The method of claim 34, wherein the conditioned medium is derived from mesenchymal stem cells.

36. The method of claim 23, wherein the mammalian fluid is selected from the group consisting of: peripheral blood, serum, plasma, ascites, urine, CSF, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, Breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, feces, hair, tears, cystic fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyle, chyle, bile, Interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stools, pancreatic juice, lavage fluids, fluids derived from cells, fluids derived from tissue samples, and cell culture fluids.

37. The method of claim 36, wherein the mammalian fluid is a conditioned medium.

38. The method of claim 37, wherein the conditioned medium is derived from mesenchymal stem cells.

39. The method of claim 1, wherein the epidermolysis bullosa is dystrophic epidermolysis bullosa.

40. The method of claim 39, wherein the dystrophic epidermolysis bullosa is recessive.

41. The method of claim 11, wherein the epidermolysis bullosa is dystrophic epidermolysis bullosa.

42. The method of claim 41, wherein the dystrophic epidermolysis bullosa is recessive.

43. The method of claim 20, wherein the epidermolysis bullosa genotype is recessive dystrophic epidermolysis bullosa.

44. The method of claim 20, wherein one or both of stimulating proliferation of the cells and enhancing resistance of the cells to trypsinization are performed.

45. The method of claim 23, wherein the cell has a recessive dystrophic epidermolysis bullosa genotype.

46. The method of claim 23, wherein one or both of stimulating proliferation of the cells and enhancing resistance of the cells to trypsinization are performed.