EP3684336A1 - Méthodes et compositions pour le traitement d'une épidermolyse bulleuse - Google Patents

Méthodes et compositions pour le traitement d'une épidermolyse bulleuse

Info

Publication number
EP3684336A1
EP3684336A1 EP18859166.3A EP18859166A EP3684336A1 EP 3684336 A1 EP3684336 A1 EP 3684336A1 EP 18859166 A EP18859166 A EP 18859166A EP 3684336 A1 EP3684336 A1 EP 3684336A1
Authority
EP
European Patent Office
Prior art keywords
fluid
microvesicles
cells
cell
isolated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP18859166.3A
Other languages
German (de)
English (en)
Other versions
EP3684336A4 (fr
Inventor
Evangelos V. BADIAVAS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Miami
Original Assignee
University of Miami
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US15/712,294 external-priority patent/US20180104186A1/en
Application filed by University of Miami filed Critical University of Miami
Publication of EP3684336A1 publication Critical patent/EP3684336A1/fr
Publication of EP3684336A4 publication Critical patent/EP3684336A4/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders

Definitions

  • the present invention relates to the fields of medicine, cell biology, molecular biology and genetics.
  • the present invention relates to compositions and methods for treating epidermolysis bullosa.
  • EB Epidermolysis bullosa
  • Dystrophic epidermolysis bullosa is an inherited variant affecting the skin and other organs. Children born with this disease are referred to as "butterfly children" as their skin is described to be as delicate and fragile as a butterfly's wings.
  • the skin of DEB patients is highly susceptible to severe blistering. Open wounds on the skin heal slowly or not at all, often scarring extensively, and are particularly susceptible to infection. Many individuals bathe in a bleach and water mixture to fight off these infections. The chronic inflammation leads to errors in the DNA of the affected skin cells, which in turn causes squamous cell carcinoma (SCC).
  • SCC squamous cell carcinoma
  • DEB is caused by mutations within the human COL7A1 gene encoding the protein type VII collagen (collagen VII).
  • DEB-causing mutations can be either autosomal dominant (DDEB) or autosomal recessive (RDEB).
  • DDEB autosomal dominant
  • RDEB autosomal recessive
  • COL7A1 is located on the short arm of human chromosome 3, in the chromosomal region denoted 3p21.31. The gene is approximately 31,000 base pairs in size and is remarkable for the extreme fragmentation of its coding sequence into 118 exons.
  • COL7A1 is transcribed into an mRNA of 9,287 base pairs.
  • the type VII collagen protein is synthesized by keratinocytes and dermal fibroblasts.
  • Collagen VII is a 300 kDa protein that dimerizes to form a semicircular looping structure: the anchoring fibril.
  • Anchoring fibrils are thought to form a structural link between the epidermal basement membrane and the fibrillar collagens in the upper dermis.
  • Collagen VII is also associated with the epithelium of the esophageal lining, and DEB patients may suffer from chronic scarring, webbing, and obstruction of the esophagus. 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 uncertain origin, which leads to chronic fatigue.
  • the present invention provides methods to isolate micro vesicles (MVs), e.g., extracellular vesicles (MVs) from biological fluids without damaging the structural and/or functional integrity of the microvesicles.
  • MVs micro vesicles
  • the present invention also provides methods to isolate ectosomes, microparticles, microvesicles, nanovesicles, shedding vesicles, apoptotic bodies, or membrane particles from biological fluids without damaging their structural and/or functional integrity.
  • the present invention further provides MVs (e.g., EVs) and methods of using MVs(e.g., EVs) for the treatment of EB (e.g., RDEB and/or DDEB).
  • a method of treating epidermolysis bullosa in a subject in need thereof comprising administering a pharmaceutical composition comprising isolated microvesicles purified by precipitation from a biological fluid to the subject, and alleviating or reducing one or more symptoms of epidermolysis bullosa in the subject is provided.
  • the epidermolysis bullosa is dystrophic epidermolysis bullosa, e.g., recessive dystrophic epidermolysis bullosa.
  • the isolated microvesicles are extracellular vesicles that are optionally precipitated from a 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.
  • 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 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, broncheo alveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluid, fluid derived from a cell, fluid derived from a tissue sample, and cell culture fluid.
  • the biological fluid is from mammalian (e.g., human) cells.
  • CSF cerebrospinal fluid
  • sputum saliva
  • the precipitating agent is polyethylene glycol, that optionally has a molecular weight of about 6,000 Da, about 8,000 Da, about 10,000 Da or about 20,000 Da.
  • the one or more symptoms of epidermolysis bullosa are selected from the group consisting of any combination of thickened calluses, epidermal blistering (e.g., of the hands, the feet, the elbows and/or the knees), blistering of oral mucosa, thickened fingernails and/or toenails, sepsis, malnutrition, dehydration, electrolyte imbalance, obstructive airway complications, defective collagen VII expression, anemia, esophageal strictures, growth retardation, webbing or fusion of fingers and/or toes, malformation of teeth, microstomia and corneal abrasions.
  • treatment comprises increasing collagen VII expression in the subject.
  • a method of treating epidermolysis bullosa in a subject in need thereof comprising administering a pharmaceutical composition comprising isolated extracellular vesicles to the subject and alleviating or reducing one or more symptoms of epidermolysis bullosa in the subject is provided.
  • the epidermolysis bullosa is dystrophic epidermolysis bullosa, e.g., recessive dystrophic epidermolysis bullosa.
  • the isolated microvesicles are extracellular vesicles that are optionally precipitated from a 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.
  • 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, broncheo alveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluid, fluid derived from a cell, fluid derived from a tissue sample, and cell culture fluid.
  • the biological fluid is from mammalian (e.g., human) cells.
  • CSF cerebrospinal fluid
  • sputum saliva
  • the precipitating agent is polyethylene glycol, that optionally has a molecular weight of about 6,000 Da, about 8,000 Da, about 10,000 Da or about 20,000 Da.
  • the one or more symptoms of epidermolysis bullosa are selected from the group consisting of any combination of thickened calluses, epidermal blistering (e.g., of the hands, the feet, the elbows and/or the knees), blistering of oral mucosa, thickened fingernails and/or toenails, sepsis, malnutrition, dehydration, electrolyte imbalance, obstructive airway complications, defective collagen VII expression, anemia, esophageal strictures, growth retardation, webbing or fusion of fingers and/or toes, malformation of teeth, microstomia and corneal abrasions.
  • treatment comprises increasing collagen Vfl expression in the subject.
  • a method of increasing collagen VII levels in a cell comprising contacting the cell with an isolated extracellular vesicle from a mammalian fluid, wherein the cell expresses an epidermolysis bullosa genotype, is provided.
  • 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, broncheo alveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluid, fluid derived from a cell, fluid derived from a tissue sample, and cell culture fluid.
  • the mammalian fluid is a conditioned medium.
  • the conditioned medium is derived from mes
  • the cell comprises a mutation in the COL7A1 gene.
  • the epidermolysis bullosa genotype is recessive dystrophic epidermolysis bullosa.
  • one or both of proliferation of the cell is stimulated, and resistance of the cell to trypsin digestion is enhanced.
  • the isolated extracellular vesicle delivers collagen VII protein and/or COL7A1 mRNA to the cell.
  • a method of delivering one or more bioactive agents to a cell comprising contacting the cell with an isolated extracellular vesicle from a mammalian fluid is provided.
  • 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, broncheo alveolar lavage fluid, semen, prostatic fluid, Cowper's fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluid, fluid derived from a cell, fluid derived from a tissue sample, and cell culture fluid.
  • the mammalian fluid is a conditioned medium.
  • the conditioned medium is derived from mes
  • the cell comprises a mutation in the COL7A1 gene.
  • the one or more bioactive agents are selected from the group consisting of collagen VII protein, collagen VII mRNA, a STAT3 signalling activator (e.g., an interferon, epidermal growth factor, interleukin-S, interleukin-6, a MAP kinase, and/or a c-src non-receptor tyrosine kinase), and a canonical Wnt activator.
  • a STAT3 signalling activator e.g., an interferon, epidermal growth factor, interleukin-S, interleukin-6, a MAP kinase, and/or a c-src non-receptor tyrosine kinase
  • STAT3 is phosphorylated.
  • the one or more bioactive agents are one or more pharmaceutical compounds.
  • the cell has a recessive dystrophic epidermolysis bullosa genotype.
  • one or both of proliferation of the cell is stimulated, and resistance of the cell to trypsin digestion is enhanced.
  • a method for isolating and/or purifying microvesicles from cell culture supernatants or biological fluids utilizing precipitation agent that precipitates the microvesicle from the cell culture supernatant or biological fluid by displacing the water of solvation is provided.
  • an isolated preparation of microvesicles is provided.
  • the isolated preparation of microvesicles is subsequently purified.
  • the isolated preparation of microvesicles is subsequently purified to yield a preparation of ectosomes.
  • the isolated preparation of microvesicles is subsequently purified to yield a preparation of microparticles.
  • the isolated preparation of microvesicles is subsequently purified to yield a preparation of nanovesicles.
  • the isolated preparation of microvesicles is subsequently purified to yield a preparation of shedding vesicles.
  • the isolated preparation of microvesicles is subsequently purified to yield a preparation of membrane particles.
  • the isolated preparation of microvesicles is subsequently purified to yield a preparation of apoptotic bodies.
  • an isolated preparation of microvesicles that promotes or enhances angiogenesis is provided.
  • the isolated preparation of microvesicles promotes or enhances angiogenesis in a patient.
  • an isolated preparation of microvesicles that promotes or enhances neuronal regeneration is provided.
  • the isolated preparation of microvesicles promotes or enhances neuronal regeneration in a patient.
  • an isolated preparation of microvesicles that promotes or enhances cellular proliferation is provided.
  • the isolated preparation of microvesicles promotes or enhances cellular proliferation in a patient.
  • an isolated preparation of microvesicles that promotes or enhances cellular migration is provided.
  • the isolated preparation of microvesicles promotes or enhances cellular migration in a patient.
  • the present invention provides an isolated preparation of microvesicles that promotes or enhances wound healing.
  • the wound is a full-thickness burn.
  • the wound is a second-degree burn.
  • an isolated preparation of microvesicles that reduces scar formation in a patient is provided.
  • an isolated preparation of microvesicles that reduces wrinkle formation in the skin of a patient is provided.
  • an isolated preparation of microvesicles that is used to diagnose the presence and/or progression of a disease in a patient.
  • the disease is metastatic melanoma.
  • the disease in an inflammatory/autoimmune disorder such as rheumatoid arthritis.
  • the disease is graft versus host disease.
  • an isolated preparation of microvesicles that can promote functional regeneration and organization of complex tissue structures.
  • an isolated preparation of microvesicles that can regenerate hematopoietic tissue in a patient with aplastic anemia is provided.
  • an isolated preparation of microvesicles that can regenerate at least one tissue in a patient with diseased, damaged or missing skin selected from the group consisting of: epithelial tissue, stromal tissue, nerve tissue, vascular tissue and adnexal structures is provided.
  • the present invention provides an isolated preparation of microvesicles that can regenerate tissue and/or cells from all three germ layers.
  • an isolated preparation of microvesicles that is used to modulate the immune system of a patient is provided.
  • an isolated preparation of microvesicles that enhances the survival of tissue or cells that is transplanted into a patient.
  • the patient is treated with the isolated preparation of microvesicles prior to receiving the transplanted tissue or cells.
  • the patient is treated with the isolated preparation of microvesicles after receiving the transplanted tissue or cells.
  • the tissue or cells is treated with the isolated preparation of microvesicles.
  • the tissue or cells is treated with the isolated preparation of microvesicles prior to transplantation.
  • an isolated preparation of microvesicles containing at least one molecule selected from the group consisting of RNA, DNA, and protein from a host cell is provided.
  • the host cell is engineered to express at least one molecule selected from the group consisting of RNA, DNA, and protein.
  • the isolated preparation of microvesicles containing at least one molecule selected from the group consisting of RNA, DNA, and protein from a host cell is used as a therapeutic agent.
  • Fig. 1 shows a schematic outline of a protocol used to isolate microvesicles by ultracentrifugation.
  • Fig. 2 shows one embodiment of a microvesicle isolation method of the present invention.
  • FIG. 3 shows an alternate embodiment of a microvesicle isolation method of the present invention.
  • Fig. 4 shows one embodiment of an apparatus of the present invention that facilitates the clarification of the biological fluid and the collection of the precipitated microvesicles by filtration.
  • FIGs. 5A - Fig. 5D show electron micrographs of microvesicles derived from medium conditioned using human bone marrow-derived mesenchymal stem cells isolated by the ultracentrifuge method described in Example 1 (Fig. 5A & Fig. 5B) and isolated according to the methods of the present invention (Fig. 5C & Fig. 5D) at the magnifications shown in the panels.
  • Figs. 6A - Fig. 6D show electron micrographs of microvesicles derived from medium conditioned using porcine bone marrow-derived mesenchymal stem cells isolated by the ultracentrifuge method described in Example 1 (Fig. 6A & Fig. 6B) and isolated according to the methods of the present invention (Fig. 6C & Fig. 6D) at the magnifications shown in the panels.
  • FIGs. 7 A - Fig. 7D show electron micrographs of microvesicles derived from medium conditioned using murine bone marrow-derived mesenchymal stem cells isolated by the ultracentrifuge method described in Example 1 (Fig. 7A & Fig. 7B) and isolated according to the methods of the present invention (Fig. 7C & Fig. 7D) at the magnifications shown in the panels.
  • Figs. 8A— Fig. 8C show electron micrographs of microvesicles isolated from human plasma according to the methods of the present invention.
  • Fig. 8A through Fig. 8C show the microvesicles under increasing magnification, as shown by the scale bars in the panels.
  • Figs. 9A - Fig. 9C show electron micrographs of microvesicles isolated from porcine plasma according to the methods of the present invention.
  • Fig. 9 A through Fig. 9C show the microvesicles under increasing magnification, as shown by the scale bars in the panels.
  • i s. 10A - Fig. 1OC show electron micrographs of microvesicles isolated from human urine according to the methods of the present invention.
  • Fig. 10A through Fig. IOC show the microvesicles under increasing magnification, as shown by the scale bars in the panels.
  • FIG. 11 shows a Western blot, reporting the expression of HSP70, CD63, STAT 3 and phosphorylated STAT3 in lysates of human bone marrow-derived mesenchymal stem cells, microvesicles isolated from medium conditioned using human bone marrow-derived stem cells, prepared by ultracentrifugation (hMSC MV Ultracentrifuge), or the methods of the present invention, as described in Example 3 (hMSC PEG Precipitation). Microvesicles derived from human plasma and human urine, prepared by the methods of the present invention, as described in Example 3 were also analyzed. (Human plasma PEG Precipitation) and (human urine PEG Precipitation) respectively.
  • Figs. 12A - Fig. 12C show the effect of microvesicles isolated from medium conditioned using human bone marrow-derived mesenchymal stem cells on the proliferation of normal human dermal fibroblasts (Fig. 12A), dermal fibroblasts obtained from a diabetic foot ulcer (Fig. 12B), and dermal fibroblasts obtained from a pressure foot ulcer (Fig. 12C).
  • the effect of microvesicles isolated by ultracentrifugation (MV U/C) and microvesicles isolated by the methods of the present invention (MV PEG) were compared. Fibroblasts treated with PBS or microvesicle depleted culture medium were included as a control. Proliferation was determined using an MT T assay.
  • Figs. 13A— Fig. 13G show the effect of microvesicles isolated from medium conditioned using human bone marrow-derived mesenchymal stem cells on the migration of human dermal fibroblasts, as determined by the ability of the fibroblasts to migrate into a region that had been scratched off.
  • the panel labeled "pretreatment” shows a representative area of a cell culture plate where the cells were removed, prior to the addition of the test treatments.
  • the effect of fibroblast migration was tested using microvesicles isolated according to the methods of the present invention (PEG precipitation) and microvesicles isolated by ultracentrifugation (Ultracentrifuge) at the concentrations shown. Fibroblasts treated with PBS or micro vesicle depleted culture medium were included as a control.
  • Figs. 14A— Fig. 14G show the effect of microvesicles isolated from medium conditioned using human bone marrow-derived mesenchymal stem cells on the migration of human dermal fibroblasts obtained from a diabetic foot ulcer, as determined by the ability of the fibroblasts to migrate into a region that had been scratched off.
  • the panel labeled "pretreatment” shows a representative area of a cell culture plate where the cells were removed, prior to the addition of the test treatments.
  • the effect of fibroblast migration was tested using microvesicles isolated according to the methods of the present invention (PEG precipitation) and microvesicles isolated by ultracentrifugation (Ultracentrifuge) at the concentrations shown. Fibroblasts treated with PBS or microvesicle depleted culture medium were included as a control.
  • Figs. ISA - Fig. 15D show the uptake of the microvesicles of the present invention into human dermal fibroblasts.
  • Cell nuclei, resolved using Hoechst 33342 dye are shown in the panels labeled "Hoechst33342.”
  • Cells, resolved using vybrant dye are shown in the panel labeled "Vybrant-Dio.”
  • Microvesicles, resolved using PKH dye are shown in the panel labeled "PKH labeled MV”.
  • a panel where images obtained from all three dyes are overlaid is seen in the panel labeled "Composite.”
  • Figs. 16A - Fig. 16D show the uptake of the microvesicles of the present invention into human dermal fibroblasts.
  • Cell nuclei, resolved using Hoechst 33342 dye are shown in the panels labeled "Hoechst33342.”
  • Cells, resolved using vybrant dye are shown in the panel labeled "Vybrant- Dio.”
  • Microvesicles, resolved using PKH dye are shown in the panel labeled "PKH labeled MV”.
  • a panel where images obtained from all three dyes are overlaid is seen in the panel labeled "Composite.”
  • FIG. 17 shows a Western blot of lysates of human dermal fibroblasts treated with: microvesicles isolated according to the methods of the present invention from plasma obtained from a patient suffering from rheumatoid arthritis (Human Plasma MV PEG Precipitation); microvesicles isolated according to the methods of the present invention from medium conditioned with bone marrow-derived mesenchymal stem cells (Human hMSC MV PEG Precipitation); microvesicles isolated via ultracentrifugation from medium conditioned with bone marrow-derived mesenchymal stem cells (Human hMSC MV ultracentrifugation); PBS control; and a depleted medium control (hMSC conditioned medium depleted of MV).
  • Fig. 18 shows the presence of the region containing 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); micro vesicles isolated according to the methods of the present invention from medium conditioned with SK-MEL28 cells, from RNA amplified using primer 1 (lane S); micro vesicles isolated according to the methods of the present invention from medium conditioned with SK-MEL28 cells, 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 according to the methods of the present invention from medium conditioned with SK-MEL28 cells, from DNA amplified using primer 1 (lane 9); and microvesicles isolated according to the methods of the present invention from medium conditioned with SK
  • Fig. 19 shows the presence of V600E BRAF in a lysate of SK-MEL28 cells and a lysate of microvesicles isolated according to the methods of the present invention from medium conditioned with SK-MEL28 cells.
  • FIGs. 20 A - Fig. 20D show the uptake of the microvesicles isolated according to the methods of the present invention from culture medium conditioned using bone marrow- derived stem cells obtained from a green fluorescent protein (GFP) expressing mouse into human dermal fibroblasts.
  • GFP green fluorescent protein
  • Cell nuclei, resolved using Hoechst 33342 dye are shown in the panels labeled "Hoechst33342.”
  • Cells, resolved using vybrant dye are shown in the panel labeled "Vybrant- Dio.”
  • GFP-labeled microvesicles are shown in the panel labeled "GFP.”
  • a panel where images obtained from all three dyes are overlaid is seen in the panel labeled "Composite.”
  • Figs. 21A - Fig. 21D show the uptake of the microvesicles isolated according to the methods of the present invention from culture medium conditioned using bone marrow- derived stem cells obtained from a GFP expressing mouse into human dermal fibroblasts.
  • Cell nuclei, resolved using Hoechst 33342 dye are shown in the panels labeled "Hoechst33342.”
  • Cells, resolved using vybrant dye are shown in the panel labeled "Vybrant- Dio.”
  • GFP-labeled microvesicles are shown in the panel labeled "GFP.”
  • a panel where images obtained from all three dyes are overlaid is seen in the panel labeled "Composite.”
  • Figs. 22A - Fig. 22D show histological sections of full-thickness wounds from: Fig. 22A - untreated animals; Fig. 22B - microvesicles isolated from medium conditioned using autologous bone marrow-derived mesenchymal stem cells according to the methods of the present invention; Fig. 22C - saline; and Fig 22D - microvesicles isolated from autologous bone marrow-derived mesenchymal stem cells by ultracentrifugation, S days post wound.
  • Figs. 23A- Fig. 23D show pictures of second degree burns on animals treated with: Fig. 23A - microvesicles isolated from medium conditioned using autologous bone marrow- derived mesenchymal stem cells by ultracentrifugation; Fig. 23B - microvesicles isolated from medium conditioned using autologous bone marrow-derived mesenchymal stem cells according to the methods of the present invention; and Fig. 23C - untreated animals, 7 days post wound.
  • Fig 23D - shows a full thickness wound in an animal treated with microvesicles isolated from medium conditioned using autologous bone marrow-derived mesenchymal stem cells by ultracentrifugation 7 days post wound. Arrows indicate abscess formation in a full thickness wound treated with microvesicles isolated by ultracentrifugation at Day 7 (40X). This was not observed in full thickness wounds treated with microvesicles prepared according to the methods of the present invention.
  • Fig. 24 shows a histological slide of a second degree wound, 28 days post wound, from an animal treated with microvesicles isolated from medium conditioned using autologous bone marrow-derived mesenchymal stem cells according to the methods of the present invention.
  • Fig. 25 shows a histological slide of a second-degree wound, 28 days post wound, from an animal treated with saline.
  • Fig. 26 shows a histological slide of a full-thickness wound, 28 days post wound, from an animal treated with microvesicles isolated from medium conditioned using autologous bone marrow-derived mesenchymal stem cells according to the methods of the present invention.
  • Figs. 27 A. - Fig. 27C show a histological slide of a full-thickness wound, 28 days post wound, from an animal treated with microvesicles isolated from medium conditioned using autologous bone marrow-derived mesenchymal stem cells according to the methods of the present invention.
  • Fig. 27 A shows new nerve growth (arrows) and angiogenesis (circles).
  • Fig. 27B shows new nerve growth (arrows).
  • Fig. 27C shows new blood vessel growth (arrows).
  • Fig. 28 shows a histological slide of a full-thickness wound, 7 days post wound in an animal treated with microvesicles derived from medium conditioned using autologous bone marrow-derived mesenchymal stem cells.
  • Figs. 29A - Fig. 29B show the presence or absence of chimerism in irradiated animals following administration of GFP-labeled bone marrow.
  • Figs. 30A - Fig. 30C show the effects of MSC treatment on hair growth following gamma irradiation (Fig. 30A and Fig. 30B), and the absence of chimerism in irradiated animals following administration of GFP-labeled bone marrow (Fig. 30C).
  • Fig. 31A - Fig. 31F show the effect of bone marrow-derived microvesicles obtained using the method of the present invention on blood vessel formation, using an in vitro assay of angiogenesis.
  • the upper three panels are representative images taken using an epifluorescent microscope of cultures of HUVEC cells treated with bone marrow-derived microvesicles obtained using the method of the present invention ("Bone Marrow Aspirate MV").
  • the lower three panels are representative images taken using an epifluorescent microscope of cultures of HUVEC cells treated with vehicle control ("Vehicle Control").
  • Figs. 32A - Fig. 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 using an epifluorescent microscope of cultures of normal adult fibroblasts treated with bone marrow-derived microvesicles obtained using the method of the present invention ("Bone Marrow MV") or PBS (“PBS”), three days post treatment.
  • Fig. 32B shows the average cell number in cultures of normal adult fibroblasts treated with bone marrow-derived microvesicles obtained using the method of the present invention (“Bone Marrow MV”) or PBS (“PBS”), three days post treatment.
  • Fig. 32C graphically depicts cell numbers.
  • Figs. 33 A - Fig. 33B show the results of chronic wound treatment with bone marrow stem cells (including BM-MSCs).
  • Fig. 33A Prior to treatment and before wound debridement. A necrotic Achilles tendon is visible.
  • Fig. 33B Healed post-administration (i.e., topical administration) of bone marrow cells.
  • Figs. 34A - Fig. 34 C show dermal rebuilding in wounds treated with bone marrow stem cells.
  • FIG. 34A pre-treatment biopsy of a fibrotic, scarred wound. Post-treatment biopsies with the generation of numerous reticulin fibers (Fig. 34B) and elastic fibers (Fig. 34C) are shown.
  • FIG. 3SC show a deep second degree burn injury.
  • the patient was given two administrations of BM-MSCs 11 days apart.
  • Fig. 35 A Deep second degree burn injury day 0 (prior to treatment).
  • the circled area represents the deepest portion of the burn injury.
  • Fig. 35B Hair follicle accentuation 11 days after the first administration (i.e., topical administration) of BM-MSCs.
  • the accentuated follicles are noted in the circled area of A.
  • Fig. 35C Hair growth in in the circled area of Fig. 35 A, 34 days after the second administration of BM-MSCs.
  • Figs. 36A - Fig. 36C show the healing of a burn patient treated with two topical administrations of MSCs given ten days apart.
  • Fig. 36A Prior to treatment.
  • Fig. 36B 10 days post-treatment (i.e., topical administration) with first dose of MSCs.
  • Fig. 36CA 7 days post-treatment with second dose of BM-MSCs (i.e., 17 days after Fig. 36A).
  • i s. 37 A - Fig. 37B show no evidence of scarring in burn patient assessed one year post-treatment with BM-MSCs.
  • Figs. 38A - Fig. 38B show full thickness wounds (day 5) created on Yorkshire pigs.
  • Fig. 38B Wound treated with BM-MSC EVs according to certain embodiments of the invention.
  • Arrows indicate areas of increased dermal remodeling according to certain embodiments of the invention.
  • Figs. 39 A - Fig. 39C show full thickness wounds (day 28) created on Yorkshire pigs treated with BM-MSC EVs according to certain exemplary embodiments.
  • Fig. 39A Arrows highlight nerve growth and stars illustrate vascular growth.
  • Fig. 39B Higher magnification illustrating vascular growth (arrows).
  • Fig. 39C Higher magnification illustrating nerve growth (arrows).
  • Figs. 40A— Fig. 40B show second degree burn wounds in pigs 5 days post-treatment with intralesional injection of porcine BM-MSC EVs according to certain exemplary embodiments.
  • Figs. 41 A - Fig. 41B graphically depict enrichment of COL7A1 mRNA in BM-MSC EVs (middle bars in each panel). EV treatment increased COL7A1 expression in RDEB fibroblasts. Left panel shows COL7A1 expression detected with primer pair 1; right panel shows COL7A1 expression detected with primer pair 2. Gene expression was normalized by beta-actin expression, a common EV housekeeping gene.
  • Fig. 42 graphically depicts a chemoselective ligation assay (utilizing "click iT” reaction chemistry) that revealed production of new collagen VII from RDEB fibroblasts following co-treatment with BM-MSC EVs (10 ⁇ g/mL) and the L-methionine analog L- homopropargylglycine (HPG) (a modified amino acid) which incorporates into newly synthesized proteins.
  • BM-MSC EVs 10 ⁇ g/mL
  • HPG L-methionine analog L- homopropargylglycine
  • Figs. 43A - Fig. 43B graphically depict that BM-MSC EVs significantly promote both RDEB proliferation (Fig. 43A) and resistance to trypsin digestion (Fig. 43B), both standard in vitro assays to assess gain-of-function support the pro-wound healing potential of RDEB dermal fibroblasts.
  • Figs. 44A - Fig. 44C show the validation of an in vitro cell line derived from an infant diagnosed as having RDEB (Hallopeau -Siemens type).
  • the RDEB fibroblasts expressed significantly less COL7A1 compared to fibroblasts derived from non-affected subjects (NHF).
  • Fig. 44A - Primer pairs 1 and 2 designed near 3' end of cDNA corresponding to 5' end of mRNA.
  • Fig. 44C - RDEB cells secreted low levels of collagen VII protein relative to normal (control) human fibroblasts.
  • Figs. 4SA - Fig. 4SB show vesicle exchange between BM-MSCs and RDEB fibroblasts.
  • RDEBFs stained with lipid dye Dil (red)
  • BM-MSCs stained with lipid dye DiO (green)
  • Scale bar 10 ⁇ .
  • Figs. 46A - Fig. 46D show that collagen VII protein co-isolated with BM-MSC extracellular vesicles (EVs).
  • Fig. 46A Transmission electron micrograph of an extracellular vesicle isolated from BM-MSC serum-free conditioned media (CM).
  • Fig. 46B NanoSight image of BM-MSC EVs, diluted 1:500.
  • Fig. 46C Histogram of size vs concentration (diluted 1:500). Inset shows EVs contain CD63 exosome marker.
  • Fig. 46D Collagen VII protein in BM-MSC CM and associated with purified BM-MSC EVs.
  • Figs. 47 A - Fig. 47B show enrichment of COL7A1 mRNA in BM-MSC EVs (middle bars in each panel). EV treatment increased COL7A1 expression in RDEB fibroblasts. Left panel shows COL7A1 expression detected with primer pair 1 ; right panel shows COL7A1 expression detected with primer pair 2. Gene expression was normalized by beta-actin expression, a common EV housekeeping gene.
  • Figs. 48A - Fig. 48C show that RDEB fibroblasts treated with BM-MSC EVs contained more collagen VII protein in media 3 days after washing.
  • Fig. 48A Treatment schematic.
  • Fig. 48B Western blot of collagen VII in RDEB media.
  • Fig. 48C Densitometry quantification of Fig. 48B (above baseline collagen VII detection).
  • Figs. 49A - Fig. 49C depict a chemoselective ligation assay (utilizing "click iT" reaction chemistry) (Fig. 49A and Fig, 49B) that revealed production of new collagen VII from RDEB fibroblasts following co-treatment with BM-MSC EVs (10 ⁇ g mL) and L- methionine analog L-homopropargylglycine (HPG) (a modified amino acid) which incorporates into newly synthesized proteins (Fig. 49C).
  • BM-MSC EVs 10 ⁇ g mL
  • HPG L- methionine analog L-homopropargylglycine
  • Figs. 50A - Fig. 50B show that BM-MSC EVs increased in vitro surrogate assays related to wound healing (proliferation and trypsin-resistance) of RDEB fibroblasts.
  • Fig. 50A Proliferation (MTT) assay.
  • Fig. 50B Trypsin resistance assay.
  • Figs. 51A - Fig. 51E show BM-MSCs that were delivered in saline to burn patients in a clinical trial. BM-MSCs secreted large numbers of EVs (CD63 positive) in saline within hours (shown, 4 hours).
  • Fig. 52 depicts a model according to certain exemplary embodiments of the invention in which the secretome of BM-MSCs contains EV-associated and non-EV-associated proteins that deliver multiple pro-wound healing functions to RDEB fibroblasts, including collagen VII protein, collagen VII mRNA, STAT3-signaling activators, and canonical Wnt activators.
  • EV-associated and non-EV-associated proteins that deliver multiple pro-wound healing functions to RDEB fibroblasts, including collagen VII protein, collagen VII mRNA, STAT3-signaling activators, and canonical Wnt activators.
  • microvesicles refers to vesicles comprising lipid bilayers, formed from the plasma membrane of cells, and are heterogeneous in size, ranging from about 2 nm to about 5000 nm.
  • the cell from which a microvesicle is formed is herein referred to as "the host cell”
  • Microvesicles are a heterogeneous population of vesicles and include, but are not limited to, extracellular vesicles (EVs), ectosomes, microparticles, microvesicles, nanovesicles, shedding vesicles, membrane particles and the like.
  • Microvesicles exhibit membrane proteins from their host cell on their membrane surface, and may also contain molecules within the microvesicle from the host cell, such as, for example, mRNA, miRNA, tRNA, RNA, DNA, lipids, proteins or infectious particles. These molecules may result from, or be, recombinant molecules introduced into the host cell Microvesicles play a critical role in intercellular communication, and can act locally and distally within the body, inducing changes in cells by fusing with a target cell, introducing the molecules transported on and/or in the microvesicle to the target cell.
  • microvesicles have been implicated in anti-tumor reversal, cancer, tumor immune suppression, metastasis, tumor-stroma interactions, angiogenesis and tissue regeneration.
  • Microvesicles may also be used to diagnose disease, as they have been shown to carry bio- markers of several diseases, including, for example, cardiac disease, HIV and leukemia.
  • microvesicles are isolated from a biological fluid containing microvesicles in a method comprising the steps of: a) obtaining a biological fluid containing microvesicles, b) clarifying the biological fluid to remove cellular debris, c) precipitating the microvesicles by adding a precipitating agent to the clarified biological fluid, d) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, and e) suspending the washed microvesicles in a solution for storage or subsequent use.
  • the biological fluid is clarified by centrifugation. In an alternate embodiment, the biological fluid is clarified by filtration.
  • the precipitated microvesicles are collected by centrifugation. In an alternate embodiment, the precipitated microvesicles are collected by filtration.
  • microvesicles are isolated from a biological fluid containing microvesicles in a method comprising the steps of: a) obtaining a biological fluid containing microvesicles, b) clarifying the biological fluid to remove cellular debris, c) precipitating the microvesicles by adding a precipitating agent to the clarified biological fluid, d) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, e) suspending the washed microvesicles in a solution, and f) processing the micro vesicles to analyze the nucleic acid, carbohydrate, lipid, small molecules and/or protein content.
  • the biological fluid is clarified by centrifugation. In an alternate embodiment, the biological fluid is clarified by filtration.
  • the precipitated microvesicles are collected by centrifugation. In an alternate embodiment, the precipitated microvesicles are collected by filtration.
  • the present invention provides reagents and kits to isolate microvesicles from biological fluids according to the methods of the present invention.
  • the biological fluid may be peripheral blood, sera, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheo alveolar lavage fluid, semen (including prostatic fluid), Cowper's fluid or pre-ejaculatory fluid, female ejaculate, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates or other lavage fluids.
  • CSF cerebrospinal fluid
  • the biological fluid may also be derived from the blastocyl cavity, 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 cultures of plant cells.
  • the biological fluid may be derived from yeast cells or cultures of yeast cells.
  • the biological fluid is cell culture medium.
  • the cell culture medium is conditioned using tissues and/or cells prior to the isolation of microvesicles according to the methods of the present invention.
  • conditioned refers to medium, wherein a population of cells or tissue, or combination thereof is grown, and the population of cells or tissue, or combination thereof contributes factors to the medium. In one such use, the population of cells or tissue, or combination thereof is removed from the medium, while the factors the cells produce remain. In one embodiment, the factors produced are microvesicles.
  • Medium may be conditioned via any suitable method selected by one of ordinary skill in the art. For example, medium may be cultured according to the methods described in EP1780267A2.
  • microvesicles are isolated from cells or tissue that have been pre- treated prior to the isolation of the microvesicles.
  • Pretreatment may include, for example, culture in a specific medium, a medium that contains at least one additive, growth factor, medium devoid of serum, or a combination thereof.
  • pretreatment may comprise contacting cells or tissues with additives (e.g. interleukin, VEGF, inducers of transcription factors, transcription factors, hormones, neurotransmitters, pharmaceutical compounds, microRNA), transforming agents (e.g. liposome, viruses, transfected agents, etc.).
  • additives e.g. interleukin, VEGF, inducers of transcription factors, transcription factors, hormones, neurotransmitters, pharmaceutical compounds, microRNA
  • transforming agents e.g. liposome, viruses, transfected agents, etc.
  • pretreatment may comprise exposing cells or tissue to altered physical conditions (e.g. hypoxia, cold shock, heat shock and the like).
  • microvesicles are isolated from medium conditioned using cells or tissue that have been pre-treated prior to the isolation of the microvesicles.
  • Pretreatment may include, for example, culture in a specific medium, a medium that contains at least one additive, growth factor, medium devoid of serum, or a combination thereof.
  • pretreatment may comprise contacting cells or tissues with additives (e.g. interleukin, VEGF, inducers of transcription factors, transcription factors, hormones, neurotransmitters, pharmaceutical compounds, microRNA), transforming agents (e.g. liposome, viruses, transfected agents, etc.).
  • pretreatment may comprise exposing cells or tissue to altered physical conditions (e.g. hypoxia, cold shock, heat shock and the like).
  • the biological fluid is an extract from a plant.
  • the biological fluid is a cell culture medium from a culture of plant cells.
  • the biological fluid is yeast extract.
  • the biological fluid is a cell culture medium from a culture of yeast cells.
  • the methods of the present invention may be carried out at any temperature, one of ordinary skill in the art can readily appreciate that certain biological fluids may degrade, and such degradation is reduced if the sample is maintained at a temperature below the temperature at which the biological fluid degrades.
  • the method of the present invention is carried out at 4 °C.
  • at least one step of the method of the present invention is carried out at 4 °C.
  • the biological fluid may be diluted prior to being subjected to the methods of the present invention. Dilution may be required for viscous biological fluids, to reduce the viscosity of the sample, if the viscosity of the sample is too great to obtain an acceptable yield of microvesicles.
  • the dilution may be a 1 :2 dilution.
  • the dilution may be a 1 :3 dilution.
  • the dilution may be a 1 :4 dilution.
  • the dilution may be a 1:5 dilution.
  • the dilution may be a 1 :6 dilution.
  • the dilution may be a 1:7 dilution.
  • the dilution may be a 1:8 dilution.
  • the dilution may be a 1:9 dilution.
  • the dilution may be a 1: 10 dilution.
  • the dilution may be a 1:20 dilution.
  • the dilution may be a 1:30 dilution.
  • the dilution may be a 1 :40 dilution.
  • the dilution may be a 1:50 dilution.
  • the dilution may be a 1:60 dilution.
  • the dilution may be a 1:70 dilution.
  • the dilution may be a 1:80 dilution.
  • the dilution may be a 1 :90 dilution.
  • the dilution may be a 1 : 100 dilution.
  • the biological fluid may be diluted with any diluent, provided the diluent does not affect the functional and or structural integrity of the microvesicles.
  • diluents may be, for example, phosphate buffered saline, cell culture medium, and the like.
  • the biological fluid is clarified by the application of a 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 centrifugal force applied is insufficient in magnitude, duration, or both, to remove the microvesicles.
  • the biological fluid may require dilution to facilitate the clarification.
  • the duration and magnitude of the centrifugal force used to clarify the biological fluid may vary according to a number of factors readily appreciated 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.
  • a centrifugal force of 2000 x g is applied to the biological fluid for 30 minutes.
  • the clarified biological fluid is contacted with a precipitation agent to precipitate the microvesicles.
  • the precipitation agent may be any agent that surrounds the microvesicles and displaces the water of solvation.
  • Such precipitation agents may be selected from the group consisting of polyethylene glycol, dextran, and polysaccharides.
  • the precipitation agent may cause aggregation of the microvesicles.
  • the precipitation agent is selected from the group consisting of calcium ions, magnesium ions, sodium ions, ammonium ions, iron ions, organic solvents such as ammonium sulfate, and flocculating agents, such as alginate.
  • the clarified biological fluid is contacted with the precipitation agent for a period of time sufficient to precipitate the microvesicles.
  • the period of time sufficient to precipitate the microvesicles may vary according to a number of factors readily appreciated 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.
  • the period of time sufficient to precipitate the microvesicles is 6 hours.
  • the clarified biological fluid is contacted with the precipitation agent for a period of time sufficient to precipitate the microvesicles at 4 °C.
  • the concentration of the precipitation agent used to precipitate the microvesicles from a biological fluid may vary according to a number of factors readily appreciated 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.
  • the precipitation agent is polyethylene glycol.
  • the molecular weight of polyethylene glycol used in the methods of the present invention may be from about 200 Da to about 10,000 Da. In one embodiment, the molecular weight of polyethylene glycol used in the methods of the present invention may be greater than 10,000 Da. In certain embodiments, the molecular weight of polyethylene glycol used in the methods of the present invention is 10,000 Da or 20,000 Da.
  • the choice of molecular weight may be influenced 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.
  • the molecular weight of polyethylene glycol used in the methods of the present invention may be from about 200 Da to about 8,000 Da, or is approximately any of 200 Da, 300 Da, 400 Da, 600 Da, 1000 Da, 1450 Da, 1500 Da, 2000 Da, 3000 Da, 3350 Da, 4000 Da, 6000 Da, 8000 Da, 10000 Da, 20000 Da or 35000 Da or any ranges or molecular weights in between.
  • the molecular weight of polyethylene glycol used in the methods of the present invention is about 6000 Da.
  • the average molecular weight of polyethylene glycol used in the methods of the present invention is about 8000 Da.
  • the average molecular weight of polyethylene glycol used in the methods of the present invention is about 10000 Da.
  • the average molecular weight of polyethylene glycol used in the methods of the present invention is about 20000 Da.
  • the concentration of polyethylene glycol used in the methods of the present 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 present invention may be influenced 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.
  • the polyethylene glycol is used in the concentration of the present invention at a concentration between about 5% and 25% w/v. 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.
  • the concentration of polyethylene glycol used in the methods of the present invention is about 8.5% w/v.
  • the concentration of polyethylene glycol used in the methods of the present invention is about 6% w/v.
  • polyethylene glycol having an average molecular weight of 6000 Da is used, at a concentration of 8.5% w/v. In one embodiment, the polyethylene glycol is diluted in 0.4M sodium chloride.
  • the concentration of the polyethylene glycol used in the methods of the present 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 4000 Da is used, at a concentration of 20% w/v. In an alternate embodiment, polyethylene glycol having an average molecular weight of 8000 Da is used, at a concentration of 10% w/v. In an alternate embodiment, polyethylene glycol having an average molecular weight of 20000 Da is used, at a concentration of 4% w/v.
  • the precipitated microvesicles are collected by the application of centrifugal force.
  • the centrifugal force is sufficient and applied for a duration sufficient to cause the microvesicles to form a pellet, but insufficient to damage the microvesicles.
  • the duration and magnitude of the centrifugal force used to precipitate the microvesicles from a biological fluid may vary according to a number of factors readily appreciated 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.
  • the precipitated microvesicles are collected by the application of a centrifugal force of 10000 x g for 60 minutes.
  • the precipitated microvesicles may be washed with any liquid, provided the liquid does not affect the functional and/or structural integrity of the microvesicles.
  • a suitable liquid Liquids may be, for example, phosphate buffered saline, cell culture medium, and the like.
  • the washing step removes the precipitating agent.
  • the microvesicles are washed via centrifugal filtration, using a filtration device with a 100 kDa molecular weight cut off.
  • the isolated microvesicles may be suspended with any liquid, provided the liquid does not affect the functional and/or structural integrity of the microvesicles.
  • a suitable liquid Liquids may be, for example, phosphate buffered saline, cell culture medium, and the like.
  • the isolated microvesicles may be further processed.
  • the further processing may be the isolation of a microvesicle of a specific size.
  • the further processing may be the isolation of micro vesicles of a particular size range.
  • the further processing may be the isolation of a microvesicle of a particular molecular weight.
  • the further processing may be the isolation of microvesicles of a particular molecular weight range.
  • the further processing may be the isolation of a microvesicle exhibiting or containing a specific molecule.
  • the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 run to about 1000 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 500 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 400 nm as determined by electron microscopy.
  • the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 300 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 200 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 100 nm as determined by electron microscopy.
  • the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about SO nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 20 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention are further processed to isolate a preparation of microvesicles having a size of about 2 nm to about 10 nm as determined by electron microscopy.
  • the subsequent purification is performed using a method selecting from the group consisting of immunoaffinity, HPLC, tangential flow filtration, phase separation/partitioning, and microfluidics.
  • the isolated microvesicles are further processed to analyze the molecules exhibited on, or contained within the microvesicles.
  • the molecules analyzed are selected from the group consisting of nucleic acid, carbohydrate, lipid, small molecules, ions, metabolites, protein, and combinations thereof.
  • Biological fluid comprising cell culture medium conditioned using cultured cells In one embodiment, microvesicles are obtained from medium conditioned using cultured cells. Any cultured cell, or population of cells may be used in the methods of the present invention.
  • the cells may be stem cells, primary cells, cell lines, tissue or organ explants, or any combination thereof.
  • the cells may be allogeneic, autologous, or xenogeneic in origin.
  • the cells are cells derived from bone-marrow aspirate.
  • the cells derived from bone marrow aspirate are bone marrow-derived mesenchymal stem cells.
  • the cells derived from bone marrow aspirate are mononuclear cells.
  • the cells derived from bone marrow aspirate are a mixture of mononuclear cells and bone marrow-derived mesenchymal stem cells.
  • bone marrow-derived mesenchymal stem cells are isolated from bone marrow aspirate by culturing bone marrow aspirate in plastic tissue culture flasks for a period of time of up to about 4 days, followed by a wash to remove the non-adherent cells.
  • mononuclear cells are isolated from bone marrow aspirate by low- density centrifugation using a ficoll gradient, and collecting the mononuclear cells at the interface.
  • the cells prior to isolation of microvesicles according to the methods of the present invention, are cultured, grown or maintained at an appropriate temperature and gas mixture (typically, 37 °C, 5% CO 2 for mammalian cells) in a cell incubator. Culture conditions vary widely for each cell type, and are readily determined by one of ordinary skill in the art.
  • an appropriate temperature and gas mixture typically, 37 °C, 5% CO 2 for mammalian cells
  • one, or more than one culture condition is varied. In one embodiment, this variation results in a different phenotype.
  • the cell culture medium is supplemented with microvesicle- free serum and then added to the cells to be conditioned.
  • the microvesicles are collected from the conditioned cell culture medium.
  • Serum may be depleted by any suitable method, such as, for example, ultracentrifugation, filtration, precipitation, and the like.
  • the choice of medium, serum concentration, and culture conditions are influenced by a variety of factors readily appreciated by one of ordinary skill in the art, including, for example, the cell type being cultured, the desired purity of the microvesicles, the desired phenotype of the cultured cell, and the like.
  • the cell culture medium that is conditioned for the microvesicle isolation procedure is the same type of cell culture medium that the cells were grown in, prior to the microvesicle isolation procedure.
  • the cell culture medium is removed, and serum-free medium is added to the cells to be conditioned.
  • the microvesicles are then collected from the conditioned serum free medium.
  • the choice of medium, and culture conditions are influenced by a variety of factors readily appreciated by one of ordinary skill in the art, including, for example, the cell type being cultured, the desired purity of the microvesicles, the desired phenotype of the cultured cell, and the like.
  • the serum-free medium is supplemented with at least one additional factor that promotes or enhances the survival of the cells in the serum free medium. Such factor may, for example, provide trophic support to the cells, inhibit, or prevent apoptosis of the cells.
  • the cells are cultured in the culture medium for a period of time sufficient to allow the cells to secrete 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 readily appreciated by one of ordinary skill in the art, including, for example, the cell type being cultured, the desired purity of the microvesicles, the desired phenotype of the cultured cell, desired yield of microvesicles, and the like.
  • microvesicles are then removed from the culture medium by the methods of the present invention.
  • the cells prior to the microvesicle isolation procedure, are treated with at least one agent selected from the group consisting of an anti-inflammatory compound, an anti-apoptotic compound, an inhibitor of fibrosis, a compound that is capable of enhancing angiogenesis, an immunosuppressive compound, a compound that promotes survival of the cells, a chemotherapeutic, a compound capable of enhancing cellular migration, a neurogenic compound, and a growth factor.
  • the cells while the cells are being cultured in the medium from which the microvesicles are collected, the cells are treated with at least one agent selected from the group consisting of an anti-inflammatory compound, an anti- apoptotic compound, an inhibitor of fibrosis, a compound that is capable of enhancing angiogenesis, an immunosuppressive compound, a compound that promotes survival of the cells, and a growth factor.
  • at least one agent selected from the group consisting of an anti-inflammatory compound, an anti- apoptotic compound, an inhibitor of fibrosis, a compound that is capable of enhancing angiogenesis, an immunosuppressive compound, a compound that promotes survival of the cells, and a growth factor.
  • the anti-inflammatory compound may be selected from the compounds disclosed in U. S. Patent. No. 6,509,369.
  • the anti-apoptotic compound may be selected from the compounds disclosed in U. S. Patent. No. 6,793,945.
  • the inhibitor of fibrosis may be selected from the compounds disclosed in U. S. Patent. No. 6,331,298.
  • the compound that is 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.
  • the immunosuppressive compound may be selected from the compounds disclosed in U. S. Patent Application 2004/0171623.
  • the compound that promotes survival of the cells may be selected from the compounds disclosed in U. S. Patent Application 2010/0104542.
  • the growth factor may be at least one molecule selected from the group consisting of members of the TGF- ⁇ family, including TGF- ⁇ 1, 2, and 3, bone morphogenic proteins (BMP-2, -3,-4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors- 1 and -2, platelet-derived growth factor-AA, -AB, and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growth differentiation factor (GDF-5, -6, -8, -10, -15), vascular endothelial cell- derived growth factor (VEGF), pleiotrophin, endothelin, among others.
  • Other pharmaceutical compounds can include, for example, nicotinamide, hypoxia inducible factor 1 -alpha, glucagon like peptide-1 (GLP-1), GLP-1 and GLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid, parathyroid hormone, tenascin-C, tropoelastin, thrombin- derived peptides, cathelicidins, defensins, laminin, biological peptides containing cell- and heparin- binding domains of adhesive extracellular matrix proteins such as fibronectin and vitronectin, and MAPK inhibitors, such as, for example, compounds disclosed in U. S. Patent Application 2004/ 0209901 and U. S. Patent Application 2004/0132729.
  • MAPK inhibitors such as, for example, compounds disclosed in U. S. Patent Application 2004/ 0209901 and U. S. Patent Application 2004/0132729.
  • microvesicles are isolated from a biological fluid comprising cell culture medium conditioned using a culture of bone marrow-derived mesenchymal stem cells comprising the steps of: a) obtaining a population of bone marrow-derived mesenchymal stem cells and seeding flasks at a 1 :4 dilution of cells, b) culturing the cells in medium until the cells are 80 to 90% confluent, c) removing and clarifying the medium to remove cellular debris, d) precipitating the microvesicles by adding a precipitating agent to the clarified culture medium, e) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, and f) suspending the washed microvesicles in a solution for storage or subsequent use.
  • microvesicles are isolated from a biological fluid comprising cell culture medium conditioned using a culture of bone marrow-derived mononuclear cells comprising the steps of: a) obtaining a population of bone marrow-derived mononuclear cells and seeding flasks at a 1 :4 dilution of cells, b) culturing the cells in medium until the cells are 80 to 90% confluent, c) removing and clarifying the medium to remove cellular debris, d) precipitating the microvesicles by adding a precipitating agent to the clarified culture medium, e) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, and f) suspending the washed microvesicles in a solution for storage or subsequent use.
  • the bone marrow-derived mesenchymal stem cells are cultured in medium comprising ⁇ -MEM supplemented with 20% fetal bovine serum and 1% penicillin streptomycin glutamine at 37°C in 95% humidified air and 5% CO 2 .
  • the bone marrow-derived mononuclear cells are cultured in medium comprising ⁇ -MEM supplemented with 20% fetal bovine serum and 1% penicillin/streptomycinglutamine at 37°C in 95% humidified air and 5% CO 2 .
  • the medium is clarified by centrifugation.
  • the precipitating agent is polyethylene glycol having an average molecular weight of 6000. In one embodiment, the 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 having a final concentration of 0.4 M.
  • the precipitated microvesicles are collected by centrifugation.
  • the isolated microvesicles are washed via centrifugal filtration, using a membrane with a 100 kDa molecular weight cut-off, using phosphate buffered saline.
  • Biological fluid comprising plasma In one embodiment, microvesicles are obtained from plasma.
  • the plasma may be obtained from a healthy individual, or, alternatively, from an individual with a particular disease phenotype.
  • microvesicles are isolated from a biological fluid comprising plasma comprising the steps of: a) obtaining plasma and diluting the plasma with cell culture medium, b) precipitating the microvesicles by adding a precipitating agent to the diluted plasma, c) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, and d) suspending the washed microvesicles in a solution for storage or subsequent use.
  • the plasma is diluted 1: 10 with culture medium.
  • the culture medium is a- MEM.
  • the precipitating agent is polyethylene glycol having an average molecular weight of 6000. In one embodiment, the polyethylene glycol is used at a concentration of about 8.S w/v %. In one embodiment, the polyethylene glycol is diluted in a sodium chloride solution having a final concentration of 0.4 M.
  • the precipitated microvesicles are collected by centrifugation.
  • the isolated microvesicles are washed via centrifugal filtration, using a membrane with a 100 kDa molecular weight cut-off, using phosphate buffered saline.
  • Biological fluid comprising bone marrow aspirate In one embodiment, microvesicles are obtained from bone marrow aspirate. In one embodiment, microvesicles are obtained from the cellular fraction of the bone marrow aspirate. In one embodiment, microvesicles are obtained from the acellular fraction of the bone marrow aspirate.
  • microvesicles are obtained from cells cultured from bone marrow aspirate.
  • the cells cultured from bone marrow aspirate are used to condition cell culture medium, from which the microvesicles are isolated.
  • microvesicles are isolated from a biological fluid comprising bone marrow aspirate comprising the steps of: a) obtaining bone marrow aspirate and separating the bone marrow aspirate into an acellular portion and a cellular portion, b) diluting the acellular portion, c) clarifying the diluted acellular portion to remove cellular debris, d) precipitating the microvesicles in the acellular portion by adding a precipitating agent to the diluted acellular portion, e) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, and f) suspending the washed microvesicles in a solution for storage or subsequent use.
  • the acellular portion is diluted 1 : 10 with culture medium.
  • the culture medium is ⁇ -MEM.
  • the diluted acellular portion is clarified by centrifugation.
  • the precipitating agent is polyethylene glycol having an average molecular weight of 6000. In one embodiment, the polyethylene glycol is used at a concentration of about 8.S w/v %. In one embodiment, the polyethylene glycol is diluted in a sodium chloride solution having a final concentration of 0.4 M.
  • the precipitated microvesicles are collected by centrifugation.
  • the isolated microvesicles are washed via centrifugal filtration, using a membrane with a 100 kDa molecular weight cut-off, using phosphate buffered saline.
  • the cellular portion is further processed to isolate and collect cells. In one embodiment, the cellular portion is further processed to isolate and collect bone marrow- derived mesenchymal stem cells. In one embodiment, the cellular portion is further processed to isolate and collect bone marrow-derived mononuclear cells. In one embodiment, the cellular portion is used to condition medium, from which microvesicles may later be derived.
  • microvesicles are isolated from the cellular portion.
  • the cellular portion may be incubated for a period of time prior to the isolation of the microvesicles.
  • the microvesicles may be isolated from the cellular portion immediately after the cellular portion is collected.
  • the cellular portion is also treated with at least one agent selected from the group consisting of an anti-inflammatory compound, an anti-apoptotic compound, an inhibitor of fibrosis, a compound that is capable of enhancing angiogenesis, an immunosuppressive compound, a compound that promotes survival of the cells, a chemotherapeutic, a compound capable of enhancing cellular migration, a neurogenic compound, and a growth factor.
  • at least one agent selected from the group consisting of an anti-inflammatory compound, an anti-apoptotic compound, an inhibitor of fibrosis, a compound that is capable of enhancing angiogenesis, an immunosuppressive compound, a compound that promotes survival of the cells, a chemotherapeutic, a compound capable of enhancing cellular migration, a neurogenic compound, and a growth factor.
  • the anti-inflammatory compound may be selected from the compounds disclosed in U. S. Patent. No. 6,509,369.
  • the anti-apoptotic compound may be selected from the compounds disclosed in U. S. Patent. No. 6,793,945.
  • the inhibitor of fibrosis may be selected from the compounds disclosed in U. S. Patent. No. 6,331,298.
  • the compound that is 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.
  • the immunosuppressive compound may be selected from the compounds disclosed in U. S. Patent Application 2004/0171623.
  • the compound that promotes survival of the cells may be selected from the compounds disclosed in U. S. Patent Application 2010/0104542.
  • the growth factor may be at least one molecule selected from the group consisting of members of the TGF- ⁇ family, including TGF- ⁇ , 2, and 3, bone morphogenic proteins (BMP-2, -3,-4, -5, -6, -7, -11, -12, and -13), fibroblast growth factors- 1 and -2, platelet-derived growth factor-AA, -AB, and -BB, platelet rich plasma, insulin growth factor (IGF-I, ⁇ ) growth differentiation factor (GDF-5, -6, -8, -10, -15), vascular endothelial cell- derived growth factor (VEGF), pleiotrophin, endothelin, among others.
  • TGF- ⁇ bone morphogenic proteins
  • BMP-2, -3,-4, -5, -6, -7, -11, -12, and -13 bone morphogenic proteins
  • fibroblast growth factors- 1 and -2 platelet-derived growth factor-AA, -AB, and -BB
  • platelet rich plasma platelet rich plasma
  • Other pharmaceutical compounds can include, for example, nicotinamide, hypoxia inducible factor 1 -alpha, glucagon like peptide- 1 (GLP-1), GLP-1 and GLP-2 mimetibody, and ⁇ , Exendin-4, nodal, noggin, NGF, retinoic acid, parathyroid hormone, tenascin-C, tropoelastin, thrombin- derived peptides, cathelicidins, defensins, laminin, biological peptides containing cell- and heparin-binding domains of adhesive extracellular matrix proteins such as fibronectin and vitronectin, and MAPK inhibitors, such as, for example, compounds disclosed in U. S. Patent Application 2004/0209901 and U. S. Patent Application 2004/0132729.
  • the cellular portion is cultured under hypoxic conditions. In one embodiment, the cellular portion is heat- shocked.
  • Biological fluid comprising urine In one embodiment, microvesicles are obtained from urine.
  • the urine may be obtained from a healthy individual, or, alternatively, from an individual with a particular disease phenotype.
  • microvesicles are isolated from a biological fluid comprising urine comprising the steps of: a) obtaining a urine sample, b) clarifying the urine to remove cellular debris, c) precipitating the microvesicles by adding a precipitating agent to the clarified urine, d) collecting the precipitated microvesicles and washing the material to remove the precipitating agent, and e) suspending the washed microvesicles in a solution for storage or subsequent use.
  • the urine is clarified by centrifugation.
  • the precipitating agent is polyethylene glycol having an average molecular weight of 6000. In one embodiment, the polyethylene glycol is used at a concentration of about 8.S w/v %. In one embodiment, the polyethylene glycol is diluted in a sodium chloride solution having a final concentration of 0.4 M.
  • the precipitated microvesicles are collected by centrifugation.
  • the isolated microvesicles are washed via centrifugal filtration, using a membrane with a 100 kDa molecular weight cut-off, using phosphate buffered saline.
  • the biological fluids are clarified by filtration.
  • the precipitated microvesicles are collected by filtration.
  • the biological fluids are clarified and the precipitated microvesicles are collected by filtration.
  • filtration of either the biological fluid, and/or the precipitated microvesicles required the application of an external force.
  • the external force may be gravity, either normal gravity or centrifugal force. Alternatively, the external force may be suction.
  • the present embodiment provides an apparatus to facilitate the clarification of the biological fluid by filtration. In one embodiment, the present invention provides an apparatus to facilitate collection of the precipitated microvesicles by filtration. In one embodiment, the present invention provides an apparatus that facilitates the clarification of the biological fluid and the collection of the precipitated microvesicles by filtration. In one embodiment, the apparatus also washes the microvesicles.
  • the apparatus is the apparatus shown in Figure 4.
  • the biological fluid is added to the inner chamber.
  • the inner chamber has a first filter with a pore size that enables the microvesicles to pass, while retaining any particle with a size greater than a microvesicle in the inner chamber.
  • the pore size of the filter of the inner chamber is 1 um. In this embodiment, when the biological fluid passed from the inner chamber through the filter, particles greater than 1 ⁇ are retained in the inner chamber, and all other particles collect in the region between the bottom of the inner chamber and a second filter.
  • the second filter has a pore size that does not allow microvesicles to pass.
  • the pore size of the second filter of the inner chamber is 0.01 um. In this embodiment, when the biological fluid passed through the second filter, the microvesicles are retained in the region between the bottom of the inner chamber and the second filter, and all remaining particles and fluid collect in the bottom of the apparatus.
  • the apparatus can have more than two filters, of varying pore sizes to select for microvesicles of desired sizes, for example.
  • a precipitating agent is added to the biological fluid in the inner chamber.
  • a precipitating agent is added to the filtrate after it has passed through the first filter.
  • the filter membranes utilized by the apparatus of the present invention may be made from any suitable material, provided the filter membrane does not react with the biological fluid, or bind with components within the biological fluid.
  • the filter membranes may be made from a low bind material, such as, for example, polyethersulfone, nylon6, polytetrafluoroethylene, polypropylene, zeta modified glass microfiber, cellulose nitrate, cellulose acetate, polyvinylidene fluoride, regenerated cellulose.
  • the microvesicles of the present invention have a size of about 2 nm to about 5000 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about 1000 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about 500 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about 400 nm as determined by electron microscopy.
  • the microvesicles of the present invention have a size of about 2 nm to about 300 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about 200 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about 100 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about SO nm as determined by electron microscopy.
  • the microvesicles of the present invention have a size of about 2 nm to about 20 nm as determined by electron microscopy. In an alternate embodiment, the microvesicles of the present invention have a size of about 2 nm to about 10 nm as determined by electron microscopy.
  • the microvesicles of the present invention have a molecular weight of at least 100 kDa.
  • Microvesicles isolated according to the methods of the present invention may be used for therapies. Alternatively, microvesicles isolated according to the methods of the present invention may be used for diagnostic tests. Alternatively, the microvesicles of the present invention may be used to alter or engineer cells or tissues. In the case where the microvesicles of the present invention are used to alter or engineer cells or tissues, the microvesicles may be loaded, labeled with RNA, DNA, lipids, carbohydrates, protein, drugs, small molecules, metabolites, or combinations thereof, that will alter or engineer a cell or tissue. Alternatively, the microvesicles may be isolated from cells or tissues that express and/or contain the RNA, DNA, lipids, carbohydrates, protein, drugs, small molecules, metabolites, or combinations thereof.
  • the microvesicles of the present invention can be used in a diagnostic test that detects biomarkers that identify particular phenotypes such as, for example, a condition or disease, or the stage or progression of a disease.
  • Biomarkers or markers from cell-of-origin specific microvesicles can be used to determine treatment regimens for diseases, conditions, disease stages, and stages of a condition, and can also be used to determine treatment efficacy. Markers from cell-of-origin specific microvesicles can also be used to identify conditions of diseases of unknown origin.
  • biomarker refers to an indicator of a biological state. It is a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.
  • One or more biomarkers of microvesicle can be assessed for characterizing a phenotype.
  • the biomarker can be a metabolite, a nucleic acid, peptide, protein, lipid, antigen, carbohydrate or proteoglycan, such as DNA or RNA.
  • the RNA can be mRNA, miRNA, snoRNA, snRNA, rRNAs, tRNAs, siRNA, hnRNA, or shRNA.
  • a phenotype in a subject can be characterized by obtaining a biological sample from the subject and analyzing one or more microvesicles from the sample.
  • characterizing a phenotype for a subject or individual may include detecting a disease or condition (including pre- symptomatic early stage detecting), determining the prognosis, diagnosis, or theranosis of a disease or condition, or determining the stage or progression of a disease or condition. Characterizing a phenotype can also include identifying appropriate treatments or treatment efficacy for specific diseases, conditions, disease stages and condition stages, predictions and likelihood analysis of disease progression, particularly disease recurrence, metastatic spread or disease relapse.
  • a phenotype can also be a clinically distinct type or subtype of a condition or disease, such as a cancer or tumor.
  • Phenotype determination can also be a determination of a physiological condition, or an assessment of organ distress or organ rejection, such as post-transplantation.
  • the products and processes described herein allow assessment of a subject on an individual basis, which can provide benefits of more efficient and economical decisions in treatment.
  • the phenotype can be any phenotype listed in U.S. Patent 7,897,356.
  • the phenotype can be a tumor, neoplasm, or cancer.
  • a cancer detected or assessed by products or processes described herein includes, but is not limited to, breast cancer, ovarian cancer, lung cancer, colon cancer, hyperplastic polyp, adenoma, colorectal cancer, high grade dysplasia, low grade dysplasia, prostatic hyperplasia, prostate cancer, melanoma, pancreatic cancer, brain cancer (such as a glioblastoma), hematological malignancy, hepatocellular carcinoma, cervical cancer, endometrial cancer, head and neck cancer, esophageal cancer, gastrointestinal stromal tumor (GIST), renal cell carcinoma (RCC) or gastric cancer.
  • the colorectal cancer can be CRC Dukes B or Dukes C-D.
  • the hematological malignancy can be B-Cell Chronic Lymphocytic Leukemia, B-Cell Lymphoma-DLBCL, 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 premalignant condition, such as Barrett's Esophagus.
  • the phenotype can also be an inflammatory disease, immune disease, or autoimmune disease.
  • the disease may be inflammatory bowel disease (1BD), Crohn's disease (CD), ulcerative colitis (UC), pelvic inflammation, vasculitis, psoriasis, diabetes, autoimmune hepatitis, Multiple Sclerosis, Myasthenia Gravis, Type I diabetes, Rheumatoid Arthritis, Psoriasis, Systemic Lupus Erythematosis (SLE), Hashimoto's Thyroiditis, Grave's disease, Ankylosing Spondylitis Sjorgen's Disease, CREST syndrome, Scleroderma, Rheumatic Disease, organ rejection, graft versus host disease, Primary Sclerosing Cholangitis, or sepsis.
  • the disease is EB, e.g., RDEB and/or DDEB, junctional EB, EB simplex and/or acquired forms of EB.
  • the phenotype can also be a cardiovascular disease, such as atherosclerosis, congestive heart failure, vulnerable plaque, stroke, or ischemia.
  • the cardiovascular disease or condition can be high blood pressure, 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 disease (HD), Down's syndrome, cerebrovascular disease, Rasmussen's encephalitis, viral meningitis, neuropsychiatric systemic lupus erythematosus (NPSLE), amyotrophic lateral sclerosis, Creutzfeldt- Jacob disease, Gerstmann-Straussler-Scheinker disease, transmissible spongiform encephalopathy, ischemic reperfusion damage (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.
  • 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 assessed in an exosome, to characterize a viral condition.
  • the phenotype can also be a perinatal or pregnancy related condition (e.g., preeclampsia or preterm birth), metabolic disease or condition, such as a metabolic disease or condition associated with iron metabolism.
  • the metabolic disease or condition can also be diabetes, inflammation or a perinatal condition.
  • the phenotype may be detected via any suitable assay method, such as, for example, western blots, ELISA, FCR, and the like.
  • the assay methods may be combined to perform multiplexed analysis of more than one phenotype. Examples of assay methods that may be applied to the microvesicles of the present invention are disclosed in PCT Applications WO2009092386A3 and WO2012108842A1.
  • the RNA may be isolated from the microvesicles of the present invention by the methods disclosed in U.S. Patent 8,021,847.
  • the microvesicles of the present invention are utilized in a diagnostic test for the diseases disclosed in U.S. Patent 7,897,356.
  • the microvesicles of the present invention are utilized in a diagnostic test for cancer according to the methods disclosed in U.S. Patent 8,211,653.
  • the microvesicles of the present invention are utilized in a diagnostic test for cancer according to the methods disclosed in U.S. Patent 8,216,784.
  • the microvesicles of the present invention are utilized in a diagnostic test for prostate cancer according to the methods disclosed in U.S. Patent 8,278,059. In one embodiment, the microvesicles of the present invention are utilized in a diagnostic test for the prognosis for cancer survival according to the methods disclosed in U.S. Patent 8,343,725. [0232] In one embodiment, the microvesicles of the present invention are utilized in a diagnostic test for the prognosis for cancer survival according to the methods disclosed in U.S. Patent 8,349,568.
  • the microvesicles of the present invention are utilized in a diagnostic test for acute lymphomic leukemia according to the methods disclosed in U.S. Patent 8,349,560.
  • the microvesicles of the present invention are utilized in a diagnostic test for acute lymphomic leukemia according to the methods disclosed in U.S. Patent 8,349,561.
  • the microvesicles of the present invention are utilized in a diagnostic test for hepatitis C virus.
  • hepatitis C viral RNA is extracted from the microvesicles of the present invention according to the methods described in U.S. Patent 7,807,438 to test for the presence of hepatitis C virus in a patient.
  • the microvesicles of the present invention are utilized in a diagnostic test for determining the response of a patient to cancer therapy according to the methods disclosed in U.S. Patent 8,349,574.
  • the microvesicles of the present invention are utilized in a diagnostic test for diagnosing malignant tumors according to the methods disclosed in U.S. Patent Application US20120058492A1.
  • the microvesicles of the present invention are utilized in a diagnostic test for diagnosing cancer or adverse pregnancy outcome according to the methods disclosed in U.S. Patent Application US20120238467A1.
  • the microvesicles of the present invention are utilized in a diagnostic test for HIV in urine according to the methods disclosed in U.S. Patent Application US20120214151A1. In one embodiment, the microvesicles of the present invention are utilized in a diagnostic test for cardiovascular events according to the methods disclosed in U.S. Patent Application US20120309041A1. [0240] In one embodiment, the microvesicles of the present invention are utilized in a diagnostic test for cardiovascular events according to the methods disclosed in PCT Application WO2012110099A1.
  • the microvesicles of the present invention are utilized in a diagnostic test for cardiovascular events according to the methods disclosed in PCT Application WO2012126531A1.
  • the microvesicles of the present invention are utilized in a diagnostic test for cardiovascular events according to the methods disclosed in PCT Application WO2013110253A3.
  • the microvesicles of the present invention are utilized in a diagnostic test for melanoma according to the methods disclosed in PCT Application WO2012135844A2.
  • the microvesicles of the present invention are utilized in a diagnostic test for metastatic melanoma by testing microvesicles isolated according to the methods of the present invention for the presence of the biomarker BRAF.
  • the presence of BRAF may be determined via western blot, or, alternatively, by PCR.
  • the metastatic melanoma test is capable of detecting wild type and malignant BRAF.
  • the metastatic melanoma test is capable of detecting splice variants of the malignant BRAF.
  • microvesicles that are utilized in the diagnostic test for metastatic melanoma are isolated using a method comprising the steps outlined in Figure 3.
  • microvesicles are obtained from a patient wishing to be diagnosed for the presence of metastatic melanoma. In one embodiment, the microvesicles are obtained from the patient's plasma.
  • the presence of metastatic melanoma is determined via PCR, using one of the two primer sets below:
  • the presence of metastatic melanoma is determined via western blot, using the mouse auti-BRAF V600E antibody (NewEast Biosciences, Malvern, PA).
  • microvesicles of the present invention can be used as a therapy to treat a disease.
  • the microvesicles of the present invention are used as vaccines according to the methods described in U.S. Patent Application US20030198642 Al.
  • the microvesicles of the present invention are used to modulate or suppress a patient's immune response according to the methods described in U.S. Patent Application US20060116321 Al.
  • the microvesicles of the present invention are used to modulate or suppress a patient's immune response according to the methods described in PCT Patent Application WO06007529A3.
  • the microvesicles of the present invention are used to modulate or suppress a patient's immune response according to the methods described in PCT Patent Application WO2007103572A3.
  • the microvesicles of the present invention are used to modulate or suppress a patient's immune response according to the methods described in U.S. Patent 8,288,172.
  • the microvesicles of the present invention are used as a therapy for cancer according to the methods described in PCT Patent Application WO2011000551A1. In one embodiment, the microvesicles of the present invention are used as a therapy for cancer or an inflammatory disease according to the methods described in U.S. Patent Application US20120315324A1. [0256] In one embodiment, the microvesicles of the present invention are used as a therapy for vascular injury according to the methods described in U.S. Patent 8,343,485.
  • the microvesicles of the present invention are used to deliver molecules to cells.
  • the delivery of molecules may be useful in treating or preventing a disease.
  • the delivery is according to the methods described in PCT Application WO04014954A1.
  • the delivery is according to the methods described in PCT Application WO2007126386A1.
  • the delivery is according to the methods described in PCT Application WO2009115561A1.
  • the delivery is according to the methods described in PCT Application WO2010119256A1.
  • the microvesicles of the present invention are used to promote or enhance wound healing.
  • the wound is a full-thickness burn.
  • the wound is a second-degree burn.
  • the microvesicles of the present invention are used to promote or enhance angiogenesis in a patient.
  • the microvesicles of the present invention are used to promote or enhance neuronal regeneration in a patient.
  • the microvesicles of the present invention are used to reduce scar formation in a patient.
  • the microvesicles of the present invention are used to reduce wrinkle formation in the skin of a patient.
  • the microvesicles of the present invention are used to orchestrate complex tissue regeneration in a patient.
  • the present invention provides an isolated preparation of microvesicles that can promote functional regeneration and organization of complex tissue structures.
  • the present invention provides an isolated preparation of microvesicles that can regenerate hematopoietic tissue in a patient with aplastic anemia.
  • the present invention provides an isolated preparation of microvesicles that can regenerate at least one tissue in a patient with diseased, damages or missing skin selected from the group consisting of: epithelial tissue, stromal tissue, nerve tissue, vascular tissue and adnexal structures.
  • the present invention provides an isolated preparation of microvesicles that can regenerate tissue and/or cells from all three germ layers.
  • the present invention provides an isolated preparation of microvesicles that is used to modulate the immune system of a patient.
  • the present invention provides an isolated preparation of microvesicles that is used to alleviate one or more symptoms of EB (e.g., RDEB and/or DDEB, junctional EB, EB simplex and/or acquired forms of EB) in a patient.
  • EB e.g., RDEB and/or DDEB, junctional EB, EB simplex and/or acquired forms of EB
  • the present invention provides an isolated preparation of microvesicles that is used to increase collagen VII expression in a patient having EB (e.g., RDEB and/or DDEB, junctional EB, EB simplex and/or acquired forms of EB).
  • EB e.g., RDEB and/or DDEB, junctional EB, EB simplex and/or acquired forms of EB.
  • the present invention provides an isolated preparation of microvesicles that enhances the survival of tissue or cells that is transplanted into a patient.
  • the patient is treated with the isolated preparation of microvesicles prior to receiving the transplanted tissue or cells.
  • the patient is treated with the isolated preparation of microvesicles after receiving the transplanted tissue or cells.
  • the tissue or cells is treated with the isolated preparation of microvesicles.
  • the tissue or cells is treated with the isolated preparation of microvesicles prior to transplantation.
  • the present invention provides an isolated preparation of microvesicles containing at least one molecule selected from the group consisting of RNA, DNA, lipid, carbohydrate, metabolite, protein, and combination thereof from a host cell
  • the host cell is engineered to express at least one molecule selected from the group consisting of RNA, DNA, lipid, carbohydrate, metabolite, protein, and combination thereof.
  • the isolated preparation of microvesicles containing at least one molecule selected from the group consisting of RNA, DNA, lipid, carbohydrate, metabolite, protein, and combination thereof from a host cell is used as a therapeutic agent.
  • MVs are preferably combined with a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the canier(s) should be “acceptable” in the sense of being compatible with the other ingredients of the formulations and not deleterious to the recipient.
  • Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.
  • EV compositions of the present invention can comprise at least one of any suitable excipients, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • Pharmaceutically acceptable excipients are preferred.
  • Non-limiting examples of, and methods of preparing such sterile solutions 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 can be routinely selected that are suitable for the mode of administration, solubility and/or stability of EV composition as well known in the art or as described herein.
  • compositions include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
  • amino acid/antibody molecule components which can also function in a 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 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, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like.
  • Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.
  • EV compositions can also include a buffer or a pH adjusting agent; typically, 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, or phosphate buffers.
  • EV compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycols, 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).
  • polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-P-cyclodextrin), polyethylene glycols, flavoring
  • compositions comprising MVs in a pharmaceutically acceptable formulation.
  • Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresoL, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent.
  • Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, 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, 4.0, 4.3, 4.S, 4.6, 4.7, 4.8, 4.9, or any range or value therein.
  • Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, or 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, or 2.5%), 0.001-0.5% thimerosal (e.g., 0.005 or 0.01%), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, or 1.0%), 0.0005-1.0% alkylparaben(s) (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.
  • 0.1-2% m-cresol e.g., 0.2, 0.3, 0.4,
  • compositions containing MVs as disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method.
  • a pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration.
  • routes of administration are intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, and rectal administration.
  • IV intravenous
  • transdermal intradermal
  • topical transmucosal
  • rectal administration A preferred route of administration for MVs
  • Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Remington's Pharmaceutical Sciences (1990) supra.
  • Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as EDTA
  • buffers such as acetates, citrates or phosphates
  • 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.
  • compositions are preferably sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.
  • compositions of this invention may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, and liposomes.
  • liquid solutions e.g., injectable and infusible solutions
  • dispersions or suspensions e.g., 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.
  • the preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraocular, intraperitoneal, intramuscular).
  • the preparation is administered by intravenous infusion or injection.
  • the preparation is administered by intramuscular or subcutaneous injection.
  • parenteral administration and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, subcutaneous, intraarterial, intrathecal, intracapsular, intraorbital, intravitreous, intracardiac, intradermal, intraperitoneal, transtracheal, inhaled, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the present invention provides a kit, comprising packaging material and at least one vial comprising a solution of MVs with the prescribed buffers and/or preservatives, optionally in an aqueous diluent.
  • the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative.
  • Preservatives include those selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl 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 yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
  • excipients e.g. isotonicity agents, buffers, antioxidants, preservative enhancers
  • An isotonicity agent such as glycerin, is commonly used at known concentrations.
  • a physiologically tolerated buffer can be added to provide improved pH control.
  • the formulations can cover a wide range of pHs, such as from about pH 4.0 to about pH 10.0, from about pH 5.0 to about pH 9.0, or about pH 6.0 to about pH 8.0.
  • additives such as a pharmaceutically acceptable solubilizers like TWEEN 20 (polyoxyethylene (20) sorbitan monolaurate), TWEEN 40 (polyoxyethylene (20) sorbitan monopalmitate), TWEEN 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block co-polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation. These additives are particularly useful if a pump or plastic container is used to administer the formulation. The presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
  • a pharmaceutically acceptable solubilizers like TWEEN 20 (polyoxyethylene (20) sorbitan monolau
  • MVs are administered by pulmonary delivery, e.g., by intranasal administration, or by oral inhalative administration.
  • Pulmonary delivery may be achieved via a syringe or an inhaler device (e.g., a nebulizer, a pressurized metered-dose inhaler, a multi-dose liquid inhaler, a thermal vaporization aerosol device, a dry powder inhaler or the like).
  • a syringe or an inhaler device e.g., a nebulizer, a pressurized metered-dose inhaler, a multi-dose liquid inhaler, a thermal vaporization aerosol device, a dry powder inhaler or the like.
  • Suitable methods for pulmonary delivery are well-known in the art and are commercially available.
  • any of the formulations described above can be stored in a liquid or frozen form and can be optionally subjected to a preservation process.
  • EVs described herein are used to deliver one or more bioactive agents to a target cell.
  • 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 a cell and/or in a cellular fluid and are added during the purification and/or preparation of 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 and the like, that EVs described herein are exposed to during one or more purification and/or preparation steps described herein.
  • a bioactive agent is a collagen VII protein, a collagen VII mRNA, a STAT3 signalling activator (e.g., an interferon, epidermal growth factor, interleukin-S, interleukin-6, a MAP kinase, a c- src non-receptor tyrosine kinase or another molecule that phosphorylates and/or otherwise activates STAT3) and/or a canonical Wnt activator (see, e.g., McBride et al. (2017) Transgenic expression of a canonical Wnt inhibitor, kallistatin, is associated with decreased circulating CD19+ B lymphocytes in the peripheral blood. International Journal of Hematology, 1-10. DOI: 10.1007/sl2185-017-2205-5, incorporated herein by reference in its entirety).
  • a bioactive agent is one or more pharmaceutical compounds known in the art.
  • Example 1 Isolation of Microvesicles from Cell culture Medium by Ultracentrifugation
  • FIG. 1 This example illustrates the typical method by which microvesicles are isolated from cell culture medium, or any biological fluid.
  • An outline of the method to isolate microvesicles from cell culture medium is shown in Figure 1.
  • the cells are cultured in medium supplemented with microvesicle-free serum (the serum may be depleted of microvesicles by ultracentrifugation, filtration, precipitation, etc.).
  • the medium is removed and transferred to conical tubes and centrifuged at 400 x g for 10 minutes at 4 °C to pellet the cells.
  • the supernatant is transferred to new conical tubes and centrifuged at 2000 x g for 30 minutes at 4 °C to further remove cells and cell debris. This may be followed by another centrifugation step (e.g. 10000 x g for 30 minutes to further deplete cellular debris and/or remove larger microvesicles).
  • the resultant supernatant is transferred to ultracentrifuge tubes, weighed to ensure equal weight and ultracentrifuged at 70000+ x g for 70 minutes at 4 °C to pellet the microvesicles.
  • Example 2 Isolation of Microvesicles from Cell culture Medium by die Methods of the
  • FIG. 2 An outline of the method to isolate microvesicles from medium that has cultured cells is shown in Figures 2 and 3.
  • the cells are cultured in medium supplemented with microvesicle-free serum (the serum may be depleted of microvesicles by ultracentrifugation, filtration, precipitation, etc.)- After culturing the cells for a period of time, the medium is removed and transferred to conical tubes and centrifuged at 400 x g for 10 minutes at 4 °C to pellet the cells.
  • the supernatant is transferred to new conical tubes and centrifuged at 2000 x g for 30 minutes at 4 °C to further remove cells and cell debris. This may be followed by another centrifugation step (e.g. 10000 x g for 30 minutes to further deplete cellular debris and remove larger particles).
  • Microvesicles are then precipitated at 4 °C using 8.5% w/v PEG 6000 and 0.4 M NaCL This mixture is spun at 10000 x g at 4 °C for 30 minutes. The supernatant is removed and the pellet is resuspended in an appropriate buffer (e.g. PBS). It may be used for immediate downstream reactions or further purified. Further purification procedures can include the use of centrifugal filters (e.g. MWCO of 100 kDa), immunoaffinity, HPLC, tangential flow filtration, phase separationpartitioning, microfluidics, etc.
  • centrifugal filters e.g. MWCO of 100 kDa
  • immunoaffinity e.g. MWCO of 100 kDa
  • HPLC tangential flow filtration
  • phase separationpartitioning e.g., phase separationpartitioning, microfluidics, etc.
  • Example 3 Isolation of Microvesicles from Culture Medium Conditioned Using Bone Marrow Derived Stem Cells by the Methods of the Present Invention
  • the mononuclear cells were collected at the interface, washed three times in phosphate-buffered saline (PBS) supplemented with 2% FBS (Atlanta Biologies, Atlanta, GA) , and resuspended in MSC medium consisting of alpha- minimum essential medium (a-MEM) (Mediatech Inc., Manassas, VA) and 20% FBS, 1% Penicillin/Streptomycin (Lonza, Allendale, NJ) and 1% glutamine (Lonza).
  • PBS phosphate-buffered saline
  • FBS alpha- minimum essential medium
  • cryopreserved MSC were thawed at 37 °C and immediately cultured in a-MEM supplemented with 20% microvesicle-free fetal bovine serum and 1% penicillin streptomycin/glutamine at 37 °C in 95% humidified air and 5% CO 2 . They were expanded similar to above.
  • the cells were grown in the multi-flasks until 80-90% confluence was reached.
  • the flasks were rinsed twice with PBS and a -MEM supplemented with 1% Penicillin/Streptomycin/Glutamine was added.
  • the conditioned medium transferred to 50mL conical centrifuge tubes (Thermo Fisher Scientific Inc., Weston, FL) and immediately centrifuged at 400 x g for 10 minutes at 4 °C to pellet any non-adherent cells.
  • the supernatant was transferred to new 50mL conical centrifuge tubes and centrifuged at 2000 x g for 30 minutes at 4 °C to further remove cells and cell debris.
  • the supernatants were collected and placed into 250 ml sterile, polypropylene disposable containers (Corning, Corning, NY). To the supernatant, RNase and protease free polyethylene glycol average molecular weight 6000 (Sigma Aldrich, Saint Louis, MO) at 8.5 w/v % and sodium chloride (final concentration 0.4 M) were added. The solution was placed in a cold room at 4 °C overnight with rocking. The solution was transferred to 50 mL conical centrifuge tubes and centrifuged at 10000 x g at 4 °C for 30 minutes. The supernatant was decanted and the microvesicle enriched pellet resuspended in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • microvesicle enriched solution was transferred to Amicon ultra-15 centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes.
  • the filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • the concentrated sample was recovered (approximately 200 ⁇ l) from the bottom of the filter device.
  • Protein concentration was determined by the micro BSA Protein assay kit (Pierce, Rockford, IL) and the enriched microvesicle solution was stored at -70 degrees or processed for downstream use (e.g. protein, RNA, and DNA extraction).
  • Example 4 Isolation of Microvesicles from Plasma by the Methods of the Present
  • o-MEM Sterile alpha-minimum essential medium
  • microvesicle enriched solution was transferred to Amicon ultra- 15 centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes.
  • the filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • the concentrated sample was recovered (approximately 200-400 ⁇ l) from the bottom of the filter device.
  • Protein concentration was determined by the micro BSA Protein assay kit (Pierce, Rockford, IL) and the enriched microvesicle solution was stored at -70 degrees or processed for downstream use (e.g. protein, RNA, and DNA extraction).
  • Example 5 Isolation of Micro vesicles from Bone Marrow Aspirate by the Methods of the Present Invention
  • Pig bone marrow was isolated from the iliac crest. The skin area was carefully cleaned with povidine iodine 7.5% and isopropanol 70%. An 11-gauge 3 mm trocar (Ranafac, Avon, MA) was inserted into the iliac crest. An aspiration syringe with loaded with 5000 - 1000 units of heparin to prevent clotting of the marrow sample. Approximately 20-25 ml of marrow was aspirated and the solution transferred to 50 ml conical centrifuge tubes. Alternatively, normal donor human bone marrow (approximately 50 ml) was acquired from AllCells LLC (Emeryville, CA, URL: allcells.com).
  • the 50 ml conical tubes were centrifuged at 400 x g for 30 minutes at room temperature.
  • the supernatant (the acellular portion) was collected (approximately 10-12 ml per 50 ml) and placed into new 50 ml conical centrifuge tubes (Thermo Fisher Scientific Inc., Weston, FL).
  • Sterile alpha-minimum essential medium (a- MEM) (Mediatech Inc., Manassas, VA) was added in a 1: 10 (bone marrow supernatant to medium) ratio.
  • the solution was transferred to new 50 ml conical tubes and centrifuged at 2000 x g for 30 minutes at 4 °C.
  • the supernatant was transferred to new 50 ml conical tubes and to this solution, RNase and protease free polyethylene glycol average molecular weight 6000 (Sigma Aldrich, Saint Louis, MO) at 8.S w/v % and sodium chloride (final concentration 0.4 M) were added.
  • the solution was placed in a cold room at 4 °C overnight with rocking. The solution was centrifuged at 10000 x g at 4 °C for 30 minutes. The supernatant was decanted and the microvesicle enriched pellet resuspended in phosphate-buffered saline (PBS). The microvesicle enriched solution was transferred to Amicon ultra-lS centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes. The filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • PBS phosphate-buffered saline
  • the concentrated sample was recovered (approximately 200-400 ⁇ l) from the bottom of the filter device.
  • Protein concentration was determined by the micro BSA Protein assay kit (Pierce, Rockford, IL) and the enriched microvesicle solution was stored at -70 degrees or processed for downstream use (e.g. protein, RNA, and DNA extraction).
  • the cellular portion was collected and processed for mesenchymal stem isolation or for bone marrow complete isolation.
  • Example 6 Isolation of Microvesicles from Urine by the Methods of the Present
  • the 50 ml conical tubes were centrifuged at 400 x g for 30 minutes at 4°C. The supernatant was removed and placed into new 50 ml conical centrifuge tubes (Thermo Fisher Scientific Inc., Weston, FL). The solution was transferred to new 50 ml conical tubes and centrifuged at 2000 x g for 30 minutes at 4°C. The supernatant was transferred to new 50 ml conical tubes and to this solution, RNase and protease free polyethylene glycol average molecular weight 6000 (Sigma Aldrich, Saint Louis, MO) at 8.5 w/v % and sodium chloride (final concentration 0.4 M) were added.
  • RNase and protease free polyethylene glycol average molecular weight 6000 Sigma Aldrich, Saint Louis, MO
  • sodium chloride final concentration 0.4 M
  • the solution was placed in a cold room at 4 °C overnight with rocking. The solution was centrifuged at 10000 x g at 4 °C for 30 minutes. The supernatant was decanted and the microvesicle enriched pellet resuspended in phosphate-buffered saline (PBS). The microvesicle enriched solution was transferred to Amicon ultra-15 centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes. The filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • PBS phosphate-buffered saline
  • the concentrated sample was recovered (approximately 200-400 ⁇ l) from the bottom of the filter device.
  • Protein concentration was determined by the micro BSA Protein assay kit (Pierce, Rockford, IL) and the enriched microvesicle solution was stored at -70 degrees or processed for downstream use (e.g. protein, RNA, and DNA extraction).
  • Example 7 Isolation of Microvesicles from Medium from a Long-Term Culture of Bone
  • Bone marrow was obtained from an aspirate (see Example 1) and red blood cells were lysed using 0.8% ammonium chloride solution containing 0.1 mM EDTA (Stem Cell Technologies, Vancouver, BC). The nucleated cells were pelleted under a fetal bovine serum (Atlanta Biologies, Atlanta, GA) cushion at 400 X g for 5 minutes. Nucleated cells were washed in McCoy's 5a media (Mediatech Inc., Manassas, VA) by pelleting at 400 x g for 5 min. The cells were resuspended in culture media at a density of 1 x 10 6 cells/ml and plated in 25, 75 or 225 cm 2 flasks (Coming, Corning, NY).
  • McCoy's 5a media Mediatech Inc., Manassas, VA
  • Culture media consisted of McCoy's 5a media, 1% sodium bicarbonate (Life technologies, Carlsbad, CA), 0-4% MEM non-essential amino acids (Life technologies), 0- 8% MEM essential amino acids (Life technologies), 1% L-glutamine (Lonza, Allendale, NJ), 0.1 ⁇ Hydrocortisone (Life technologies), 1% penicillin/streptomycin (Lonza), 12-5% fetal calf serum (Atlanta Biologies) and 12-5% horse serum (Stem Cell Technology). The cultures were incubated at 33°C and 5% CO 2 . Feeding was performed weekly by adding half of the original volume of media without removing any media during the first nine weeks of culture. If the cultures were grown beyond nine weeks, the volume of culture media was reduced to the original volume and half the original volume of fresh media was added each week
  • the supernatant was collected and placed into 2S0 ml sterile, polypropylene disposable containers (Corning, Corning, NY). To the supernatant, RNase and protease free polyethylene glycol average molecular weight 6000 (Sigma Aldrich, Saint Louis, MO) at 8.S w/v % and sodium chloride (final concentration 0.4 M) was added. The solution was placed in a cold room at 4 °C overnight with rocking. The solution was transferred to SO mL conical centrifuge tubes and centrifuged at 10000 x g at 4 °C for 30 minutes.
  • RNase and protease free polyethylene glycol average molecular weight 6000 Sigma Aldrich, Saint Louis, MO
  • sodium chloride final concentration 0.4 M
  • the supernatant was decanted and the microvesicle enriched pellet resuspended in phosphate-buffered saline (PBS).
  • the microvesicle enriched solution was transferred to Amicon ultra- 15 centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes.
  • the filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • the concentrated sample was recovered (approximately 200 ⁇ l) from the bottom of the filter device.
  • Protein concentration was determined by the micro BSA Protein assay kit (Pierce, Rockford, IL) and the enriched microvesicle solution stored at -70 degrees or processed for downstream use (e.g. protein, RNA, and DNA extraction).
  • Example 8 Analysis of the Micro vesicles of the Present Invention
  • Figure 5 shows electron micrographs of microvesicles derived from human bone marrow- derived mesenchymal stem cells isolated by the ultracentrifuge method described in Example 1 (panels A&B) and according to the methods of the present 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 ultracentrifuge method described in Examples 1 (panels A&B) and according to the methods of the present invention as described in Example 3 (panels C&D).
  • Figure 7 shows electron micrographs of microvesicles derived from murine bone marrow-derived mesenchymal stem cells isolated by the ultracentrifuge method described in Examples 1 (panels A&B) and according to the methods of the present invention as described in Example 3 (panels C&D).
  • Figures 5 to 7 illustrate the differences between microvesicles isolated by the methods of the present invention compared to ultracentrifuge isolation.
  • the microvesicles isolated according to the methods of the present invention have borders that are smoother, uncorrugated and appear more "intact.”
  • Figure 8 shows electron micrographs of microvesicles isolated from human plasma according to the methods of the present invention.
  • the heterogeneity of the shapes and sizes achieved with PEG isolation suggests that all types of microvesicles were isolated. Similar heterogeneity was observed in microvesicles from porcine plasma ( Figure 9) and human urine ( Figure 10) that were isolated according to the methods of the present invention.
  • PBS Phosphate buffered saline
  • MSC medium depleted of microvesicles showed little growth. See Figure 12.
  • the removed medium was replaced with fresh culture medium (10% FBS) containing either microvesicles (PEG or ultracentrifuge derived), PBS, or MSC conditioned medium depleted of microvesicles.
  • the scratched area was monitored by collecting digitized images immediately after the scratch and 3 days after treatment. Digitized images were captured with an inverted 1X81 Olympus microscope (Olympus America, Center Valley, PA, URL: olympusamerica.com) and ORCA-AG Hamamatsu digital camera (Hamamatsu Photonics K.K., Hamamatsu City, Shizuoka Pref., Japan, URL: hamamatsu.com).
  • microvesicles isolated according to the methods of the present invention showed the greatest in migration (essentially closing the wound), followed by microvesicles derived from ultracentrifuge.
  • the controls (PBS) and MSC conditioned medium depleted of microvesicles (Depleted) showed little migration. See Figure 13.
  • Figure 14 shows the effects of microvesicles on cell migration fibroblasts derived from a diabetic foot ulcer. Similar to the results in Figure 13, microvesicles isolated according to the methods of the present invention evoked the greatest migration, followed by microvesicles isolated using the ultracentrifuge method described in Example 1. The controls (PBS) and MSC conditioned medium depleted of microvesicles (Depleted) showed little migration.
  • Human MSC microvesicles isolated from conditioned medium according to the methods of the present invention were labeled with the phospholipid cell linker dye PKH-26 (red) per manufacturer's instruction (Sigma- Aldrich, St. Louis, MO).
  • Normal skin fibroblasts were labeled with Vybrant-Dio (Life technology) per manufacturer instructions.
  • Normal skin fibroblasts were plated on fibronectin (Sigma-Aldrich) coated 4-well Nunc* Lab-Tek* ⁇ Chamber Slides (Thermo Fisher Scientific Inc., Weston, FL) (5 x 10 cells per well). Cells were stained with the nuclear dye Hoechst 33342 (Life technology) per manufacturer's instructions.
  • Dio labeled fibroblasts were treated with PKH-26 labeled microvesicles for 24 hours. Images were captured with an inverted 1X81 Olympus microscope and ORCA-AG Hamamatsu digital camera. Normal dermal fibroblasts (stained with the green lipid membrane dye Dio) demonstrated uptake of PKH-26 labeled human MSC MV isolated by PEG precipitation in a peri-nuclear location. See Figures IS and 16. In Figure 16, the microvesicles are seen in a peri-nuclear location.
  • Example 11 Use of the Microvesicles of the Present Invention as a Diagnostic for
  • Normal dermal fibroblasts were plated at a density of 1 x 10 s cells/well in a 6-well tissue culture plate (BD Biosciences). Fibroblasts were serum starved overnight and treated with PBS (control), 10 micrograms of either microvesicles isolated according to the methods of the present invention from plasma obtained from a patient suffering from rheumatoid arthritis (Human Plasma MV PEG Precipitation); microvesicles isolated according to the methods of the present invention from medium conditioned with bone marrow-derived mesenchymal stem cells (Human hMSC MV PEG Precipitation); micro vesicles isolated according via ultracentrifugation from medium conditioned with bone marrow-derived mesenchymal stem cells (Human hMSC MV ultracentrifugation); PBS control; and a depleted medium control (hMSC conditioned medium depleted of MV). The amount of STAT3 phosphorylation observed in the fibroblasts was greater in the
  • Example 12 Use of the Microvesicles of the Present Invention as a Diagnostic for
  • BRAF is a human gene that makes a protein called B-Raf. More than 30 mutations of the BRAF gene associated with human cancers have been identified. We have designed per primers to amplify the mutated form of BRAF that is linked to metastatic melanoma. The mutation is a T1799A mutation in exon IS in BRAF. This leads to valine (V) being substituted for by glutamate (E) at codon 600 (now referred to as V600E). The presence of this mutation is required for treatment by the BRAF inhibitor Vemurafenib.
  • the SK-Mel28 cell line, obtained from ATCC (Washington DC, Maryland) is known to have the T1799A mutation in exon 15 in BRAF. Microvesicles, isolated according to the methods of the present invention were obtained from medium conditioned by a 3 day incubation in EMEM (ATCC) + 10% serum (Atlanta Biologies, Atlanta, Georgia).
  • RNA from SK-MEL28 cells and microvesicles were reverse transcribed using iScriptTM Reverse Transcription Supermix (BioRad, Hercules, CA). A 2 ml aliquot was used for PCR utilizing Platinum® PCR SuperMix (Life technology) per manufacturer's instructions. In addition, 80 ng of DNA from SK-MEL28 cells and microvesicles was used for PCR utilizing Platinum® PCR SuperMix per manufacturer's instructions. PCR products were run on a 3% agarose gel and visualized by Bio-Rad's gel-doc system. The results are shown in Figure 18.
  • the primers used were:
  • samples of the microvesicles were lysed in RIP A buffer and protein concentration estimated by the microBSA assay kit. Approximately 50 microgram were loaded in each lane and the membranes were probed overnight (1:1000) by mouse anti-BRAF V600E antibody (NewEast Biosciences, Malvern, PA). Secondary antibody, goat anti-mouse (Pierce) was applied at 1 : 10000 dilution for 1 hour. The Western blot shows BRAF V600E detection in SKMEL28 cell and MV lysate.
  • Example 13 Isolation of Microvesicles from Medium Conditioned Using a Culture of GFP- Labeled Bone Marrow-Derived Mesenchymal Stem Cells by the Methods of the
  • mice expressing the 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 Green Fluorescent Protein
  • GFP-Mice (approximately 3-4 weeks of age) were euthanized by CO 2 asphyxiation. The limbs were cut above the hip and below the ankle joint. The hind limbs were harvested and skin, muscle, and all connective tissue was removed. The bones were then placed in a dish of ice cold sterile IX PBS and washed several times in PBS. The ends of each bone were snipped off with scissors. A 10 cc syringe with warmed medium ( ⁇ -MEM supplemented with 20% fetal bovine serum and 1% penicillirj/streptomycirj/glutamine) was forced through the bone shaft to extract all bone marrow into a 150 mm plate.
  • warmed medium ⁇ -MEM supplemented with 20% fetal bovine serum and 1% penicillirj/streptomycirj/glutamine
  • the cell mixture was pipetted several times to dissociate cells and the cell suspension was passed through a cell strainer (70
  • cryopreserved GFP Mouse-MSC's were thawed at 37°C and immediately cultured in ⁇ -MEM supplemented with 20% fetal bovine serum and 1% penicUlin/streptomycin glutamine at 37°C in 95% humidified air and 5% CO 2 . They were expanded similar to above.
  • the cells were grown in the flasks until 100% confluence was reached (approximately 1 week).
  • the supernatant were transferred to SO ml_ conical centrifuge tubes (Thermo Fisher Scientific Inc., Weston, FL) and immediately centrifuged at 400 x g for 10 minutes at 4 °C to pellet any non-adherent cells.
  • the supernatant was transferred to new SO mL conical centrifuge tubes and centrifuged at 2000 x g for 30 minutes at 4 °C to further remove cells and cell debris.
  • the supernatants were collected and placed into 2S0 ml sterile, polypropylene disposable containers (Corning, Corning, NY).
  • microvesicle enriched solution was transferred to Amicon ultra- IS centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes.
  • the filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • the concentrated sample was recovered (approximately 200-400 ⁇ l) from the bottom of the filter device.
  • Protein concentration was determined by the micro BSA Protein assay kit (Pierce, Rockford, LL) and the enriched microvesicle solution was stored at -70 degrees or processed for downstream use (e.g. protein, RNA, and DNA extraction).
  • Example 14 Use of the Microvesicles of the Present Invention as a Therapy to Promote or Enhance Wound Healing
  • Microvesicles were isolated from culture medium conditioned using autologous bone marrow-derived mesenchymal stem cells, either according to the methods described in Example 1 (the "conventional ultracentrifugation method"), or by the methods described in Example 3. 30 micrograms of microvesicles were administered to the wounds by local injection at the time of wounding and at Days 1 and 2. Controls were treated with saline or allowed to heal air exposed. After 5 days, the animals were euthanized, and the wounds examined.
  • Figure 22 shows the histology of the wounds S days post-wounding. At 5 days, wounds treated with microvesicles isolated according to the methods of the present invention (i.e., according to the methods described in Example 3) appeared smaller than saline controls, air exposed controls and wounds treated with microvesicles prepared by ultracentrifugation. The wounds treated with microvesicles prepared by ultracentrifugation showed an enhanced inflammatory response, compared to those treated with microvesicles prepared according to the methods of the present invention and both controls.
  • microvesicles were isolated from culture medium conditioned using autologous bone marrow-derived mesenchymal stem cells, either according to the methods described in Example 1 (the "conventional ultracentrifugation method"), or by the methods described in Example 3. 30 micrograms of microvesicles were administered to the wounds by local injection at the time of wounding and at days 1 and 2. Controls were treated with saline or allowed to heal air exposed.
  • Figure 23 illustrates the difference in inflammation at Day 7 post- wounding between wounds treated with microvesicles prepared by ultracentrifugation, microvesicles prepared according to the methods of the present invention and an air exposed control. Microscopically, abscess formation was seen in both full thickness and burn wounds treated with microvesicles prepared by ultracentrifugation. Without intending to be bound by scientific theory, the inflammation noted with microvesicles prepared by ultracentrifugation was thought to be due to damaged microvesicles, which can easily stimulate an inflammatory cascade. The microvesicles of the present invention may also confer additional benefits by including additional particles.
  • Figure 24 shows a second degree porcine burn wound treated with microvesicles isolated by the methods of the present invention 28 days after burn injury. There is a significant remodeling of collagen, with the appearance of ground substance. These findings are indicative of dermal remodeling with collagen type ⁇ formation. There is also dermal epidermal induction resulting in a thickened epidermis that appears well anchored to the dermis. These findings are not observed in scar formation and are more consistent with dermal regeneration. An epidermis forming over a scar is easily subject to re-injury due to the inability to anchor well to a scarred dermis.
  • Figure 25 shows a second degree porcine burn wound treated with saline 28 days after burn injury. There is minimal dermal regeneration with a flattened epidermis. The lack of significant rete ridge formation is highly suggestive of an inadequately anchored epidermis. These findings are much more indicative of scar formation with the risk of continued injury.
  • Figure 26 shows a full thickness porcine wound treated with microvesicles isolated according to the methods of the present invention 28 days after injury.
  • a nerve illustrated by the arrows
  • the nerve grown is accompanied by an angiogenic response (circled areas).
  • the nerve appears to be a developed structure and is not due to simple axon sprouting. This is a unique finding and has never been reported and was also not observed in control wounds or wounds treated with microvesicles prepared by ultracentrifugation.
  • These observations are highly indicative of complex tissue regeneration with the ability to generate mature elements from all germ layers including epidermis, stroma, vasculature and nervous tissue.
  • These methods then appear to be widely applicable to the treatment of numerous conditions including traumatic, inflammatory, neoplastic and degenerative disorders of ectodermal, endodermal and mesodermal derived tissues.
  • Figure 27 shows a full thickness porcine wound treated with microvesicles isolated by the methods of the present invention 28 days after injury.
  • This Figure illustrates the observations described in Figure 26 at greater magnification.
  • A) the nerve growth appears to be following a path related to the angiogenic response. This finding is interesting as nerve growth is well known to follow angiogenesis in embryologic development. Again, these findings are indicative of tissue regeneration.
  • B) shows the nerve at higher power.
  • C) better illustrates the angiogenesis adjacent to the nerve growth.
  • Example 15 Use of the Microvesicles of the Present Invention as a Therapy to Repopulate Bone Marrow and Regenerate Complex Structures
  • C57/CJ6 mice were lethally irradiated with two cycles of 400 cGy gamma irradiation to ablate their host bone marrow progenitors. After irradiation, mice were treated in an approximately 2cm area with an ablative fractional Erbium: YAG laser. After laser treatment, a plastic chamber was adhered to the skin, and bone marrow derived cells obtained from a syngeneic GFP ⁇ +>transgenic mouse were added to the chamber. The GFP + bone marrow cells included, freshly harvested total bone marrow cells, lineage negative selected bone marrow cells, mesenchymal stem cells and bone marrow complete cultured cells (as described in this application).
  • Microvesicles secreted by the delivered cells are likely responsible for the recovery of the host bone marrow leading to survival of these animals.
  • fresh bone marrow which includes lineage negative cells
  • mesenchymal stem cells produce ample amounts of microvesicles that could accomplish this effect.
  • C57/CJ6 (GFP-) mice were lethally irradiated with two cycles of 400 cGy gamma irradiation to inhibit their hair growth and partially ablate their bone marrow. After irradiation, the backs of the mice were shaved and the mice were then in an approximately 2cm area with an ablative fractional Erbium: YAG laser.
  • the supernatant was decanted and the microvesicle enriched pellet resuspended in phosphate-buffered saline (PBS).
  • PBS phosphate-buffered saline
  • the microvesicle enriched solution was transferred to Amicon ultra- 15 centrifugal filter units (nominal molecular weight limit 100 kDa) (Millipore, Billerica, MA) and centrifuged at 5000 x g for 30 minutes.
  • the filter units were washed with phosphate-buffered saline and centrifuged again at 5000 x g for 30 minutes.
  • the concentrated sample was recovered (approximately 200-400 ⁇ l) from the bottom of the filter device.
  • Angiogenesis assay Angiogenesis was measured using an endothelial tube formation assay (Invitrogen Life Technologies, Grand Island, NY). Cryopreserved primary Human Umbilical Vein Endothelial cells (HUVEC) (Invitrogen Life Technologies) were grown in a 75-cm tissue-culture flask for 6 days in Medium 200PRF supplemented with 2% low serum growth supplement (Invitrogen Life Technologies). Cells were then plated at a density of 3 x 10 ⁇ 4>in a 24 well tissue culture plate containing medium without supplement. HUVEC Cells were subsequently treated with bone marrow microvesicles (approximately 100 ⁇ g). PBS was used as the vehicle control.
  • HUVEC Human Umbilical Vein Endothelial cells
  • Example 17 EV-Mediated Delivery of Bioactive Materials to Target Cells
  • EVs described herein are useful for delivering one or more bioactive agent (e.g., collagen VII proteins or peptides, collagen VII mRNA, STAT3-signalling activators, canonical Wnt activators and the like) to a target cell.
  • one or more bioactive agent e.g., collagen VII proteins or peptides, collagen VII mRNA, STAT3-signalling activators, canonical Wnt activators and the like
  • This example demonstrates delivery of EVs to RDEB fibroblast cells that were deficient in COL7A1 expression compared to wild-type fibroblast cells.
  • the EVs stimulated collagen VII expression in the RDEB fibroblasts.
  • the EVs also stimulated the expression of markers related to wound healing in the RDEB fibroblasts.
  • Figure 44 shows the validation of an in vitro cell line derived from an infant diagnosed as having RDEB (Hallopeau-Siemens type). Vesicle exchange was observed between BM-MSCs and RDEB fibroblasts ( Figure 45). Collagen VII was co-isolated with BM-MSC EVs ( Figure 46), and COL7A1 mRNA was enriched in MB-MSC EVs ( Figure 47).
  • RDEB fibroblasts were treated with BM-MSC EVs on day 1, were washed on day three, and showed an increase in collagen VII expression on day six (Figure 48).
  • a chemoselective ligation assay (utilizing "click iT” reaction chemistry) revealed the production of new collagen VII from RDEB fibroblasts following co-treatment with BM- MSC EVs ( Figure 49).
  • BM-MSC EVs were shown to increase in vitro surrogate assays related to wound healing (e.g., proliferation and trypsin-resistance) of RDEB fibroblasts ( Figure 50).
  • BM-MSCs that were delivered in saline to burn patients in a clinical trial were shown to secrete large numbers of EVs (CD63 positive) in saline within hours (shown, 4 hours) ( Figure 51).
  • a model of BM-MSC-mediated wound healing is set forth in Figure 52.
  • Example 18 Extracellular Vesicles Derived From Bone Marrow Mesenchymal Stem Cells in the Treatment of Recessive Dystrophic Epidermolysis Bullosa
  • BM-MSCs bone marrow-derived mesenchymal stem cells
  • RDEB Recessive Dystrophic Epidermolysis Bullosa
  • EVs extracellular vesicles derived from BM-MSCs
  • Col VII collagen VII
  • Treating with EVs has many advantages over cellular therapy including much lower risks of genetic instability and malignant transformation.
  • IND Investigational New Drug
  • Aim 2 Conduct an open-label, dose-escalation clinical trial of topical, allogeneic BM-MSC- derived EVs in the treatment of wounds in 30 RDEB patients
  • Dosing will be based on the Pi's successful clinical trial of topically applied BM- MSCs to burn patients. There will be 3, serially escalating, dosing groups with 10 patients to complete each group. The dosing schedule will be first dost at treatment day 0 with three additional doses given monthly (total of four treatments over three months). Primary outcomes will assess safety and tolerability of topically applied BM-MSC EV; secondary outcomes will assess wound healing, pain, itch, and cosmesis (including pigment, scar assessment and evidence of tissue regeneration). Integrium Contract Research Organization will assist in the clinical trial.
  • BM-MSC EV product characteristics will be defined. These parameters include protein concentrations, EV size distributions (e.g., using NanoSight NS300), surface marker characterization, removal of reagents used during manufacturing, and stability testing of the product. Using mass spectrometry and RNA sequencing, we will define protein and RNA cargo contents of several BM-MSC EV donors, correlating cargo with functional assay performance.
  • BM-MSC EV functional activities will be defined on recipient RDEB cells, including in vitro studies to establish potency for wound healing and reversal of phenotype, including RDEB fibroblast proliferation and trypsin resistance assays. Additionally, endothelial angiogenesis assays will be examined in vitro.
  • Aim 2 Conduct an open-label, dose-escalation, clinical trial of topical allogeneic BM- MSC-derived EVs in the treatment of wounds in 30 RDEB patients
  • the clinical trial will be an open-label, pilot study with three escalating treatment dose groups (10 patients per dose level). Investigators will identify target lesions between S and SO cm 2 for treatment. EVs in saline will be applied underneath a thin silicone sheet dressing as a primary layer with an overlying secondary standard-of-care wound dressings. Control wounds will be treated with saline underneath a silicone sheet. Treatment frequency will at baseline, 4 weeks, 8 weeks, and 12 weeks. Dose levels will be derived from levels administered in the Pi's burn trial. Digital images will be taken of treatment and control wounds. Treatment and control wounds will be measured using the Silhouette® device.
  • evaluating differences of greater than 50% could require a statistical power of less than 0.8.
  • the probability that a chance difference will be called significant is denoted by a (type I error) and typically should have a threshold of 0.05, below which a p value is deemed significant.
  • the chance of missing a real difference (type II error) is designated by ⁇ .
  • Table 1 sample size that will be required.
  • the a refers to the probability that a chance difference will be called significant.
  • the threshold is 0.05 (95% confidence level), below which a p value is deemed significant.
  • Table 1 are for two-tailed tests.
  • the sample size chosen of 10 patients per group will be more than adequate even when increasing the statistical power well beyond the recommended 0.8 to 0.95. While this is more stringent than we might need, it does ensure that we have proper statistical power with the number of subjects proposed.
  • Key inclusion criteria includes: Male or female patients, 12 years or older at time of screening, and given consent with guardian, if under 18 years of age; Have confirmed RDEB diagnosis, as defined by clinical presentation and histologic confirmation; Have at least 1 active wound between 5 and 50 cm 2 on arms and/or legs; Females of childbearing potential must have a negative urine or serum pregnancy test at screening and be using an acceptable form of birth control (oral implant injectable/transdermal contraceptives, intrauterine device, or other forms of birth control). Key exclusion criteria will include: Clinical evidence of infection; Concurrent immunosuppressive therapy of any kind for any reason.
  • BM-MSCs are known to have immune modulatory properties (Bartholomew A, Polchert D, Szilagyi E, Douglas GW, Kenyon N. Mesenchymal stem cells in the induction of transplantation tolerance. Transplantation 2009;87:S55-7; Siegel G, Schafer R, Dazzi F. The immunosuppressive properties of mesenchymal stem cells.
  • Gentamicin induces functional type VII collagen in recessive dystrophic epidermolysis bullosa patients. J Clin Invest 2017; Jones DA, Hunt SW, 3rd, Prisayanh PS, Briggaman RA, Gammon WR. Immunodominant autoepitopes of type VII collagen are short, paired peptide sequences within the fibronectin type ⁇ homology region of the noncollagenous (NCI) domain. J Invest Dermatol 1995;104:231-5; Lapiere JC, Woodley DT, Parente MG, et aL Epitope mapping of type VII collagen. Identification of discrete peptide sequences recognized by sera from patients with acquired epidermolysis bullosa. J Clin Invest 1993;92:1831-9; Pfendner E, Uitto J, Fine JD. Epidermolysis bullosa carrier frequencies in the US population. J Invest Dermatol2001;116:483-4).
  • Blister/erosion reduction based on change in body surface area index (BSAI).
  • Target wound size reduction or closure is an assessment to determine possible efficacy of the EV treatment.
  • Target wounds will be measured using Silhouette® (Aranz Medical), an FDA-approved medical device wound imaging, 3D measurement and documentation system using noninvasive laser technology providing accurate, precise and repeatable wound assessments.

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Abstract

La présente invention concerne des compositions et des méthodes pour le traitement d'une épidermolyse bulleuse.
EP18859166.3A 2017-09-22 2018-09-21 Méthodes et compositions pour le traitement d'une épidermolyse bulleuse Pending EP3684336A4 (fr)

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