# US 10,946,047 B2 · Extracellular vesicles with enhanced potency > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,946,047 B2 - Document ID: US10946047B2 - Status: Granted - Kind: B2 - Application number: 15/967,714 - Filed: 2018-05-01 - Published: 2018-10-18 - Granted: 2021-03-16 - Earliest claimed: 2016-06-17 - Assignees: United Therapeutics Corporation - Applicants: United Therapeutics Corporation - Inventors: Sarah Hogan; Roger Marquez Ilagan; Maria Pia Rodriguez; Carolina Medrano; John Cheadle - Web page: https://patentgrep.com/patent/US10946047B2 ## CPC - A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells - A61K 31/519: ortho- or peri-condensed with heterocyclic rings - A61K 49/0008: {Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure} - A61P 9/12: Antihypertensives - A61P 11/00: Drugs for disorders of the respiratory system - C07K 14/47: from mammals - C12N 5/0662: {Stem cells} - C12N 5/0669: {Bone marrow stromal cells; Whole bone marrow (isolated stem cells from bone marrow C12N5/0647, C12N5/0663)} - C12N 5/0691: {Vascular smooth muscle cells; 3D culture thereof, e.g. models of blood vessels} - C12N 9/1205: {Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases} - C12N 9/14: Hydrolases (3) - C12Y 207/0104: Pyruvate kinase (2.7.1.40) - C12Y 306/01003: Adenosine triphosphatase (3.6.1.3) - A61K 48/00: Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy - C12N 2310/141: MicroRNAs, miRNAs - C12N 2502/1394: Bone marrow stromal cells; whole marrow - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Y: ENZYMES ## Abstract Provided are methods for isolating potent extracellular vesicle or exosome populations from mesenchymal stromal cells, and the use of the isolated extracellular vesicles or exosomes in treating vasculopathy, including pulmonary hypertension, bronchopulmonary dysplasia, and disease and conditions associated with mitochondrial dysfunction. ## Claims ### Claim 1 A method of treating pulmonary hypertension, comprising administering to a subject in need thereof isolated extracellular vesicles or exosomes obtained from mesenchymal stromal cells, wherein the isolated extracellular vesicles or exosomes are selected to have increased expression of one or more expression products of genes in the glycolysis pathway as compared to the average amount of the expression products in all extracellular vesicles or exosomes obtained from the mesenchymal stromal cells. ### Claim 2 (depends on 1) The method of claim 1, wherein the extracellular vesicles or exosomes comprise at least 20% more expression of the expression products compared to the average amount of the same expression product in all extracellular vesicles or exosomes obtained from the mesenchymal stromal cells. ### Claim 3 (depends on 1) The method of claim 1, wherein the expression product of genes in the glycolysis pathway is selected from the group consisting of pyruvate kinase (PK), AGI, aldolase (ALDO), aldolase fructose-bysphosphate A (ALDOA), enolase 3 (ENO3), glucose-6-phosphate isomerase (GPI), hexokinase 2 (HK2), hexokinase 3 (HK3), phosphofructokinase 1 (PFK), phosphoglucomutase 1 (PGM), triosephosphate isomerase (TPI), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), enolase (ENO), and phosphoglycerate mutase (PGAM). ### Claim 4 (depends on 3) The method of claim 3, wherein the expression product is pyruvate kinase (PK). ### Claim 5 (depends on 1) The method of claim 1, wherein the isolated extracellular vesicles or exosomes normalize glucose oxidation in lung tissue of the subject. ### Claim 6 (depends on 1) The method of claim 1, wherein the isolated extracellular vesicles or exosomes have a pyruvate kinase (PK) activity of at least 0.15 nmol/min/mL. ### Claim 7 (depends on 1) The method of claim 1, wherein the isolated extracellular vesicles or exosomes are capable of increasing 02 consumption by smooth muscle cell (SMC) lysates subjected to a 24-hour hypoxia exposure by at least 20% compared to control SMC cell lysates subjected to a 24-hour hypoxia exposure and treated with PBS control. ### Claim 8 (depends on 1) The method of claim 1, wherein the isolated extracellular vesicles or exosomes express one or more of flotillin-1 (FLOT1), intracellular adhesion molecule (ICAM), ALIX, CD81, CD63, epithelial cell adhesion molecule (EpCAM), annexin A5 (ANXA5), and tumor susceptibility gene 101 (TSG101), and/or wherein the isolated extracellular vesicles or exosomes do not express Cis-golgi matrix protein 130 (GM130). ### Claim 9 (depends on 1) The method of claim 1, wherein the isolated extracellular vesicles or exosomes upregulate glutamate dehydrogenase (GLUD1) and/or pyruvate dehydrogenase (PDH) gene expression in the subject, downregulate pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the subject, and/or downregulate sirtuin4 (SIRT4) gene expression in the subject. ### Claim 10 (depends on 1) The method of claim 1, wherein: (a) the subject suffers from increased expression of HSP90 associated with mitochondria damage, and the isolated extracellular vesicles or exosomes downregulate the expression of HSP90; (b) the subject suffers from increased expression of tumor necrosis factor (TNF) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes downregulate the expression of TNF; (c) the subject suffers from increased expression of fas ligand TNF superfamily member 6 (FASLG) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes downregulate the expression of FASLG; (d) the subject suffers from decreased expression of cytochrome c oxidase subunit 4 (COX4) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of COX4; (e) the subject suffers from decreased expression of LIM homeobox transcription factor 1 beta (LMX1B) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of LMX1B; and (f) the subject suffers from decreased expression of tumor protein P53 (TP53) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of TP53. ### Claim 11 (depends on 1) The method of claim 1, wherein the subject suffers from increased proliferation of pulmonary arterial smooth muscle cells (PASMC), and the isolated extracellular vesicles or exosomes decrease the proliferation of PASMC. ### Claim 12 (depends on 1) The method of claim 1, wherein: (a) the subject suffers from increased right ventricular weight associated with pulmonary arterial hypertension (PAH), and the isolated extracellular vesicles or exosomes decrease the right ventricular weight of the subject; (b) the subject suffers from increased right ventricular to left ventricular and septum weight ratio associated with PAH, and the isolated extracellular vesicles or exosomes decrease the right ventricular to left ventricular and septum weight ratio of the subject; (c) the subject suffers from increased heart rate associated with PAH, and the isolated extracellular vesicles or exosomes decrease the heart rate of the subject; and (d) the subject suffers from decreased cardiac output associated with PAH, and the isolated extracellular vesicles or exosomes increase the cardiac output of the subject. ### Claim 13 (depends on 1) The method of claim 1, wherein the subject suffers from decreased expression of mitochondrial transcription factor A (TFAM) associated with mitochondria damage, and wherein the isolated extracellular vesicles or exosomes upregulate the expression of TFAM. ### Claim 14 (depends on 1) The method of claim 1, further comprising administering sildenafil to the subject. ### Claim 15 (depends on 1) The method of claim 1, wherein the isolated extracellular vesicles or exosomes are capable of reducing Right Ventricular Systolic Pressure (RVSP) of mice subjected to a three-week chronic hypoxia exposure by at least 10% compared to control mice subjected to a three-week chronic hypoxia exposure and treated with PBS. ### Claim 16 (depends on 15) The method of claim 15, wherein the isolated extracellular vesicles or exosomes express one or more of flotillin-1 (FLOT1), intracellular adhesion molecule (ICAM), ALIX, CD81, CD63, epithelial cell adhesion molecule (EpCAM), annexin A5 (ANXA5), and tumor susceptibility gene 101 (TSG101), and/or wherein the isolated extracellular vesicles or exosomes do not express Cis-golgi matrix protein 130 (GM130). ### Claim 17 (depends on 15) The method of claim 15, wherein the isolated extracellular vesicles or exosomes upregulate glutamate dehydrogenase (GLUD1) and/or pyruvate dehydrogenase (PDH) gene expression in the subject, downregulate pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the subject, and/or downregulate sirtuin4 (SIRT4) gene expression in the subject. ### Claim 18 (depends on 15) The method of claim 15, wherein: (a) the subject suffers from increased expression of HSP90 associated with mitochondria damage, and the isolated extracellular vesicles or exosomes downregulate the expression of HSP90; (b) the subject suffers from increased expression of tumor necrosis factor (TNF) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes downregulate the expression of TNF; (c) the subject suffers from increased expression of fas ligand TNF superfamily member 6 (FASLG) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes downregulate the expression of FASLG; (d) the subject suffers from decreased expression of cytochrome c oxidase subunit 4 (COX4) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of COX4; (e) the subject suffers from decreased expression of LIM homeobox transcription factor 1 beta (LMX1B) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of LMX1B; and (f) the subject suffers from decreased expression of tumor protein P53 (TP53) associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of TP53. ### Claim 19 (depends on 15) The method of claim 15, wherein the subject suffers from decreased expression of mitochondrial transcription factor A (TFAM) associated with mitochondria damage, and wherein the isolated extracellular vesicles or exosomes upregulate the expression of TFAM. ### Claim 20 (depends on 15) The method of claim 15, further comprising administering sildenafil to the subject.