Extracellular vesicles with enhanced potency

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.

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 comprise extracellular vesicles or exosomes having increased expression of one or more expression products selected from the group consisting of (a) genes in the TCA cycle, and (b) genes in the electron transport chain as compared to an average amount of the expression products in all extracellular vesicles or exosomes obtained from the mesenchymal stromal cells. 
     
     
         2 . 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. 
     
     
         3 . The method of  claim 1 , wherein (a) the gene in the TCA cycle is selected from the group consisting of MDH2, OGDH, PC, PDHA1, PDHB, SDHA, SDHC, and SUCLG2, and (b) the gene in the electron transport chain is selected from the group consisting of ETFA, ATPase, NDUFC2, NDUFB1,NDUFS5, NDUFA8, NDUFA9, NDUF S2, SDHA, SDHC, UQCRH1, Cox 6c1, and Cox10. 
     
     
         4 . The method of  claim 1 , wherein the gene is MDH2. 
     
     
         5 . The method of  claim 1 , wherein the gene is ATPase. 
     
     
         6 . The method of  claim 1 , wherein the isolated extracellular vesicles or exosomes normalize glucose oxidation in lung tissue of the subject. 
     
     
         7 . The method of  claim 1 , wherein the isolated extracellular vesicles or exosomes have a PK activity of at least 0.15 nmol/min/mL. 
     
     
         8 . 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. 
     
     
         9 . The method of  claim 1 , wherein the isolated extracellular vesicles or exosomes are capable of increasing O 2  consumption by SMC cell 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. 
     
     
         10 . A method of treating a disease or condition associated with mitochondrial dysfunction, 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 comprise extracellular vesicles or exosomes having increased expression of one or more expression products selected from the group consisting of (a) genes in the glycolysis pathway, (b) genes in the TCA cycle, and (c) genes in the electron transport chain as compared to an average amount of the expression products in all extracellular vesicles or exosomes obtained from the mesenchymal stromal cells. 
     
     
         11 . The method of  claim 10 , wherein (a) the gene in the glycolysis pathway is selected from the group consisting of PK, AGI, ALDO, ALDOA, ENO3, GPI, HK2, HK3, PFK, PGM, TPI, GAPDH, ENO, and PGAM, (b) the gene in the TCA cycle is selected from the group consisting of MDH2, OGDH, PC, PDHA1, PDHB, SDHA, SDHC, and SUCLG2, and (c) the gene in the electron transport chain is selected from the group consisting of ETFA, ATPase, NDUFC2, NDUFB1,NDUF S5, NDUFA8, NDUFA9, NDUF S2, SDHA, SDHC, UQCRH1, Cox 6c1, and Cox10. 
     
     
         12 . The method of  claim 10 , wherein the gene is PK. 
     
     
         13 . The method of  claim 13 , wherein the isolated extracellular vesicles or exosomes a PK activity of at least 0.15 nmol/min/mL. 
     
     
         14 . The method of  claim 10 , wherein the gene is ATPase. 
     
     
         15 . The method of  claim 10 , wherein the isolated extracellular vesicles or exosomes normalize glucose oxidation in lung tissue of the subject. 
     
     
         16 . The method of  claim 10 , wherein the disease or condition associated with mitochondrial dysfunction is associated with decreased mitochondrial glucose oxidation in the subject. 
     
     
         17 . The method of  claim 10 , wherein the disease or condition associated with mitochondrial dysfunction is selected from the group consisting of Friedreich's ataxia, Leber's Hereditary Optic Neuropathy, Kearns-Sayre Syndrome, Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-Like Episodes, Leigh syndrome, obesity, atherosclerosis, amyotrophic lateral sclerosis, Parkinson's Disease, cancer, heart failure, myocardial infarction (MI), Alzheimer's Disease, Huntington's Disease, schizophrenia, bipolar disorder, fragile X syndrome, and chronic fatigue syndrome. 
     
     
         18 . A method of isolating extracellular vesicles or exosomes capable of treating or preventing pulmonary hypertension, comprising the following steps:
 a. providing a culture media of mesenchymal stromal cells comprising extracellular vesicles or exosomes;   b. separating at least a portion of the extracellular vesicles or exosomes from the other components of the culture media by size exclusion chromatography;   c. isolating a extracellular vesicle or exosome population from other extracellular vesicle or exosome populations, wherein the population has increased expression of one or more expression products selected from the group consisting of (a) genes in the glycolysis pathway, (b) genes in the TCA cycle, and (c) genes in the electron transport chain as compared to an average amount of the expression products in all extracellular vesicles or exosomes obtained from the mesenchymal stromal cells.   
     
     
         19 . The method of  claim 18 , wherein the extracellular vesicle or exosome population is isolated by phospholipid detection. 
     
     
         20 . The method of  claim 18 , wherein (a) the gene in the glycolysis pathway is selected from the group consisting of PK, AGI, ALDO, ALDOA, ENO3, GPI, HK2, HK3, PFK, PGM, TPI, GAPDH, ENO, and PGAM, (b) the gene in the TCA cycle is selected from the group consisting of MDH2, OGDH, PC, PDHA1, PDHB, SDHA, SDHC, and SUCLG2, and (c) the gene in the electron transport chain is selected from the group consisting of ETFA, ATPase, NDUFC2, NDUFB1,NDUF S5, NDUFA8, NDUFA9, NDUF S2, SDHA, SDHC, UQCRH1, Cox 6c1, and Cox10. 
     
     
         21 . The method of  claim 18 , wherein the gene is PK. 
     
     
         22 . The method of  claim 18 , wherein the gene is ATPase. 
     
     
         23 . A composition comprising isolated extracellular vesicles or exosomes obtained according to  claim 18 . 
     
     
         24 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes have a mean diameter of about 100 nm. 
     
     
         25 . The composition of  claim 23 , wherein at least 70% of the isolated extracellular vesicles or exosomes have a size between 50 nm and 150 nm. 
     
     
         26 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes express FLOT and/or ANXA2. 
     
     
         27 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes have increased expression of mir204, compared to the average amount of mir204 in all extracellular vesicles or exosomes of the mesenchymal stromal cells. 
     
     
         28 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes are secreted from MSCs containing increased expression of CD105, GAPDH, DLST, and/or ATP5A1, compared to the average amount of CD105, GAPDH, DLST, and/or ATP5A1 in all the mesenchymal stromal cells. 
     
     
         29 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes have increased RNA expression of SORCS1, FHIT and/or ANKRD30BL, compared to the average amount of SORCS1, FHIT and/or ANKRD30BL in all extracellular vesicles or exosomes of the mesenchymal stromal cells. 
     
     
         30 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes are substantially free of MHCII contaminants. 
     
     
         31 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes are substantially free of fibronectin. 
     
     
         32 . The composition of  claim 23 , wherein the isolated extracellular vesicles or exosomes express one or more of FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, ANXA5, and TSG101, and/or wherein the isolated extracellular vesicles or exosomes substantially do not express GM130. 
     
     
         33 . The method of  claim 1 , wherein the isolated extracellular vesicles or exosomes express one or more of FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, ANXA5, and TSG101, and/or wherein the isolated extracellular vesicles or exosomes substantially do not express GM130. 
     
     
         34 . The method of  claim 8 , wherein the isolated extracellular vesicles or exosomes express one or more of FLOT1, ICAM, ALIX, CD81, CD63, EpCAM, ANXA5, and TSG101, and/or wherein the isolated extracellular vesicles or exosomes substantially do not express GM130. 
     
     
         35 . The method of  claim 1 , wherein the isolated extracellular vesicles or exosomes upregulate GLUD1 and/or PDH gene expression in the subject, downregulate PDK4 gene expression in the subject, and/or downregulate SIRT4 gene expression in the subject. 
     
     
         36 . The method of  claim 8 , wherein the isolated extracellular vesicles or exosomes upregulate GLUD1 and/or PDH gene expression in the subject, downregulate PDK4 gene expression in the subject, and/or downregulate SIRT4 gene expression in the subject. 
     
     
         37 . 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 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 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 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 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 TP53 associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of TP53.   
     
     
         38 . The method of  claim 8 , 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 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 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 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 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 TP53 associated with mitochondria damage, and the isolated extracellular vesicles or exosomes upregulate the expression of TP53.   
     
     
         39 . The method of  claim 1 , wherein the subject suffers from increased proliferation of PASMC, and the isolated extracellular vesicles or exosomes downregulate the proliferation of PASMC. 
     
     
         40 . The method of  claim 1 , wherein:
 (a) the subject suffers from increased right ventricular weight associated with PAH, and the isolated extracellular vesicles or exosomes downregulate 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 downregulate 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 downregulate the heart rate of the subject;   (d) the subject suffers from decreased cardiac output associated with PAH, and the isolated extracellular vesicles or exosomes upregulate the cardiac output of the subject.   
     
     
         41 . The method of  claim 1 , wherein the subject suffers from decreased expression of TFAM associated with mitochondria damage, and wherein the isolated extracellular vesicles or exosomes upregulate the expression of TFAM. 
     
     
         42 . The method of  claim 8 , wherein the subject suffers from decreased expression of TFAM associated with mitochondria damage, and wherein the isolated extracellular vesicles or exosomes upregulate the expression of TFAM. 
     
     
         43 . The method of  claim 1 , further comprising administering sildenafil to the subject. 
     
     
         44 . The method of  claim 8 , further comprising administering sildenafil to the subject. 
     
     
         45 . A method of isolating extracellular vesicles or exosomes capable of treating or preventing pulmonary hypertension or bronchopulmonary dysplasia, comprising the following steps:
 a. providing a culture media of mesenchymal stromal cells comprising extracellular vesicles or exosomes;   b. separating at least a portion of the extracellular vesicles or exosomes from the other components of the culture media, optionally by size exclusion chromatography;   c. separating different populations of extracellular vesicles or exosomes based on molecular size;   d. treating hypoxia-exposed mice with the different populations of extracellular vesicles or exosomes;   e. measuring Right Ventricular Systolic Pressure (RVSP) of normoxia mice, hypoxia-exposed mice and hypoxia exposed mice treated with the extracellular vesicles or exosomes;   f. identifying a potent population of extracellular vesicles or exosomes based on the RVSP.   
     
     
         46 . The method of  claim 45 , wherein a population of extracellular vesicles or exosomes is potent if the ratio of RVSP of hypoxia-exposed mice treated with the extracellular vesicles or exosomes to RVSP of hypoxia-exposed mice is 0.85 or less. 
     
     
         47 . The method of  claim 45 , wherein a population of extracellular vesicles or exosomes is potent if delta RVSP is less than 5, wherein delta RVSP is RVSP of hypoxia-exposed mice treated with extracellular vesicles or exosomes minus RVSP of normoxia mice. 
     
     
         48 . The method of  claim 45 , wherein in step c, different populations of extracellular vesicles or exosomes are separated by phospholipid detection. 
     
     
         49 . The method of  claim 45 , wherein the potent population of extracellular vesicles or exosomes have increased expression of one or more expression products selected from the group consisting of (a) genes in the glycolysis pathway, (b) genes in the TCA cycle, and (c) genes in the electron transport chain as compared to an average amount of the expression products in all extracellular vesicles or exosomes obtained from the mesenchymal stromal cells. 
     
     
         50 . The method of  claim 45 , wherein (a) the gene in the glycolysis pathway is selected from the group consisting of PK, AGI, ALDO, ALDOA, ENO3, GPI, HK2, HK3, PFK, PGM, TPI, GAPDH, ENO, and PGAM, (b) the gene in the TCA cycle is selected from the group consisting of MDH2, OGDH, PC, PDHA1, PDHB, SDHA, SDHC, and SUCLG2, and (c) the gene in the electron transport chain is selected from the group consisting of ETFA, ATPase, NDUFC2, NDUFB1,NDUF S5, NDUFA8, NDUFA9, NDUF S2, SDHA, SDHC, UQCRH1, Cox 6c1, and Cox10. 
     
     
         51 . The method of  claim 45 , wherein the gene is PK. 
     
     
         52 . The method of  claim 45 , wherein the gene is ATPase. 
     
     
         53 . A method of treating or preventing bronchopulmonary dysplasia, comprising administering to a subject in need thereof isolated extracellular vesicles or exosomes according to  claim 45 . 
     
     
         54 . The method of  claim 53 , wherein the isolated extracellular vesicles or exosomes increase immunomodulatory capacity of the subject. 
     
     
         55 . The method of  claim 54 , wherein the isolated extracellular vesicles or exosomes reduces IL-6 and/or TNFα expression in the subject. 
     
     
         56 . The method of  claim 53 , wherein the isolated extracellular vesicles or exosomes promote angiogenesis of the subject. 
     
     
         57 . The method of  claim 56 , wherein the isolated extracellular vesicles or exosomes reduce hyperoxia-induced apoptosis in the subject. 
     
     
         58 . The method of  claim 56 , wherein the isolated extracellular vesicles or exosomes reduces Cytochrome C level in the subject. 
     
     
         59 . The method of  claim 53 , wherein the isolated extracellular vesicles or exosomes increase mitochondrial metabolism of the subject, and/or restore tube formation in the subject. 
     
     
         60 . The method of  claim 53 , wherein the isolated extracellular vesicles or exosomes upregulate GLUD1 and/or PDH gene expression in the subject, downregulate PDK4 gene expression in the subject, and/or downregulate SIRT4 gene expression in the subject.