Allogeneic mitochondrial delivery platform

Abstract

Provided are erythroid progenitor cell-derived extracellular vesicles comprising functional mitochondria, compositions comprising same, methods of making the mitochondriarich extracellular vesicles, and related therapeutic methods.

Description

ALLOGENEIC MITOCHONDRIAL DELIVERY PLATFORMFIELD OF THE INVENTION

[0001] The invention relates to compositions and methods for mitochondrial transplantation therapy.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with Government support under contract TR003142 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0003] Diseases caused by mutations in mitochondrial DNA represent a growing health challenge that may be addressed in part by mitochondrial transplantation therapy. In addition, another leading cause of morbidity and mortality is heart failure which may also benefit from mitochondrial transplantation therapy. Current approaches to heart failure include pharmacologic therapies that seek to reduce cardiac workload, specifically energy demand. But these therapies do not address the underlying need to increase energy production.Mitochondrial deficits are directly related to disease progression in heart failure patients. Transplantation of isolated mitochondria extracted from homogenized cells have been demonstrated to reduce infarct size in murine models of myocardial injury (MI).

[0004] Despite its promise, significant challenges remain to be overcome before mitochondrial transplantation therapy can be successfully translated into clinical practice.These challenges include a need for improved methods for mitochondrial isolation that preserve the functional characteristics of the organelles as well as improved methods for delivery of mitochondria to target cells. Current methods for isolating mitochondria include extracting the mitochondria from healthy donor cells by disrupting the cell membrane and separating the mitochondria from other cellular components. Common separation methods include density gradient centrifugation using sucrose or percoll and affinity-based purifications. Current delivery methods include direct injection, or the use of a delivery vehicle that may comprise, for example, cell-penetrating peptides or polymers.BRIEF SUMMARY

[0005] Provided are erythroid progenitor cell-derived extracellular vesicles comprising functional mitochondria and compositions comprising same. Also provided are methods of making the mitochondria-rich extracellular vesicles and related therapeutic methods.

[0006] In one aspect, provided is a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells, and a buffer. In aspects, the extracellular vesicles are of erythroid progenitor cells comprising one or more of CD34+ erythroid progenitor cells, proerythroblast cells, basophilic normoblast cells, polychomatic normoblast cells, and orthochromatic normoblast cells. In aspects, less than 0.5% or less than 1% of the mitochondria-rich extracellular vesicles of the plurality express HLA class 1 antigen. In aspects, less than 5% of the mitochondria-rich extracellular vesicles of the plurality express the erythrocyte maturation marker CD235a. In aspects, at least 15-50% or at least 20-40% of the mitochondria-rich extracellular vesicles of the plurality comprise functional mitochondria. The composition may also include where the mitochondria-rich extracellular vesicles do not express either the A or B blood group antigen. The composition may also include where the composition is sterile. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0007] Also provided are pharmaceutical compositions. In one aspect, provided is a pharmaceutical composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells according to any of the preceding embodiments, and a pharmaceutically acceptable carrier. Also provided is a pharmaceutical composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells according to any of the preceding embodiments, for use in a method of treating a disease or disorder characterized by a mitochondrial abnormality. Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.

[0008] Also provided are methods of treating a subject having a disease or disorder characterized by a mitochondrial abnormality, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as described herein. In aspects, the disease or disorder is advanced heart failure, sarcopenia, or a mitochondrial disease or disorder. In aspects, the mitochondrial disease or disorder is encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Leber hereditary optic neuropathy (LHON),Leigh syndrome, Kearns-Sayre syndrome (KSS), myoclonic epilepsy or ragged-red fiber disease (MERRF). The method may also include where the mitochondrial disease or disorder is secondary to another disease or disorder such as Alzheimer’s disease, Parkinson's disease, muscular dystrophy, Type 1 diabetes, multiple sclerosis (MS), or cancer. The method may also include where the mitochondrial disease or disorder is secondary to a neurodegenerative disease or disorder. The method may also include where the neurodegenerative disease or disorder is Alzheimer’s disease or Parkinson's disease. The method may also include where the mitochondrial abnormality manifests as reduced cellular energy metabolism in cells of at least one tissue of the subject.

[0009] Also provided are methods for treating a subject having myocardial injury, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as described herein. In aspects, the myocardial injury is a result of myocardial ischemia, cardiac surgery, or circulatory arrest. In aspects, the myocardial injury is myocardial ischemia-reperfusion (IR) injury.

[0010] Also provided are methods for treating a subject having cerebral infarction or ischemic stroke, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as described herein.

[0011] In one aspect, the methods may comprise, prior to administering to the subject a therapeutically effective amount of mitochondria-rich erythroid progenitor cell-derived extracellular vesicles, isolating erythroid progenitor cells from type-0 donor blood, differentiating the erythroid progenitor cells into red blood cells in a cell culture medium in vitro, and isolating mitochondria-rich extracellular vesicles from the cell culture medium during differentiation. The method may also include where the mitochondria-rich extracellular vesicles are isolated from the cell culture medium over a period of from 4 to 14 days from initiation of differentiation. The method may also include where the assay includes flow cytometry.

[0012] Also provided are methods for producing mitochondria-rich erythroid progenitor cell extracellular vesicles. In one aspect, the method includes isolating erythroid progenitor cells from type-0 donor blood, differentiating the erythroid progenitor cells into red blood cells in a cell culture medium in vitro, and isolating mitochondria-rich extracellular vesicles from the cellculture medium. The method may also include where the mitochondria-rich extracellular vesicles are isolated from the cell culture medium over a period of from 4 to 14 days from initiation of differentiation. The method may also include where differentiation is initiated on day zero by addition of stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO) to the medium. The method may also include where the isolated extracellular vesicles are assayed for expression of one or more antigens, including a red blood cell differentiation marker, an HLA class 1 antigen, and / or a blood group antigen. In aspects, the method may also include where the isolated extracellular vesicles are assayed for functional mitochondria.

[0013] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. l is a schematic illustrating the process of erythroid differentiation in accordance with one embodiment.

[0015] FIG. 2A is a bar graph showing percentage of extracellular vesicles positive for CD235a at days 4, 7, 9, 11, and 14 of differentiation.

[0016] FIG. 2B is a bar graph showing percentage of extracellular vesicles positive for HLA class 1 antigen at days 4, 7, 9, 11, and 14 of differentiation.

[0017] FIG. 2C is a bar graph showing the number (xlO8) of extracellular vesicles isolated from culture medium at days 4, 7, 9, 11, and 14 of differentiation.

[0018] FIG. 3A is a bar graph showing percentage of extracellular vesicles positive for Mitotracker Deep Red, a marker of functional mitochondria, at days 4, 7, 9, 11, and 14 of differentiation.

[0019] FIG. 3B is a bar graph showing percentage of extracellular vesicles positive for Mitotracker Green, a marker of mitochondria, at days 4, 7, 9, 11, and 14 of differentiation.

[0020] FIG. 4A is a bar graph showing improvement in cardiac function, LVEF (%), by EPC- MEVs administered in a murine mode of acute myocardial infarction.

[0021] FIG. 4B is a bar graph showing improvement in cardiac function, LVEDV (ul), by EPC-MEVs administered in a murine mode of acute myocardial infarction.DETAILED DESCRIPTION

[0022] Provided are compositions comprising mitochondria-rich extracellular vesicles of erythroid progenitor cells, methods of making the mitochondria-rich extracellular vesicles, and related therapeutic methods.

[0023] The mitochondria-rich extracellular vesicles (mEVs) described herein are nanosized lipid membrane-bound vesicles released from donor erythroid progenitor cells. Accordingly, the mEVs described here are physiologically secreted by the parent erythroid progenitor cells and contain whole mitochondria derived from the parent cells. As described in more detail infra, the mEVs are useful in mitochondrial transplantation therapy. In accordance with such therapy, the parent cells from which the mEVs are collected may be referred to as “donor” cells. The terms “parent” and “donor” are used interchangeably herein to refer to the cells from which the mEVs are collected. In aspects, the parent or donor erythroid progenitor cells may include one or more of CD34+ erythroid progenitor cells, proerythroblast cells, basophilic normoblast cells, polychomatic normoblast cells, orthochromatic normoblast cells.

[0024] In aspects, the erythroid progenitor cells are isolated from type-0 donor blood. Accordingly, in aspects, the extracellular vesicles are characterized by an absence of expression of both the A and B blood group antigens. In aspects, the extracellular vesicles are characterized by expression of the cluster of differentiation 34 (CD34) antigen. In aspects, the extracellular vesicles are characterized by the absence of expression of HLA class 1 antigen on at least about 95-99% of the extracellular vesicles in a plurality. In aspects, at least 15-50% or at least 20-40% of extracellular vesicles in a plurality comprise functional mitochondria. In aspects, the extracellular vesicles are characterized by an average diameter of between about 100 nanometers (nm) and 1000 nm, or between about 200-800 nm or about 200-600 nm, or about 200-400 nm.

[0025] In aspects, a composition comprising a plurality of extracellular vesicles as described herein is characterized by a very low expression of HLA class 1 antigen. In this context, the plurality is considered to have a “very low” expression of HLA class 1 antigen where less than 0.5% or less than 1% of the extracellular vesicles in the composition express HLA class 1 antigen, for example as determined using flow cytometry. In aspects, the plurality of extracellular vesicles is characterized by low expression of the erythrocyte maturation marker CD235a. In aspects, the plurality is considered to have a “low” expression of CD235a where less than 5% of the extracellular vesicles express CD235a.

[0026] Accordingly, in aspects provided are compositions comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells where the plurality is characterized as at least 15-50% comprising functional mitochondria and by one or more of an absence of expression of both the A and B blood group antigens, low expression of HLA class 1 antigen, and low expression of CD235a.

[0027] In aspects, the plurality is characterized by an absence of expression of both the A and B blood group antigens, less than 1% expression of HLA class 1 antigen, less than 5% expression of CD235a, and at least 15-50% comprising functional mitochondria. In this context “less than 1% expression of HLA class 1 antigen” means that less than 1% of the extracellular vesicles of the plurality express the antigen. Similarly, “less than 5% expression of CD235a” means that less than 5% of the extracellular vesicles of the plurality express CD235a, and “at least 15-50% comprising functional mitochondria” means that at least 15-50% of the extracellular vesicles of the plurality comprise functional mitochondria.

[0028] In other aspects, it may be desirable to match the donor mitochondria-rich extracellular vesicles to a recipient blood time. Thus, in aspects, the erythroid progenitor cells are isolated from type-A, type-B, or type-AB donor blood, according to the blood type of the recipient.Also provided are pharmaceutical compositions comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells isolated from type-A, type-B, or type-AB blood, for use in a method of mitochondria transplantation therapy where the recipient is different from the donor and the donor blood type is selected to match the blood type of the recipient.

[0029] In aspects the composition is formulated as an aqueous buffered solution having a suitable pH, e.g., a pH in the range of 7-7.4, and isotonic with physiological fluids such as blood, serum, or plasma. In aspects, the composition is sterile.

[0030] In aspects, the composition is a pharmaceutical composition. In aspects, the pharmaceutical composition comprises a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as discussed above, including for example where the plurality is characterized as at least 15-50% comprising functional mitochondria and by one or more of an absence of expression of both the A and B blood group antigens, low expression of HLA class 1 antigen, and low expression of CD235a. In aspects, the plurality is characterized by an absence of expression of both the A and B blood group antigens, less than 1% expression of HLA class 1 antigen, less than 5% expression of CD235a, and at least 15-50% comprisingfunctional mitochondria. The pharmaceutical compositions comprise a pharmaceutically acceptable carrier, excipient or diluent. In aspects, the carrier, excipient or diluent is a buffered aqueous solution. In aspects, the pharmaceutical composition is sterile.

[0031] The pharmaceutical compositions may be formulated with one or more pharmaceutically acceptable ingredients including, for example, pharmaceutically acceptable carriers, diluents, excipients, adjuvants, fillers, buffers, preservatives, antioxidants, lubricants, stabilizers, solubilizers, and surfactants. In aspects, the pharmaceutical composition is sterile.

[0032] The term “extracellular vesicle” or “EV” is used herein in accordance with its ordinary and customary meaning in the biomedical arts to refer to nanosized, membrane-bound vesicles released from cells. In organisms, extracellular vesicles may transport cargo, including DNA, RNA, and proteins, between cells as a form of intercellular communication. Different types extracellular vesicles, including microvesicles and exosomes, have been characterized based on their biogenesis or release pathways. The content of extracellular vesicles may include lipids, nucleic acids, proteins, and organelles from donor cells. Microvesicles bud directly from the plasma membrane, are typically 100 nanometers (nm) to 1 micrometer (um) in size, and contain cytoplasmic cargo. Exosomes are formed by a fusion of multivesicular bodies and the plasma membrane in which multivesicular bodies release smaller vesicles (exosomes) whose diameters typically range from 40 to 120 nm.

[0033] The mitochondria-rich extracellular vesicles described herein may be collected from cell culture medium of donor erythroid progenitor cells, for example by differential centrifugation (e. ., 10,000g for 30 min). In aspects, the extracellular vesicles may be isolated by a method comprising one or more of differential centrifugation, ultrafiltration, density gradient / cushion centrifugation, and immunoaffinity-based capture.

[0034] Importantly, the mitochondria-rich extracellular vesicles described here are not prepared by a method comprising disruption of cellular membranes. Instead, the mitochondria- rich extracellular vesicles described here are collected following their secretion from intact cells. Accordingly, the methods for producing the mitochondria-rich extracellular vesicles described here do not include processes typical of the isolation and subfractionation of mitochondria such as homogenization and syringe / needle transfer.

[0035] Erythroid progenitor cells may be obtained using methods known in the art. For example, by isolation from human peripheral blood, human cord blood or human bone marrow using one or more of centrifugation, flow cytometry, density gradient centrifugation, and / or aCD34 affinity purification reagent. Affinity reagents for isolation of CD34+ cells are commercially available, for example from Stemcell Technologies and ThermoFisher Scientific.

[0036] The erythroid progenitor cells may be cultured for several days, for example to expand the population, while maintaining their pluripotency using methods known in the art. For example, CD34+ cells may cultured in serum-free culture medium supplemented with a combination of growth factors and cytokines including one or more of thrombopoietin (TPO), stem cell factor (SCF), Flt-3 ligand (Flt-3L), and PIXY321 (IL-3 / GM-CSF fusion protein), with or without added serum, e.g., human serum. Suitable basal serum free cell culture media are commercially available, including for example Stemline™ HSC media available from Sigma- Aldrich. Exemplary methods of culturing CD34+ cells for maintaining pluripotency are described in Yaday et al., Int J Stem Cells 2020 13(3): 326-334.

[0037] The erythroid progenitor cells may be differentiated into red blood cells using methods known in the art. For example, Baek et al. describe differentiation using Iscove’s Modified Dulbecco’s Medium (IMDM) media with L-Glutamine (L-G), L-monothioglycerol (L-MT), SCF., 1% Pen / Strep, 1% human serum albumin, ferric nitrate, transferrin, hydrocortisone (HC), interleukin-3 (IL-3), erythropoietin (EPO), insulin and BM-MSCs. Transfusion. 2008;48:2235- 2245. Exemplary methods for differentiating CD34+ cells are described in Yaday et al., Int J Stem Cells 2020 13(3): 326-334. Provided below and illustrated in FIG. 1 is an exemplary differentiation protocol.

[0038] Also provided are methods of therapy. In one aspect, provided is a method of treating a subject having a disease or disorder characterized by a mitochondrial abnormality, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising mitochondria-rich extracellular vesicles of erythroid progenitor cells, and a pharmaceutically acceptable carrier.

[0039] In one aspect, provided is a method of treating a subject having a mitochondrial disease or disorder, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising mitochondria-rich extracellular vesicles of erythroid progenitor cells, and a pharmaceutically acceptable carrier.

[0040] In aspects, the mitochondrial disease or disorder may be selected from mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, Kearns-Sayre syndrome (KSS), myoclonic epilepsy and ragged-red fiber disease (MERRF).

[0041] In aspects, the mitochondrial disease or disorder may include mitochondrial dysfunction secondary to a chronic disease or disorder such as Alzheimer’s disease, Parkinson's disease, muscular dystrophy, Type 1 diabetes, multiple sclerosis (MS), or cancer of the trachea, bronchus, or lung. In aspects, the mitochondrial disease or disorder may include mitochondrial dysfunction secondary to a chronic disease or disorder such as ischemic heart disease, chronic obstructive pulmonary disease, dementia, diabetes, particularly type 2 diabetes, or sarcopenia.

[0042] In aspects, the mitochondrial disease or disorder may include mitochondrial dysfunction secondary to a neurodegenerative disease or disorder. In aspects, the neurodegenerative disease or disorder is Alzheimer’s disease or Parkinson's disease.

[0043] In one aspect, provided is a method of treating a subject having advanced heart failure, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising mitochondria-rich extracellular vesicles of erythroid progenitor cells, and a pharmaceutically acceptable carrier.

[0044] In one aspect, provided is a method of treating a subject having sarcopenia, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising mitochondria-rich extracellular vesicles of erythroid progenitor cells, and a pharmaceutically acceptable carrier.

[0045] In one aspect, provided is a method of treating a subject having myocardial injury, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as described herein. In aspects, the myocardial injury is a result of myocardial ischemia, cardiac surgery, or circulatory arrest. In aspects, the myocardial injury is myocardial ischemia-reperfusion (IR) injury.

[0046] In one aspect, provided is a method of treating a subject having cerebral infarction or ischemic stroke, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as described herein.

[0047] In aspects, provided are methods of mitochondrial transplantation therapy for the treatment of a degenerative disease or disorder, wherein the methods comprise administering to a subject in need of such therapy a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cellsas described herein. In aspects, the degenerative disease or disorder is Alzheimer’s disease, Parkinson's disease, muscular dystrophy, Type 1 diabetes, or multiple sclerosis (MS).

[0048] In aspects, provided is a method of reversing a symptom of aging or reducing frailty due to age, the methods comprising administering to the subject a therapeutically effective amount of a composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells as described herein.

[0049] In accordance with aspects of the foregoing methods of therapy, the subject is a human subject. In aspects, the human is an adult human, a pediatric human, or a geriatric human, as those terms are understood by the medical practitioner, for example as defined by the U.S. Food and Drug Administration. In some aspects, the human subject is one in need of therapy for the disease, disorder, or condition being treated, for example one who has received a clinical diagnosis for the disease, disorder, or condition.

[0050] The present inventors previously developed a mitochondrial transplantation therapy using extracellular vesicles secreted from autologous induced pluripotent stem cell (iPSC)- derived cardiomyocytes, described in W02020232301. While the advantage of this autologous treatment lies in its low immunogenicity, challenges include the time, cost, and complexity associated with establishing patient-derived iPSCs and their differentiation into cardiomyocytes.

[0051] The present inventors unexpectedly discovered that erythroid progenitor cells (EPCs) release extracellular vesicles containing functional mitochondria during the process of differentiation from hematopoietic stem cells into red blood cells, and that these EPC-derived extracellular vesicles could be utilized in allogenic mitochondrial transplantation therapy. Accordingly, the EPC-derived mitochondria-rich extracellular vesicles of the present invention address the limitations of the earlier autologous iPSC-derived cardiomyocyte therapy. In particular, the EPC-derived extracellular vesicles are more readily produced in a less time consuming and cost efficient process than the autologous iPSC-derived cardiomyocytes. In addition, the EPC-derived mitochondria-rich extracellular vesicles advantageously exhibit very low expression of HLA antigen, which is advantageous for their use in allogenic mitochondrial transplantation therapy. As discussed below, EPC-derived mitochondria-rich extracellular vesicles were successfully isolated from culture medium and tested in a murine model of myocardial infarction where they demonstrated efficacy.

[0052] FIG. 1 illustrates a process of erythroid differentiation in accordance with one embodiment. Differentiation proceeds in three phases. During Phase I, CD34+ cells are cultured for 7 days in serum free basal medium, such as Stempro™ (Thermofi scher, Gibco) supplemented with hydrocortisone (HC, 10'6M), stem cell factor (SCF, 100 ng / ml), interleukin-3 (IL-3, 10 ng / ml), and erythropoietin (EPO, 6 U / ml). After 7 days, the cells are cultured for another 7 days in serum free basal medium supplemented with SCF (50 ng / ml), IL- 3 (10 ng / ml), and EPO (3 U / ml). This is followed by 4 days in serum free basal medium supplemented with SCF (50 ng / ml), EPO (2 U / ml), and Pluronic F68 of the PEO76PPO29PEO76 formula, also known as poloxamer 188 (F68, 0.05%) followed by 3 days in serum free basal medium supplemented with F68 (0.05%).

[0053] FIG. 2A is a bar graph showing percentage of extracellular vesicles positive for CD235a at days 4, 7, 9, 11, and 14 of differentiation. The figure illustrates the differentiation of the progenitor cells along the red blood cell lineage.

[0054] FIG. 2B is a bar graph showing percentage of extracellular vesicles positive for HLA class 1 antigen at days 4, 7, 9, 11, and 14 of differentiation. The figure illustrates an important characteristic of the EPC-derived mitochondria-rich extracellular vesicles, namely that they exhibit extremely low expression of HLA antigen, especially after 4 days of differentiation.

[0055] FIG. 2C is a bar graph showing the number (xlO8) of extracellular vesicles isolated from culture medium at days 4, 7, 9, 11, and 14 of differentiation.

[0056] FIG. 3A is a bar graph showing percentage of extracellular vesicles positive for Mitotracker Deep Red, a marker of functional mitochondria, at days 4, 7, 9, 11, and 14 of differentiation. The data indicate that at least about 15-30% of the extracellular vesicles contain functional mitochondria.

[0057] FIG. 3B is a bar graph showing percentage of extracellular vesicles positive for Mitotracker Green, a marker of mitochondria, at days 4, 7, 9, 11, and 14 of differentiation. The data indicate that at about 20-60% of the extracellular vesicles include mitochondria.

[0058] EPC-derived extracellular vesicles were successfully isolated from the culture medium of EPCs and demonstrated to provide an effective allogeneic mitochondrial transplant platform in a mouse model of acute myocardial infarction (MI). FIG. 4A is a bar graph showing improvement in cardiac function, LVEF (%), for mice administered sham treatment (sham), phosphate buffered saline (PBS), extracellular vesicles from iPSC-derived cardiomyocytes(iCM-EV), or EPC-derived extracellular vesicles (EPC-EV). Both iCM-EVs and EPC-EVs show improvement in cardiac function.

[0059] The experiment was carried out by injecting, directly into the cardiac infarct border zone, either PBS, l.OxlO8EPC-MEVs, or l.OxlO8iCM-EVs. Sham treatment included subjecting the mice to a similar surgery as the MI mice, but without surgical ligation. Mice were evaluated by MRI at week 4. Statistical significance is shown by 1-way ANOVA followed by Tukey's test.

[0060] FIG. 4B is a bar graph showing a different measure of cardiac function, LVEDV (ul) in the same experiment. In sum, the data represented in FIG. 4 A and FIG. 4B shows that intramyocardial injection of 1.0 x 108EPC-derived MEVs significantly improved left ventricular ejection fraction (LVEF) and left ventricular end-diastolic volume (LVEDV) four weeks after treatment in a mouse model of myocardial infarction.

[0061] While the invention has been described by means of specific embodiments and applications thereof, modifications and variations could be made thereto by those skilled in the art without departing from the scope set forth in the claims.

[0062] The present invention is set forth in various levels of detail. In certain instances, details not necessary for one of ordinary skill in the art to understand the invention may have been omitted.

[0063] Section headings are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0064] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting beyond the scope of the appended claims. An “embodiment” may refer to an illustrative representation of a method or article in which a disclosed concept or feature may be provided or embodied, or a representation of a manner in which a concept or feature may be provided or embodied. Such illustrated embodiments are to be understood as examples (unless otherwise stated), and other manners of embodying the described concepts or features, such as may be understood by one of ordinary skill in the art upon learning the concepts or features from the present disclosure, are within the scope of the disclosure. Accordingly, disclosed embodiments are to be considered in all respects as illustrative and not restrictive, the scope of the claimed subject matter being indicated by the appended claims, and not limited to the foregoing description or particular embodiments or arrangements described or illustrated herein. It is intended that the present subject mattercovers such modifications and variations as come within the scope of the appended claims and their equivalents.

[0065] Unless defined otherwise, technical terms used herein are to be understood as commonly understood by one of ordinary skill in the art to which the disclosure belongs.

[0066] The phrases “at least one”, “one or more”, and “and / or”, as used herein, are open- ended expressions that are both conjunctive and disjunctive in operation. The terms “a”, “an”, “the”, “first”, “second”, etc., do not preclude a plurality. For example, the term “a” or “an” entity, as used herein, refers to one or more of that entity. As such, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein.

[0067] The term “about” when used before a numerical designation, e.g., temperature, time, amount, concentration, and such other, including a range, indicates approximations which may vary by ( + ) or ( - ) 10%, 5%, 1%, or any subrange or subvalue there between. Preferably, the term “about” means that the value may vary by + / - 10%.

[0068] The term “comprises / comprising” does not exclude the presence of other elements, components, features, regions, integers, steps, operations, etc. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. By contrast, the transitional phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps “and those that do not materially affect the basic and novel character! stic(s)” of the claimed invention.

[0069] As used herein, a “subject” refers to a mammal, including non-human primates and humans. In aspects, the subject is a human, a non-human primate, swine, sheep, cow, equine, canine, feline or rodent, including a mouse, rat, a guinea pig or other rodent.

[0070] The terms “treatment” or “treating” as used herein pertain generally to describe the management and care of a subject, including human and animal subjects (e.g., in veterinary applications), to achieve a therapeutic effect in relation to a disease or disorder, for example, to alleviate, ameliorate, or mitigate one or more symptoms or complications of the disease or disorder, to slow the progression of the disease or disorder, including a reduction in the rate of progression or a halt in the progression, and cure of the condition. In the context of the present invention, treating may include improving mitochondrial function. In aspects, improved mitochondrial function may include increased intracellular ATP levels, and / or increasedexpression of PGC-Ia. In aspects, treating advanced heart failure may include reducing or ameliorating one or more symptoms of advanced heart failure including exercise intolerance, unintentional weight loss, refractory volume overload, recurrent ventricular arrhythmias, hypotension and inadequate perfusion for example manifested as low pulse pressure.

[0071] The term “effective amount” or “therapeutically effective amount” or “pharmaceutically effective amount” refers to an amount effective, at dosages and for periods of time necessary to achieve the desired result. For example in the context of treating a subject with a disease, disorder, or condition characterized by a mitochondrial abnormality, an effective amount is an amount that, for example, provides some alleviation, amelioration, mitigation and / or decrease in one or more symptoms of the disease, disorder, or condition experienced by a subject.

[0072] Effective amounts may vary according to factors such as the disease state, age, sex and / or weight of the subject. The amount of a given therapeutic agent that will correspond to a therapeutically effective amount will vary depending upon factors including the pharmaceutical formulation, the route of administration, the type or stage of condition, disease or disorder, and subject-specific factors that may include sex, age, race, age, weight, comorbidities, etc. Methods for determining an effective dose are known in the art.

[0073] In aspects, the route of administration may be an intravenous or parenteral route. Parenteral administration includes, e.g, intramyocardial, intracoronary, intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial.

Claims

CLAIMSWhat is claimed is:

1. A composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells, and a buffer.

2. The composition of claim 1, wherein the extracellular vesicles are of a erythroid progenitor cell selected from one or more of CD34+ erythroid progenitor cells, proerythroblast cells, basophilic normoblast cells, polychomatic normoblast cells, and / or orthochromatic normoblast cells.

3. The composition of claim 1 or 2, wherein less than 0.5% or less than 1% of the mitochondria-rich extracellular vesicles of the plurality express HLA class 1 antigen.

4. The composition of any one of claims 1 to 3, wherein less than 5% of the mitochondria-rich extracellular vesicles of the plurality express the erythrocyte maturation marker CD235a.

5. The composition of any one of claims 1 to 4, wherein at least 15-50% or at least 20-40% of the mitochondria-rich extracellular vesicles of the plurality comprise functional mitochondria.

6. The composition of any one of claims 1 to 5, wherein the mitochondria-rich extracellular vesicles do not express either the A or B blood group antigen.

7. The composition of any one of claims 1 to 6, wherein the composition is sterile.

8. A pharmaceutical composition comprising a plurality of mitochondria-rich extracellular vesicles of erythroid progenitor cells according to any one of claims 1 to 7, and a pharmaceutically acceptable carrier.

9. The composition of any one of claims 1 to 7, for use in a method of treating a disease or disorder characterized by a mitochondrial abnormality.

10. A method of treating a subject having a disease or disorder characterized by a mitochondrial abnormality, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of claims 1 to 8.

11. The method of claim 10, wherein the disease or disorder is advanced heart failure, sarcopenia, vision loss, hearing loss, liver dysfunction, or a mitochondrial disease or disorder.

12. The method of claim 11, wherein the mitochondrial disease or disorder is mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome, Leber hereditary optic neuropathy (LHON), Leigh syndrome, Kearns-Sayre syndrome (KSS), myoclonic epilepsy or ragged-red fiber disease (MERRF).

13. The method of claim 11, wherein the mitochondrial disease or disorder is secondary to a chronic disease or disorder selected from, muscular dystrophy, type 1 diabetes, type 2 diabetes, multiple sclerosis (MS), lung cancer, tracheal cancer, bronchus cancer, sarcopenia, ischemic heart disease and stroke, or chronic obstructive pulmonary disease.

14. The method of claim 11, wherein the mitochondrial disease or disorder is secondary to a neurodegenerative disease or disorder, optionally wherein the neurodegenerative disease or disorder is dementia, Alzheimer’s disease or Parkinson's disease.

15. The method of claim 10, wherein the mitochondrial abnormality manifests as reduced cellular energy metabolism in cells of at least one tissue of the subject.

16. The method of any one of claims 10 to 15, wherein the method comprises, prior to administering to the subject a therapeutically effective amount of mitochondria-rich erythroid progenitor cell-derived extracellular vesicles, isolating erythroid progenitor cells from type-0 donor blood, differentiating the erythroid progenitor cells into red blood cells in a cell culture medium in vitro, and isolating mitochondria-rich extracellular vesicles from the cell culture medium during differentiation.

17. The method of any one of claims 10 to 15, wherein the method comprises, prior to administering to the subject a therapeutically effective amount of mitochondria-rich erythroid progenitor cell-derived extracellular vesicles, isolating erythroid progenitor cells from donor blood, differentiating the erythroid progenitor cells into red blood cells in a cell culture medium in vitro, and isolating mitochondria-rich extracellular vesicles from the cell culture medium during differentiation18. The method of claim 17, wherein the donor blood is type- A, type-B, or type AB.

19. The method of any one of claims 16 to 18, wherein the mitochondria-rich extracellular vesicles are isolated from the cell culture medium over a period of from 4 to 14 days from initiation of differentiation.

20. A method for producing mitochondria-rich erythroid progenitor cell extracellular vesicles, the method comprising isolating erythroid progenitor cells from type-0 donor blood, differentiating the erythroid progenitor cells into red blood cells in a cell culture medium in vitro, and isolating mitochondria-rich extracellular vesicles from the cell culture medium.

21. The method of claim 20, wherein the mitochondria-rich extracellular vesicles are isolated from the cell culture medium over a period of from 4 to 14 days from initiation of differentiation.

22. The method of claim 20 or 21, wherein differentiation is initiated on day zero by addition of stem cell factor (SCF), interleukin-3 (IL-3), and erythropoietin (EPO) to the medium.

23. The method of any one of claims 20 to 22, wherein the isolated extracellular vesicles are assayed for expression of one or more of a red blood cell differentiation marker, an HLA class 1 antigen, and functional mitochondria.

24. The method of claim 23, wherein the assay comprises flow cytometry.

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