Mitochondrial enhancement therapy for myelodysplastic syndromes

Abstract

The present disclosure provides mitochondria-enriched cells for treating myelodysplastic syndromes (MDS), diseases and disorders. The present disclosure provides pharmaceutical compositions of mitochondrially-enriched stem and / or progenitor cells, methods of treating myelodysplastic syndromes, and methods of using mitochondrially-enriched stem and / or progenitor cells to alleviate symptoms of MDS and / or prevent progression of MDS.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. Provisional Application No. 63 / 289,069, filed December 13, 2021. The disclosure of said prior application is deemed to be part of the disclosure of this application and is incorporated by reference in its entirety into this application.

[0002] FIELD OF THEINVENTION The present invention relates generally to mitochondria-enriched cells, and more specifically to mitochondrial-enriched stem and / or progenitor cell compositions and methods of use thereof for treating myelodysplastic syndromes. [Background technology]

[0003] Background information Mitochondria are membrane-bound organelles 0.5-1.0 μm in diameter found in most eukaryotic cells. Mitochondria are found in almost all eukaryotic cells, varying in number and location depending on the cell type. Mitochondria contain their own DNA (mtDNA) and their own machinery for synthesizing RNA and proteins. Because mtDNA contains only 37 genes, most gene products in mammals are encoded by nuclear DNA.

[0004] Mitochondria play many important roles in eukaryotic cells, including pyruvate oxidation, the Krebs cycle, and amino acid, fatty acid, and steroid metabolism. However, the primary function of mitochondria is to generate energy as adenosine triphosphate (ATP) through the electron transport chain and oxidative phosphorylation system (the "respiratory chain"). Other processes in which mitochondria participate include heat production, calcium ion accumulation, calcium signaling, programmed cell death (apoptosis), and cell proliferation.

[0005] ATP in cells, typically at a concentration of 1-10 mM ATP, can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as energy sources. To be synthesized into ATP, complex energy sources must first be broken down into smaller, simpler molecules. Complex carbohydrates are hydrolyzed into simple sugars such as glucose and fructose. Fats (triglycerides) are metabolized into fatty acids and glycerol.

[0006] The entire process of oxidizing glucose to carbon dioxide is known as cellular respiration and can produce about 30 molecules of ATP from a single molecule of glucose. ATP can be produced by several different cellular processes. The three main pathways for generating energy in eukaryotes are glycolysis and the citric acid cycle / oxidative phosphorylation (both components of cellular respiration), and beta-oxidation.

[0007] The majority of this ATP production by non-photosynthetic eukaryotes occurs in mitochondria, which can occupy nearly 25% of the total volume of a typical cell.

[0008] Myelodysplastic syndromes (MDS) are defined by ineffective hematopoiesis resulting in blood cytopenias and clonal instability with the risk of clonal evolution to acute myeloid leukemia (AML). Patients with MDS have a high overall symptom burden and are at risk of death from cytopenias and AML complications. The goal of therapy for patients with MDS is to improve both quality and length of life by reducing disease-related symptoms and the risk of disease progression and death. Unfortunately, treatment options are still limited to blood transfusions, growth factors, and supportive care with the limited number of approved drugs currently available.

[0009] Currently available treatments are primarily limited in response rate, short duration of effect, limited treatment options for neutropenia and thrombocytopenia, and virtually no curative treatments other than hematopoietic cell transplantation (HCT). The majority of patients eventually relapse, and progression is not prevented by current treatments. Therefore, new therapies, especially with novel mechanisms of action, as monotherapy or combination therapy, are highly needed.

[0010] Mitochondrial enhancement therapy (MAT) is a cellular therapy that includes autologous stem and / or progenitor cells (HSPCs) enriched with healthy mitochondria. MAT provides a therapeutic modality to restore hematopoietic lineage function by colonizing the mitochondria-enriched cells in the bone marrow to establish new hematopoietic colonies of stem and / or progenitor cells with healthy mitochondria. MAT can be used as a monotherapy or in combination with current standard of care to reduce overall disease burden and healthcare service utilization and improve quality of life. Thus, MAT addresses a significant unmet medical need for patients with myelodysplastic syndromes. Summary of the Invention

[0011] The present invention is based on the seminal discovery that mitochondria-enriched cells are useful for the treatment of myelodysplastic syndromes (MDS), diseases and disorders. The present invention provides pharmaceutical compositions of mitochondrially-enriched stem and / or progenitor cells, methods of treating myelodysplastic syndromes, and methods of using mitochondrially-enriched stem and / or progenitor cells to alleviate symptoms of and / or prevent the progression of MDS.

[0012] In one embodiment, the invention provides a pharmaceutical composition comprising stem and / or progenitor cells enriched in exogenous mitochondria and a pharma- ceutically acceptable carrier, wherein the stem and / or progenitor cells are obtained from a subject having a myelodysplastic syndrome (MDS) disease, disorder, or symptoms thereof, and the exogenous mitochondria are obtained from a donor free of MDS disease, disorder, or symptoms thereof, or a mitochondrial disease.

[0013] In one embodiment, the exogenous mitochondria-enriched stem and / or progenitor cells are produced by a method comprising contacting the stem and / or progenitor cells with exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the stem and / or progenitor cells. In some embodiments, the conditions that allow the exogenous mitochondria to enter the stem and / or progenitor cells comprise incubating the stem and / or progenitor cells with the exogenous mitochondria for a time period ranging from about 0.5 to 30 hours at a temperature ranging from about 4 to 37° C. In one embodiment, the conditions that allow the exogenous mitochondria to enter the target cells comprise incubating the target cells with the exogenous mitochondria for a time period ranging from about 0.5 to 30 hours at a temperature ranging from about 4 to 37° C. 6 In some embodiments, the conditions that allow the exogenous mitochondria to enter the target cells include incubating the target cells with the exogenous mitochondria at a ratio of about 0.044 to 176 mU of citrate synthase (CS) activity per cell. 6 In various embodiments, the exogenous mitochondria are incubated at a concentration of about 1 to 50 mU of citrate synthase (CS) activity per cell. 6 The citrate synthase (CS) activity is about 4.4, 17.6, or 35 mU per cell. In one embodiment, the conditions that allow the exogenous mitochondria to enter the target cells include incubating the target cells with the exogenous mitochondria at a ratio of about 1 to up to 200 mitochondrial particles per cell. In a particular embodiment, the conditions that allow the exogenous mitochondria to enter the target cells include incubating the target cells with the exogenous mitochondria at a ratio of about 10 to up to 50 mitochondrial particles per cell.

[0014] In another embodiment, the stem and / or progenitor cells enriched with exogenous mitochondria have an increased content of at least one mitochondrial protein; an increased rate of oxygen (O2) consumption; an increased level of activity of citrate synthase, succinate, or tryptamine; an increased rate of adenosine triphosphate (ATP) production; an increased mitochondrial DNA content; an increased colony-forming unit activity in liquid or solid medium; an increased rate of proliferation; an increased rate of differentiation; or any combination thereof, compared to the stem and / or progenitor cells prior to mitochondria enrichment. In one embodiment, the exogenous mitochondria constitute at least 0.5% of the total mitochondria in the stem and / or progenitor cells enriched with exogenous mitochondria. In another embodiment, the exogenous mitochondria are isolated, derived, or partially purified from human placenta. In another embodiment, the composition is frozen / thawed. In some embodiments, the stem and / or progenitor cells are frozen / thawed prior to enrichment. In other embodiments, the exogenous mitochondria are frozen / thawed prior to enrichment of the stem and / or progenitor cells. In one embodiment, the stem and / or progenitor cells have been subjected to at least one freeze-thaw cycle after being enriched with exogenous mitochondria. In another embodiment, the stem and / or progenitor cells are selected from the group consisting of pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, hematopoietic progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, CD34 + cells, CD34 + In various embodiments, the stem and / or progenitor cells are selected from the group consisting of CD34 + Cells or CD34 + In one embodiment, the stem and / or progenitor cells are derived from whole blood, a blood fraction, peripheral blood, umbilical cord blood, bone marrow, or blood mobilized bone marrow cells. In one embodiment, the exogenous mitochondria are isolated, derived, or purified from placenta, placental cells grown in culture, or blood cells.

[0015] In another embodiment, the MDS disease or disorder is selected from the group of myelodysplastic syndrome with single lineage dysplasia (MDS-SLD), myelodysplastic syndrome with multilineage dysplasia (MDS-MLD), myelodysplastic syndrome with ringed sideroblasts (MDS-RS), myelodysplastic syndrome with isolated del(5q), myelodysplastic syndrome with excess blasts (MDS-EB), unclassifiable myelodysplastic syndrome (MDS-U), and acute myeloid leukemia (AML). In one embodiment, the MDS symptoms are selected from the group consisting of shortness of breath, weakness, fatigue, pallor, anemia, thrombocytopenia, leukopenia, bruising, bleeding, petechiae, ineffective hematopoiesis, blood cytopenia, clonal instability, and any combination thereof. In another embodiment, the subject is being treated or has been treated with an MDS therapy. In some embodiments, the MDS treatment is a hypomethylating agent, an erythropoietin stimulating agent (ESA), a granulocyte colony stimulating factor (G-CSF), azacitidine, decitabine, immunosuppressive therapy (IST), luspatercept, or a combination thereof.

[0016] In another embodiment, mitochondrial disease is a group of disorders caused by dysfunctional mitochondria. Mitochondrial disease can be caused by mutations in mitochondrial DNA that affect mitochondrial function. Other causes of mitochondrial disease are mutations in genes in nuclear DNA whose gene products are imported into mitochondria (mitochondrial proteins) and acquired mitochondrial conditions. Mitochondrial diseases have unique characteristics due to both the way in which the disease is often inherited and the extremely important importance of mitochondria to cellular function. A subclass of these diseases with neuromuscular disease symptoms is often called mitochondrial myopathy.

[0017] In another embodiment, the present invention provides a method of reducing symptoms associated with myelodysplastic syndrome (MDS) disease or disorder in a subject, comprising administering exogenous mitochondrial enriched stem and / or progenitor cells to the subject. In one embodiment, the subject has one or more symptoms associated with MDS disease or disorder. In various embodiments, the one or more symptoms associated with MDS disease or disorder are selected from the group consisting of shortness of breath, weakness, fatigue, pallor, anemia, thrombocytopenia, leukopenia, subcutaneous bleeding, bleeding, petechiae, ineffective hematopoiesis, blood cytopenia, myelodysplasia, lymphopenia, clonal instability, and any combination thereof.

[0018] In an additional embodiment, the present invention provides a method of preventing progression of a myelodysplastic syndrome (MDS) disease or disorder in a subject, comprising administering to the subject exogenous mitochondrial enriched stem and / or progenitor cells.

[0019] In a further embodiment, the present invention provides a method of treating a myelodysplastic syndrome (MDS) disease or disorder in a subject, comprising administering to the subject exogenous mitochondrial enriched stem and / or progenitor cells.

[0020] In one embodiment, administration of exogenous mitochondria-enriched stem and / or progenitor cells prevents disease progression and / or improves survival.

[0021] In one embodiment, the invention provides a method of restoring hematopoietic lineage function or cell numbers in a subject in need thereof, comprising administering to the subject exogenous mitochondrial-enriched stem and / or progenitor cells. In certain embodiments, restoration of hematopoietic function or cell numbers is characterized by improved cell differentiation, amelioration of anemia, reduction in the number of blasts, reduction in the number of ringed sideroblasts, and / or reduction in the need for blood transfusions.

[0022] In one embodiment, the improved cell differentiation is achieved by improved CD34+ Includes erythroid differentiation.

[0023] In one embodiment, the stem and / or progenitor cells are autologous. In one embodiment, the stem and / or progenitor cells are allogeneic. In another embodiment, the exogenous mitochondria-enriched stem and / or progenitor cells colonize bone marrow to establish new hematopoietic colonies. In another embodiment, the method further comprises administering to the subject an MDS treatment selected from hypomethylating agents, erythropoietin stimulating agents (ESAs), erythroid maturation agents, immunosuppressive therapy (IST), growth factors, immunomodulatory agents, nucleoside analogs, transfusions, bone marrow transplants, or combinations thereof. In some embodiments, the MDS treatment is selected from the group consisting of azacitidine, decitabine, cedazuridine, luspatercept, granulocyte colony stimulating factor (G-CSF), lenalidomide, epoetin, and darbepoetin, or combinations thereof. In one embodiment, the exogenous mitochondria constitute at least 1% of the total mitochondrial content in the mitochondria-enriched stem and / or progenitor cells. In another embodiment, administration of the mitochondria-enriched stem and / or progenitor cells is by intravenous, intraperitoneal, intraarterial or intramuscular administration, or by direct injection into bone marrow. In one embodiment, at least 5×10 5 ~5×10 9 The mitochondrial-enriched stem and / or progenitor cells are administered to the subject. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram of the whole bone marrow augmentation protocol described herein. [Diagram 2] 1 shows the effect of MAT augmentation on MDS cell differentiation and proliferation. [Figure 3A] Figures 3A-3B show red blood cell counts in low-risk MDS cells and intermediate-risk MDS cells before and after MAT augmentation. Figure 3A shows red blood cell counts in CD34 purified cells. Figure 3B shows red blood cell counts in total bone marrow cells. [Figure 3B]See legend to Figure 3A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Detailed Description of the Invention The present invention is based on the seminal discovery that mitochondria-enriched cells are useful for the treatment of myelodysplastic syndromes (MDS), diseases and disorders. The present invention provides pharmaceutical compositions of mitochondrially-enriched stem and / or progenitor cells, methods of treating myelodysplastic syndromes, and methods of using mitochondrially-enriched stem and / or progenitor cells to alleviate symptoms of and / or prevent the progression of MDS.

[0026] Before the compositions and methods of the present invention are described, it is to be understood that the invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0027] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein, which will become apparent to those of skill in the art upon reading this disclosure, or otherwise.

[0028] As used herein, the term "about" in connection with a numerical value is meant to include any additional numerical values ​​reasonably close to the indicated numerical value. For example, based on the context, the value may vary up or down by 5-10%. For example, a value of about 100 means 90-110 (or any value between 90-110).

[0029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but it will be understood that modifications and variations are within the spirit and scope of this disclosure. The following describes preferred methods and materials.

[0031] In one embodiment, the invention provides a pharmaceutical composition comprising stem and / or progenitor cells enriched in exogenous mitochondria and a pharma- ceutically acceptable carrier, wherein the stem and / or progenitor cells are obtained from a subject having a myelodysplastic syndrome (MDS) disease, disorder, or symptoms thereof, and the exogenous mitochondria are obtained from a donor without MDS disease, disorder, or symptoms thereof.

[0032] As used herein, the term "pharmaceutical composition" refers to a formulation containing an active ingredient and, optionally, a pharma- ceutically acceptable carrier, diluent or excipient. The term "active ingredient" may refer interchangeably to "effective ingredient" and is intended to refer to any agent capable of inducing a desired effect upon administration. Examples of active ingredients include, but are not limited to, cells or biological tissues, compounds, drugs, therapeutic agents, small molecules, and the like.

[0033] The pharmaceutical compositions described herein include stem cells and / or progenitor cells enriched with exogenous mitochondria. As used herein, the term "stem cells and / or progenitor cells enriched with exogenous mitochondria" can be used interchangeably with the term "mitochondria-enriched stem cells" or "mitochondria-enriched progenitor cells" and refers to a population of stem cells that have been contacted with exogenous mitochondria, resulting in some or all of the stem cells containing exogenous mitochondria.

[0034] As used herein, the term "stem cell" generally refers to any mammalian stem cell. Stem cells are undifferentiated cells that can differentiate into other types of cells and divide to produce more stem cells of the same type. Stem cells can be totipotent or pluripotent. "Stem cells" generally refer to all stem cells naturally found in a subject and all stem cells produced or derived from outside the body. "Progenitor cells", such as stem cells, tend to differentiate into a specific type of cell, but are more specific than stem cells already and can be encouraged to differentiate into their "target" cells. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times. As used herein, the term "human stem cells" further includes "progenitor cells" and "non-fully differentiated stem cells".

[0035] By "pharmacologically acceptable" it is meant that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation, not deleterious to the recipient thereof, and not deleterious to the activity of the active ingredients of the formulation. Pharmaceutically acceptable carriers, excipients or stabilizers are well known in the art, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, m-cresol, and the like), low molecular weight (less than about 10 residues) polypeptides, serum The carrier may include proteins such as albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.Examples of excipients include, but are not limited to, anti-adherents such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins such as gelatin and synthetic polymers, lubricants such as talc and silica, and preservatives such as antioxidants, vitamin A, vitamin E, vitamin C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0036] As used herein, the term "donor" refers to a provider who provides exogenous cells or mitochondria. In some embodiments, the donor does not suffer from a disease or disorder or does not suffer from the same disease or disorder as the subject. In certain embodiments, the donor is a subject and the cells and / or mitochondria are autologous. In other embodiments, the donor is not a subject and the cells and mitochondria are allogeneic.

[0037] The term "exogenous" or "isolated exogenous" with respect to mitochondria refers to mitochondria from a source outside of the recipient cells. For example, in some embodiments, the exogenous mitochondria are derived from or isolated from donor cells that are different from the donor of the recipient cells. In some embodiments, the exogenous mitochondria are derived from or isolated from donor cells from the same subject as the recipient cells. For example, the exogenous mitochondria can be purified, isolated or obtained from donor cells and then introduced into recipient cells from the same subject or a different donor as the donor cells, making the exogenous mitochondria autologous and allogeneic, respectively. In certain embodiments, the exogenous mitochondria are whole mitochondria.

[0038] In some embodiments, the exogenous donor cells are stem and / or progenitor cells, hematopoietic blasts, or any combination thereof.

[0039] In some embodiments, the present invention provides compositions comprising stem cells derived from MDS patients enriched with ex vivo isolated healthy mitochondria. The term "myelodysplastic syndrome" or "MDS" refers to a heterogeneous group of closely related clonal hematopoietic disorders that are common in the elderly. It is known that during aging, mitochondria accumulate mutations and deletions in mitochondrial DNA, leading to dysfunction in multiple organ systems, including bone marrow. All of them are characterized by one or more peripheral blood cytopenias. Bone marrow is usually highly cellular, although hypocellular bone marrow resembling aplastic anemia can rarely be seen. Bone marrow cells exhibit abnormal morphology and maturation (dysmyelopoiesis), resulting in ineffective blood cell production. MDS affects hematopoiesis at the stem cell level, as indicated by chromosomal abnormalities, molecular mutations, and morphological and physiological abnormalities in the maturation and differentiation of one or more of the hematopoietic cell lineages.

[0040] Myelodysplastic syndromes (MDS) are a group of diseases in which immature blood cells in the bone marrow fail to mature and become healthy blood cells. The term "MDS" encompasses multiple diseases and disorders, some of which may progress to acute myeloid leukemia.

[0041] The terms "disease" and "disorder" refer to any affliction that is not considered normal or different from a physiological state. Diseases and disorders can affect virtually any organ, tissue, or function in the body. Non-limiting examples of diseases and conditions include, for example, cancer and blood diseases and disorders. As used herein, the term "subject suffering from a disease or disorder" or "subject having a disease or disorder" refers to a human subject experiencing the debilitating effects caused by a particular condition. The disorder can refer to cancer, age-related disorders, or blood disorders, as well as other diseases or disorders.

[0042] The term "MDS disease" or "MDS disorder" is understood to refer to any of the diseases and disorders caused by blood cells that are insufficiently formed or do not function properly. Non-limiting examples of MDS diseases include: - Myelodysplastic syndrome with single lineage dysplasia (MDS-SLD) occurs when one type of blood cell, such as white blood cells, red blood cells, or platelets, is in low numbers and appears abnormal under a microscope. This type of MDS is uncommon and rarely progresses to AML; - Myelodysplastic syndrome with multilineage dysplasia (MDS-MLD) occurs when two or three types of blood cells are in small or abnormal amounts. This is the most common form of MDS; - Myelodysplastic syndrome with ringed sideroblasts (MDS-RS) occurs when red blood cells in the bone marrow contain excess iron rings in combination with a low number of one or more types of blood cells. MDS-RS is diagnosed when at least 15% of early red blood cells are ringed sideroblasts or at least 5% of cells also have a mutation in the SF3B1 gene; - Myelodysplastic syndrome with isolated del(5q) chromosomal abnormality occurs when a small number of red blood cells are missing part of chromosome 5. This type of MDS is uncommon and rarely progresses to AML; - Myelodysplastic syndrome with excess blasts (MDS-EB) occurs when small numbers of any of the three types of blood cells, which look abnormal under a microscope and are very immature (blasts) are found in the blood and bone marrow. MDS-EB1 occurs when blasts make up 5%-9% of the cells in the bone marrow or 2%-4% of the cells in the blood. MDS-EB2 occurs when blasts make up 10%-19% of the cells in the bone marrow or 5%-19% of the cells in the blood. This type of MDS is fairly common and is more likely to progress to AML; - Myelodysplastic syndrome, undifferentiated (MDS-U) occurs when the number of one or more types of mature blood cells is reduced and the cells may look abnormal under a microscope. Often, blood cells appear normal, but analysis may reveal that the cells have DNA changes associated with myelodysplastic syndrome; - Acute myeloid leukemia (AML); and - Chronic myelomonocytic leukemia (CMML) Includes.

[0043] As used herein, "leukemia" refers to a blood cancer caused by the rapid production of abnormal white blood cells. Examples of leukemia include acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia, chronic myeloid leukemia, and hairy cell leukemia.

[0044] In one embodiment, stem and / or progenitor cells enriched in exogenous mitochondria are produced by a method comprising contacting stem and / or progenitor cells with exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the stem and / or progenitor cells.

[0045] As used herein, the term "contacting" refers to bringing mitochondria and a cell (e.g., stem and / or progenitor cells) into sufficient proximity to facilitate or stimulate entry of exogenous mitochondria into the cell. The terms introducing or inserting mitochondria into a cell (e.g., stem and / or progenitor cells) are used interchangeably with the term contacting.

[0046] As used herein, the phrases "conditions that allow exogenous mitochondria to enter stem cells" and "conditions that allow exogenous mitochondria to enter progenitor cells" generally refer to parameters such as time, temperature, centrifugation, culture medium, and proximity between mitochondria and recipient cells. For example, human cells and human cell lines are typically incubated in liquid medium and kept in a sterile environment, such as a tissue culture incubator, at 37° C. and 5% CO2 atmosphere. According to alternative embodiments disclosed and exemplified herein, cells may be incubated at room temperature in saline supplemented with human serum albumin.

[0047] In some embodiments, the conditions that allow exogenous mitochondria to enter the stem and / or progenitor cells include incubating the stem and / or progenitor cells with the exogenous mitochondria for a time ranging from about 0.5 to 30 hours at a temperature ranging from about 4 to 37° C.

[0048] In certain embodiments, the cells are incubated with the exogenous mitochondria for a time ranging from 0.5 to 30 hours at a temperature ranging from about 4 to 37° C. In certain embodiments, the cells are incubated with the exogenous mitochondria for a time ranging from about 0.5 to 30 hours or about 5 to 25 hours. In certain embodiments, the incubation is for about 0.5 to 20 hours. In certain embodiments, the incubation is for about 20 to 30 hours. In some embodiments, the incubation is for at least about 0.5, 1, 5, 10, 15, 20, 21, 22, 23, or 24 hours. In other embodiments, the incubation is for up to 5, 10, 15, 20, or 30 hours. In certain embodiments, the incubation is for 24 hours. In certain embodiments, the incubation is for until the mitochondrial content in the cells is increased by an average of 1% to 45% compared to their initial mitochondrial content.

[0049] In some embodiments, incubation is at room temperature (16° C.-30° C.). In other embodiments, incubation is at 37° C. In certain embodiments, incubation is at 4° C. In some embodiments, incubation is in a 5% CO2 atmosphere. In other embodiments, incubation does not include the addition of CO2 above the level present in air.

[0050] In yet further embodiments, the incubation is performed in culture medium. In some embodiments, the culture medium is supplemented with human serum albumin (HSA). In certain embodiments, the incubation is performed in a medium that maintains mitochondrial integrity. In additional embodiments, the incubation is performed in saline supplemented with HSA. According to certain exemplary embodiments, the conditions that allow exogenous mitochondria to enter human stem cells, thereby enriching said human exogenous mitochondria in said human stem cells, include incubation at room temperature in saline supplemented with 4.5% human serum albumin.

[0051] In certain embodiments, the incubation is at 37° C. In certain embodiments, the incubation is for at least 1 hour. In certain embodiments, the incubation is for at least 6 hours. In certain embodiments, the incubation is for at least 12 hours. In certain embodiments, the incubation is for 12-24 hours.

[0052] As used herein, the term "enrich" refers to any action designed to increase the mitochondrial content of a cell or cell population, e.g., the number of intact mitochondria, or the mitochondrial functionality of a mammalian cell or cell population. In some embodiments, stem and / or progenitor cells enriched with exogenous mitochondria exhibit enhanced function compared to the same stem and / or progenitor cells prior to enrichment.

[0053] The terms "enrich" and "enrichment" as used herein refer to any action taken ex vivo that increases the mitochondrial content of a human cell or cell population, e.g., the number of intact, functional, or healthy mitochondria. According to the principles of the present invention, exogenous mitochondria are introduced into human stem and / or progenitor cells, such that these cells are enriched with exogenous mitochondria. According to some embodiments, the exogenous mitochondria constitute more than 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, or 20% of the total mitochondria in the mitochondria-enriched stem and / or progenitor cells.

[0054] As used herein, the term "mitochondrial content" refers to the amount of mitochondria in a cell or the average amount of mitochondria in a plurality of cells. As used herein, the term "increased mitochondrial content" refers to a mitochondrial content that is detectably higher than the mitochondrial content of the cell prior to mitochondrial enrichment.

[0055] In certain embodiments, the mitochondrial content of cells enriched with exogenous mitochondria is detectably higher than that of naive cells. According to various embodiments, the mitochondrial content of the mitochondrial-enriched stem cells is at least 0.5%, 1%, 5%, 10%, 25%, 50%, 100%, 200% or more higher than that of the cells prior to mitochondrial enrichment. In certain embodiments, the stem cells are freshly used. In certain embodiments, the stem and / or progenitor cells are frozen and thawed. In certain embodiments, the term "detectably higher" as used herein refers to a statistically significant increase between normal and increased values. In certain embodiments, the term "detectably higher than" as used herein refers to a non-pathological increase, i.e., a level that does not reveal pathological symptoms associated with substantially higher values. In certain embodiments, the term "increased" as used herein refers to a value that is 1.05-fold, 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold or more higher than the corresponding value found in the corresponding cells prior to enrichment or in the corresponding mitochondria of a healthy subject or plurality of healthy subjects (e.g., stem and progenitor cells) prior to mitochondrial enrichment.

[0056] In certain embodiments, the mitochondrial content of cells or mitochondria-enriched stem and / or progenitor cells is determined by determining the content of citrate synthase. In certain embodiments, the mitochondrial content of naive cells or enriched cells is determined by determining the activity level of citrate synthase. In certain embodiments, the mitochondrial content of naive cells or enriched cells is correlated with the content of citrate synthase. In certain embodiments, the mitochondrial content of naive cells or enriched cells is correlated with the activity level of citrate synthase. CS activity can be measured by commercially available kits.

[0057] In one embodiment, the conditions that allow exogenous mitochondria to enter the target cells are determined by subjecting the target cells to at least one of the following conditions: 6It involves incubation with exogenous mitochondria at a ratio of approximately 0.044 to 176 mU of citrate synthase (CS) activity per cell.

[0058] Citrate synthase (CS) is localized in the mitochondrial matrix but is encoded by nuclear DNA. It is involved in the first step of the Krebs cycle and is commonly used as a quantitative enzyme marker to indicate the presence of intact mitochondria (Larsen S. et al., J. Physiol., 2012, Vol. 590(14), pages 3349-3360; Cook GA et al., Biochim. Biophys. Acta., 1983, Vol. 763(4), pages 356-367).

[0059] Mitochondrial dosage, as interpreted herein, can be expressed in CS activity units or mtDNA copy number, other quantifiable measures of mitochondrial mass. A "CS activity unit" is defined as the amount that allows the conversion of 1 micromole of substrate in 1 minute in a reaction volume of 1 mL.

[0060] In some embodiments, exogenous mitochondrial enrichment of cells (e.g., stem and / or progenitor cells) provides an increase in citrate synthase (CS) activity of at least 0.044 up to 176 milliunits (mU) per million cells, an increase in CS activity of at least 0.088 up to 176 mU per million cells, an increase in CS activity of at least 0.2 up to 150 mU per million cells, an increase in CS activity of at least 0.4 up to 100 mU per million cells, or an increase in CS activity of at least 0.5 up to 100 mU per million cells. The method includes introducing mitochondria into a cell at a dose of CS activity, at least 0.6 to a maximum of 80 mU of CS activity per million cells, at least 0.7 to a maximum of 50 mU of CS activity per million cells, at least 0.8 to a maximum of 20 mU of CS activity per million cells, at least 0.88 to a maximum of 17.6 mU of CS activity per million cells, or at least 0.44 to a maximum of 17.6 mU of CS activity per million cells.

[0061] In some embodiments, the conditions that allow exogenous mitochondria to enter the target cells are selected from the group consisting of: inducing the target cells to undergo 10 6 In various embodiments, the concentration of exogenous mitochondria is between about 10 and 50 mU of citrate synthase (CS) activity per cell. 6 Citrate synthase (CS) activity of approximately 0.88, 4.4, 17.6, or 35 mU per cell.

[0062] In some embodiments, the concentration of exogenous mitochondria is at least 1 million to up to 400 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 1 million to up to 200 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 5 million to up to 150 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 5 million to up to 150 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 5 million to up to 100 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 5 million to up to 75 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 10 million to up to 100 million mitochondrial particles per million cells. In some embodiments, the concentration of exogenous mitochondria is at least 10 million to up to 75 million mitochondrial particles per million cells.In some embodiments, the concentration of exogenous mitochondria is at least 10 million to up to 50 million mitochondrial particles per million cells.In some embodiments, the concentration of exogenous mitochondria is 1 million mitochondrial particles per million cells as measured by MTG method.

[0063] In another aspect, stem and / or progenitor cells enriched with exogenous mitochondria have an increased content of at least one mitochondrial protein; an increased rate of oxygen (O2) consumption; an increased activity level of citrate synthase, succinate, or tryptamine; an increased rate of adenosine triphosphate (ATP) production; an increased mitochondrial DNA content; increased colony forming unit activity in liquid or solid medium; an increased proliferation rate; an increased differentiation rate; or any combination thereof, compared to stem and / or progenitor cells prior to mitochondria enrichment.

[0064] Mitochondrial DNA content can be measured by quantitative or digital PCR of mitochondrial genes before and after mitochondrial enrichment, normalized to nuclear genes.

[0065] In certain circumstances, prior to mitochondrial enrichment, the same cells serve as a control to determine the enrichment level.

[0066] The term "increased mitochondrial DNA content" as used herein refers to a content of mitochondrial DNA that is detectably higher than the mitochondrial DNA content in cells prior to mitochondrial enrichment. "Normal mitochondrial DNA" in the specification and claims refers to mitochondrial DNA that does not have / does not have mutations or deletions known to be associated with mitochondrial disease. The term "normal oxygen (O2) consumption rate" as used herein refers to the average O2 consumption of cells from a healthy individual. The term "normal citrate synthase activity level" as used herein refers to the average activity level of citrate synthase in cells from a healthy individual. The term "normal adenosine triphosphate (ATP) production rate" as used herein refers to the average ATP production rate in cells from a healthy individual.

[0067] According to some embodiments, the mitochondrial content is determined by measuring the content of mitochondrial proteins. According to some embodiments, the mitochondrial proteins are proteins encoded by mitochondrial DNA. According to some embodiments, the mitochondrial proteins are proteins encoded by nuclear DNA and localized in mitochondria. Non-limiting examples are citrate synthase (CS), cytochrome C oxidase (COX1), succinate dehydrogenase complex flavoprotein subunit A (SDHA), tryptamine, succinate.

[0068] In some embodiments, the conditions that allow exogenous mitochondria to enter the target cells include incubating the target cells with exogenous mitochondria at a ratio of about 1 million to 100 million mitochondrial particles per million cells. In certain embodiments, the conditions that allow exogenous mitochondria to enter the target cells include incubating the target cells with exogenous mitochondria at a ratio of about 10 million to 50 million mitochondrial particles per million cells.

[0069] In some embodiments, identification / distinguishing of endogenous from exogenous mitochondria can be performed by various means after the exogenous mitochondria are introduced into a target cell, including, but not limited to, identifying differences in mtDNA sequences between endogenous and exogenous mitochondria, e.g., different haplotypes.

[0070] In certain embodiments, the endogenous and exogenous mitochondria are from the same haplogroup.

[0071] In other embodiments, the endogenous and exogenous mitochondria are from different haplogroups.

[0072] The degree of exogenous mitochondrial enrichment of cells can be measured by functional and / or enzymatic assays, including but not limited to oxygen (O2) consumption rate, citrate synthase content or activity level, adenosine triphosphate (ATP) production rate. In some embodiments, the degree of exogenous mitochondrial enrichment of cells can be confirmed by detection of mitochondrial DNA. According to some embodiments, the degree of exogenous mitochondrial enrichment of cells can be determined by the level of heteroplasmy change and / or the copy number of mtDNA per cell.

[0073] Heteroplasmy is the presence of more than one type of mitochondrial DNA in a cell or individual. Heteroplasmy level is the ratio of mutant mtDNA molecules to wild type / functional mtDNA molecules and is an important factor in considering the severity of mitochondrial disease. Lower heteroplasmy levels (sufficient amount of functioning mitochondria) are associated with a healthy phenotype, whereas higher heteroplasmy levels (insufficient amount of functioning mitochondria) are associated with disease states. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 1% lower than the heteroplasmy level of the stem cells before enrichment. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 3% lower than the heteroplasmy level of the stem cells before enrichment. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 5% lower than the heteroplasmy level of the stem cells before enrichment. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 10% lower than the heteroplasmy level of the stem cells before enrichment. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 20% lower than the heteroplasmy level of the stem cells before enrichment. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 30% lower than the heteroplasmy level of the stem cells before enrichment. In some embodiments, the heteroplasmy level of the mitochondrially enriched stem cells is at least 50% lower than the heteroplasmy level of the stem cells before enrichment.

[0074] TMRM (tetramethylrhodamine methyl ester) or the related TMRE (tetramethylrhodamine ethyl ester) are cell-permeable fluorogenic dyes commonly used to assess mitochondrial function in live cells by identifying changes in mitochondrial membrane potential. According to some embodiments, the level of enrichment can be determined by staining with TMRE or TMRM. Other fluorogenic dyes known in the art can also be used.

[0075] According to some embodiments, the mitochondria comprise intact mitochondria, disrupted mitochondria, and / or mitochondrial components selected from the group consisting of mitochondrial proteins, mitochondrial nucleic acids, mitochondrial lipids, and mitochondrial sugars.

[0076] According to some embodiments, mitochondrial membrane integrity can be determined by any method known in the art. In a non-limiting example, mitochondrial membrane integrity is measured using cytochrome c release test, tetramethylrhodamine methyl ester (TMRM), or tetramethylrhodamine ethyl ester (TMRE) fluorescent probe. Each possibility represents a separate embodiment of the present invention. Mitochondria that show TMRM or TMRE staining under a microscope have an intact mitochondrial outer membrane. As used herein, the term "mitochondrial membrane" refers to a mitochondrial membrane selected from the group consisting of inner mitochondrial membrane, outer mitochondrial membrane, and both.

[0077] In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched stem cells and / or progenitor cells is determined by sequencing at least a statistically representative portion of the total mitochondrial DNA in the cells and determining the relative levels of host / endogenous mitochondrial DNA and exogenous mitochondrial DNA. In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched stem cells and / or progenitor cells is determined by single nucleotide polymorphism (SNP) analysis. In certain embodiments, the largest mitochondrial population and / or the largest mitochondrial DNA population is the host / endogenous mitochondrial population and / or the host / endogenous mitochondrial DNA population, and / or the second largest mitochondrial population and / or the second largest mitochondrial DNA population is the exogenous mitochondrial population and / or the exogenous mitochondrial DNA population.

[0078] According to certain embodiments, the exogenous mitochondrial enrichment of stem cells and / or progenitor cells can be determined by conventional assays recognized in the art. In certain embodiments, the level of mitochondrial enrichment in mitochondrially enriched human stem cells and / or progenitor cells is determined by (i) the level of host / endogenous mitochondrial DNA and exogenous mitochondrial DNA, (ii) the level of mitochondrial protein, (iii) the level of CS activity, or (iv) any combination of (i), (ii) and (iii). Methods for determining these various parameters are well known in the art.

[0079] In certain embodiments, the level of mitochondrial enrichment in the mitochondrial-enriched stem and / or progenitor cells is determined by at least one of: (i) the level of host mitochondrial DNA and exogenous mitochondrial DNA, (ii) the level of citrate synthase activity, (iii) the level of succinate dehydrogenase complex flavoprotein subunit A (SDHA) or cytochrome C oxidase (COX1), (iv) oxygen (O2) consumption rate, (v) adenosine triphosphate (ATP) production rate, or (vi) any combination thereof. Each possibility represents a separate embodiment of the present invention. Methods for measuring these various parameters are well known in the art.

[0080] In some embodiments, the exogenous human mitochondria enrichment of cells comprises washing the mitochondria-enriched cells (e.g., mitochondria-enriched stem and / or progenitor cells) after incubation of the cells with the exogenous human mitochondria. This step provides mitochondria-enriched cells that are substantially free of cellular debris or mitochondrial membrane debris and mitochondria that did not enter the stem and / or progenitor cells. In some embodiments, washing comprises centrifugation of the mitochondria-enriched cells after incubation of the human cells with the exogenous human mitochondria. According to some embodiments, the method produces mitochondria-enriched cells separated from free mitochondria, i.e., mitochondria that did not enter the cells or other cellular debris, and the pharmaceutical composition contains the mitochondria-enriched cells separated from free mitochondria. According to some embodiments, the method produces mitochondria-enriched stem and / or progenitor cells that do not contain detectable amounts of free mitochondria, and the pharmaceutical composition contains the same.

[0081] As used herein, the term "increased oxygen (O2) consumption rate" refers to an oxygen (O2) consumption rate that is detectably higher than the oxygen (O2) consumption rate prior to mitochondrial enrichment.

[0082] As used herein, the term "increased content of at least one mitochondrial protein" refers to a content of nuclear- or mitochondrially-encoded mitochondrial proteins, such as CS, COX1 and SDHA, that is detectably higher than the content of said mitochondrial proteins in cells prior to mitochondrial enrichment.

[0083] As used herein, the term "increased citrate synthase content or activity level" refers to a citrate synthase content or activity level that is detectably higher than the citrate synthase content or activity level in a cell prior to mitochondrial enrichment.

[0084] As used herein, the term "increased adenosine triphosphate (ATP) production rate" refers to an adenosine triphosphate (ATP) production rate that is detectably higher than the adenosine triphosphate (ATP) production rate prior to mitochondrial enrichment.

[0085] In one embodiment, the exogenous mitochondria constitute at least 0.5% of the total mitochondria in the stem and / or progenitor cells enriched for exogenous mitochondria.

[0086] In certain embodiments, the exogenous mitochondria constitute at least 0.5% of the total mitochondrial content in the mitochondria-enriched cells. In certain embodiments, the exogenous mitochondria constitute at least 10% of the total mitochondrial content in the mitochondria-enriched target cells. In some embodiments, the exogenous mitochondria constitute at least about 0.5%, 1%, 2%, 3%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, or 50% of the total mitochondrial content in the mitochondria-enriched target cells. In certain embodiments, the total amount of mitochondrial protein in the isolated mitochondria is 10-90%, 20-80%, 20-70%, 40-70%, 20-40%, or 20-30% of the total amount of cellular protein. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the total amount of mitochondrial protein in the isolated mitochondria is 20%-80% of the total amount of cellular protein in the sample. In certain embodiments, the total amount of mitochondrial protein in the isolated mitochondria is between 10% and 80% of the combined weight of mitochondria and other subcellular fractions, while in other embodiments, the total amount of mitochondrial protein in the isolated mitochondria is greater than 80% of the combined weight of mitochondria and other subcellular fractions.

[0087] In another embodiment, the exogenous mitochondria are isolated, derived or partially purified human mitochondria.

[0088] As used herein, the terms "isolated," "derived," and "partially purified" in the context of mitochondria include exogenous mitochondria that have been at least partially purified from other cellular components. The total amount of mitochondrial protein in exogenous isolated or partially purified mitochondria is about 10%-90% of the total amount of cellular protein in the sample.

[0089] As used herein, the term "functional mitochondria" refers to mitochondria that exhibit normal mitochondrial DNA (mtDNA) and / or parameters indicative of normal, non-pathological levels of activity. Mitochondrial activity, such as membrane potential, O2 consumption, ATP production, and citrate synthase (CS) activity levels, can be measured by a variety of methods well known in the art.

[0090] In one embodiment, the composition is frozen / thawed. In some embodiments, the stem cells and / or progenitor cells are frozen / thawed prior to enrichment. In other embodiments, the exogenous mitochondria are frozen / thawed prior to enrichment of the stem cells and / or progenitor cells. In another embodiment, the stem cells and / or progenitor cells are subjected to at least one freeze-thaw cycle after the exogenous mitochondria are enriched.

[0091] In some embodiments, the stem and / or progenitor cells are cultured and expanded in vitro. In certain embodiments, the stem and / or progenitor cells are subjected to at least one freeze-thaw cycle before or after mitochondrial enrichment.

[0092] In certain embodiments, the stem and / or progenitor cells are frozen and stored and used after thawing. In further embodiments, the exogenous mitochondria are frozen and stored and thawed before use. In further embodiments, the mitochondria-enriched stem and / or progenitor cells are used without freezing and storage. In yet further embodiments, the mitochondria-enriched stem and / or progenitor cells are used after freezing, storage and thawing. Suitable methods for freezing and thawing cell preparations to retain viability are well known in the art.

[0093] As used herein, the term "freeze-thaw cycle" refers to freezing exogenous mitochondria to a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a period of time, and thawing the exogenous mitochondria to room temperature or body temperature or any temperature above 0°C that allows for treatment of cells with the exogenous mitochondria. The term "room temperature" as used herein typically refers to a temperature between 18°C ​​and 25°C. The term "body temperature" as used herein refers to a temperature between 35.5°C and 37.5°C, preferably 37°C.

[0094] In another embodiment, the mitochondria subjected to freeze-thaw cycles are frozen at temperatures below -20°C, below -4°C, or below -70°C. According to another embodiment, the mitochondria are frozen gradually. According to one embodiment, the mitochondria are frozen by flash freezing. As used herein, the term "flash freezing" refers to rapid freezing of mitochondria by exposure to extremely low temperatures.

[0095] In another embodiment, the mitochondria subjected to the freeze-thaw cycle are frozen for at least 30 minutes before thawing. According to another embodiment, the freeze-thaw cycle includes freezing the exogenous mitochondria for at least 30, 60, 90, 120, 180, 210 minutes before thawing. Each possibility represents a separate embodiment of the invention. In another embodiment, the mitochondria subjected to the freeze-thaw cycle are frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48, 72, 96, or 120 hours before thawing. In another embodiment, the mitochondria subjected to the freeze-thaw cycle are frozen for at least 4, 5, 6, 7, 30, 60, 120, 365 days before thawing. According to another embodiment, the freeze-thaw cycle includes freezing the exogenous mitochondria for at least 1, 2, 3 weeks before thawing. According to another embodiment, the freeze-thaw cycle includes freezing the exogenous mitochondria for at least 1, 2, 3, 4, 5, 6, 12 months before thawing. Each possibility represents a separate embodiment of the present invention.

[0096] According to certain embodiments, thawing is performed at room temperature. In other embodiments, thawing is performed at body temperature. According to other embodiments, thawing is performed at a temperature that allows administration of mitochondria according to the methods of the present invention. According to other embodiments, thawing is performed gradually.

[0097] In another embodiment, the stem and / or progenitor cells are pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, hematopoietic progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, CD34 + cells, CD34 + a subset of cells, and any combination thereof.

[0098] In certain embodiments, the stem cells are pluripotent stem cells (PSCs). In other embodiments, the PSCs are non-embryonic stem cells. In some embodiments, the stem cells are induced PSCs (iPSCs). In certain embodiments, the stem cells are embryonic stem cells. In certain embodiments, the stem cells are derived from bone marrow cells. In certain embodiments, the stem cells are CD34 + In yet another embodiment, the stem cells are derived from blood. In a further embodiment, the stem cells are derived from umbilical cord blood. In certain embodiments, the stem cells obtained from a subject suffering from a disease or disorder or from a healthy subject are bone marrow cells or bone marrow derived stem cells.

[0099] As used herein, the term "pluripotent stem cells (PSCs)" refers to cells that can grow indefinitely and give rise to multiple cell types in the body. Totipotent stem cells refer to cells that can give rise to all other cell types in the body. Embryonic stem cells (ESCs) are totipotent stem cells, and induced pluripotent stem cells (iPSCs) are pluripotent stem cells.

[0100] As used herein, the term "induced pluripotent stem cells (iPSCs)" refers to a type of pluripotent stem cell that can be generated from human adult somatic cells. Some non-limiting examples of somatic cells from which iPSCs can be generated herein include hematopoietic stem cells or their progenitor cells.

[0101] As used herein, the term "embryonic stem cell (ESC)" refers to a type of totipotent stem cell derived from the inner cell mass of a blastocyst.

[0102] As used herein, the term "bone marrow cells" refers to all human cells naturally found in human bone marrow and to all cell populations naturally found in human bone marrow. The terms "bone marrow stem cells" and "bone marrow-derived stem cells" refer to stem and / or progenitor stem cell populations derived from bone marrow.

[0103] In some embodiments, the autologous or allogeneic human stem cells are pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs).

[0104] According to some embodiments, the human stem cells are derived from blood, umbilical cord blood, bone marrow, or blood mobilized bone marrow cells. Each possibility represents a separate embodiment of the present invention. In certain embodiments, the human stem cells are derived from bone marrow.

[0105] In certain embodiments, bone marrow-derived stem cells include bone marrow hematopoietic cells. As used herein, the term "bone marrow hematopoietic cells" refers to cells involved in myelopoiesis, e.g., cells involved in the production of bone marrow and all cells arising from bone marrow, i.e., all blood cells.

[0106] In certain embodiments, bone marrow-derived stem cells include erythropoietic cells. As used herein, the term "erythropoietic cells" refers to cells involved in erythropoiesis, e.g., cells involved in the production of red blood cells (erythrocytes).

[0107] In certain embodiments, bone marrow-derived stem cells include multipotent hematopoietic stem cells (HSCs). As used herein, the term "multipotent hematopoietic stem cells" or "hemopoietic cells" refers to stem cells that give rise to all other blood cells by the process of hematopoiesis.

[0108] In certain embodiments, bone marrow derived stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, bone marrow derived stem cells comprise mesenchymal stem cells. As used herein, the term "common myeloid progenitor cells" refers to cells that give rise to myeloid cells. As used herein, the term "common lymphoid progenitor cells" refers to cells that give rise to lymphocytes.

[0109] In certain embodiments, bone marrow-derived stem cells further comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof, with each possibility representing a separate embodiment of the present invention.

[0110] In certain embodiments, the bone marrow derived stem cells comprise bone marrow hematopoietic cells. In certain embodiments, the bone marrow derived stem cells comprise erythropoietic hematopoietic cells. In certain embodiments, the bone marrow derived stem cells comprise multipotent hematopoietic stem cells (HSCs). In certain embodiments, the bone marrow derived stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow derived stem cells comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. In certain embodiments, the stem cells comprise a plurality of human bone marrow stem cells obtained from peripheral blood.

[0111] Hematopoietic stem cells (HSCs) are stem cells that give rise to other blood cells. This process is called hematopoiesis. Hematopoietic stem cells give rise to different types of blood cells in lineages called myeloid and lymphoid. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes to platelets. Lymphoid cells include T cells, B cells, and natural killer cells.

[0112] In one embodiment, the stem and / or progenitor cells are derived from whole blood, blood fractions, peripheral blood, umbilical cord blood, bone marrow, or blood mobilized bone marrow cells.

[0113] In various embodiments, the stem and / or progenitor cells are CD34 + It is a cell.

[0114] The hematopoietic progenitor cell antigen CD34, known as the CD34 antigen, is a protein that in humans is encoded by the CD34 gene. CD34 is a group of differentiation antigens within cell surface glycoproteins that function as intercellular adhesion molecules. In certain embodiments, stem cells express the myeloid progenitor cell antigen CD34 (CD34 + In certain embodiments, the stem cells do not express CD34 (CD34 -In certain embodiments, the stem cells present the myeloid progenitor antigen CD34 on their outer membrane. + The cells are from umbilical cord blood. As used herein, the term "CD34 + "Hematopoietic stem cells" refers to hematopoietic stem cells, regardless of origin, that are characterized as CD34 positive. In certain embodiments, CD34 + The cells may be obtained from bone marrow, blood mobilized bone marrow cells, or umbilical cord blood.

[0115] CD34 is used to identify and isolate human hematopoietic stem and progenitor cells, for example for use in bone marrow transplantation. The compositions described herein contain CD34 enriched with exogenous mitochondria. + Contains stem and progenitor cells. Upon engraftment, mitochondria-enriched CD34 + The stem and progenitor cells can proliferate to form new colonies and repopulate the subject's bone marrow with healthy hematopoietic cells that differentiate from the stem and progenitor cells.

[0116] In certain embodiments, stem cells, including hematopoietic stem cells, are obtained from the peripheral blood of a subject suffering from a disease or disorder. In certain embodiments, stem cells are obtained from the peripheral blood of a healthy donor. As used herein, the term "peripheral blood" refers to blood circulating through the blood system.

[0117] As used herein, the term "autologous cells" or "cells that are autologous" refers to cells that are the subject's own. The term "autologous mitochondria" refers to mitochondria obtained from a subject's own cells or maternally related cells. The term "allogeneic cells" or "allogeneic mitochondria" refers to cells or mitochondria from a different donor individual.

[0118] The stem cells used in the composition are CD34 + Cells or CD34 + It is a subset of cells.

[0119] In some embodiments, the MDS disease or disorder is selected from the group consisting of myelodysplastic syndrome with single lineage dysplasia (MDS-SLD), myelodysplastic syndrome with multilineage dysplasia (MDS-MLD), myelodysplastic syndrome with ringed sideroblasts (MDS-RS), myelodysplastic syndrome with isolated del(5q), myelodysplastic syndrome with excess blasts (MDS-EB), myelodysplastic syndrome, unclassifiable (MDS-U), and acute myeloid leukemia (AML).

[0120] The subject with myelodysplastic syndrome may not show signs and symptoms at first.Over time, myelodysplastic syndrome may cause various non-specific symptoms.In one embodiment, the symptoms of MDS are selected from the group consisting of shortness of breath, weakness, fatigue, paleness, anemia, thrombocytopenia, leukopenia, subcutaneous bleeding, bleeding, petechiae, ineffective hematopoiesis, blood cytopenia, clonal instability, and any combination thereof.

[0121] The compositions described herein include stem cells obtained from a subject with MDS, regardless of the subject's medical history. The stem cells can be from a patient who has previously been treated for MDS but is no longer receiving treatment, a patient who is currently receiving treatment for symptoms of MDS, or a patient who has not yet received treatment.

[0122] In one embodiment, the subject is being treated or has been treated with an MDS therapy.

[0123] For subjects currently undergoing treatment, the compositions can be administered in combination with one or more MDS treatments. "Combination therapy," "in combination with," and the like refer to the simultaneous use of more than one pharmaceutical agent or treatment to increase response. The compositions of the present invention may be used in combination with other drugs or treatments used, for example, in the treatment of cancer. In particular, the compositions of the present invention can be administered in combination with any anti-MDS therapy. Such therapy may be administered before, simultaneously with, or after administration of the compositions of the present invention.

[0124] Treatment in lower-risk MDS (LR-MDS) focuses primarily on improving cytopenias and quality of life (QoL). Early intervention with current approaches has not shown any improvement in mortality or impact on reducing clonal evolution in LR-MDS. The goal of treatment in higher-risk MDS (HR-MDS) is to prevent disease progression and increase survival. For eligible patients, allogeneic hematopoietic cell transplantation (HCT) is the only potentially curative treatment. The standard of care for HR-MDS patients who are not candidates for HCT is the use of DNA hypomethylating agents (HMA) until disease progression or intolerance. Available MDS treatments are well known in the art (see, for example, myelodysplastic syndrome current treatment algorithm 2018 at www.nature.com / articles / s41408-018-0085-4.pdf) and include the orally administered immunomodulatory agent lenalidomide, the DNA hypomethylating agent nucleoside analogs (azacitidine, decitabine, and cedazuridine / decitabine), the erythrocyte maturation agent luspatercept, and the oral hypomethylating agent cedazuridine / decitabine. In addition to these agents, the erythropoietin-stimulating agents (ESAs) epoetin and darbepoetin are widely used off-label in MDS. Hematopoietic cell transplantation (HCT) is the only curative option, but the majority of patients are ineligible due to age and comorbidities. Other alternative or adjunctive treatments include the combination of coenzyme Q10 and carnitine, enhancing mitochondrial function, and IV / SC injections of ESAs (such as lenalidomide and luspatercept).

[0125] In some embodiments, the MDS treatment is a hypomethylating agent, an erythropoietin stimulating agent (ESA), a granulocyte colony stimulating factor (G-CSF), azacitidine, decitabine, immunosuppressive therapy (IST), luspatercept, or a combination thereof.

[0126] In another aspect, the exogenous mitochondria are isolated, derived or purified from human cells, hi some embodiments, the exogenous mitochondria are isolated, derived or purified from placenta, placental cells grown in culture, or blood cells.

[0127] In certain embodiments, the exogenous mitochondria are obtained from human cells or human tissue. In certain embodiments, the cells are selected from the group consisting of placenta, placental cells grown in culture, or blood cells. In some embodiments, the mitochondria are obtained from human stem cells. In some embodiments, the human cells are human somatic cells. In some embodiments, the human cells are cells grown in culture.

[0128] In some aspects, the present invention provides stem cells derived from MDS patients that are enriched for ex vivo isolated healthy mitochondria and methods of using the enriched cells in the treatment of MDS diseases, disorders and symptoms thereof.

[0129] In one embodiment, the present invention provides a method of alleviating symptoms associated with myelodysplastic syndrome (MDS) disease or disorder in a subject, comprising administering to the subject exogenous mitochondrial-enriched stem and / or progenitor cells.

[0130] In one embodiment, the subject has one or more of the symptoms associated with a disease or disorder of MDS.

[0131] In various embodiments, one or more of the MDS disease or disorder associated symptoms are selected from the group consisting of shortness of breath, weakness, fatigue, pallor, anemia, thrombocytopenia, leukopenia, bruising, hemorrhage, petechiae, ineffective hematopoiesis, blood cytopenias, myelodysplasia, lymphopenia, clonal instability, and any combination thereof.

[0132] In an additional embodiment, the present invention provides a method of preventing progression of a myelodysplastic syndrome (MDS) disease or disorder in a subject, comprising administering to the subject exogenous mitochondrial enriched stem and / or progenitor cells.

[0133] In a further embodiment, the present invention provides a method of treating a myelodysplastic syndrome (MDS) disease or disorder in a subject, comprising administering to the subject exogenous mitochondrial enriched stem and / or progenitor cells.

[0134] The term "treatment" is used interchangeably herein with the term "therapeutic method" and refers to both (1) therapeutic procedures or measures that cure, slow, alleviate, and / or halt the progression of a diagnosed condition or disorder, and (2) prophylactic / preventative measures. Those in need of treatment can include individuals who already have a particular medical disorder as well as those who may eventually suffer from the disorder (i.e., those in need of preventative measures).

[0135] The terms "therapeutically effective amount", "effective dose", "therapeutically effective dose", or "effective amount", etc., refer to an amount of a subject compound that induces a biological or medical response in a tissue, system, or human that is desired by a researcher, physician, or other clinician. Generally, the response is either an improvement in the patient's condition or a desired biological outcome (e.g., a reduction in symptoms associated with a myelodysplastic syndrome (MDS) disease or disorder). Such an amount should be sufficient to reduce symptoms associated with a myelodysplastic syndrome (MDS) disease or disorder. An effective amount can be determined as described herein.

[0136] The terms "administration of" and / or "administration" should be understood to mean providing a therapeutically effective amount of the pharmaceutical composition to a subject in need of treatment. The route of administration may be enteral, topical or parenteral. Thus, routes of administration include, but are not limited to, intravenous, intraperitoneal, intraarterial, intramuscular, infusion and direct injection into bone marrow. As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration. The pharmaceutical composition may be administered in various unit dosage forms depending on the method of administration. Suitable unit dosage forms include, but are not limited to, capsules, injections, implantable sustained release formulations, and lipid complexes. In another embodiment, administration of the mitochondria-enriched stem and / or progenitor cells is by intravenous, intraperitoneal, intraarterial or intramuscular administration.

[0137] In one embodiment, administration of exogenous mitochondria-enriched stem and / or progenitor cells prevents disease progression and / or improves survival.

[0138] "Preventing disease progression" and "enhancing survival" mean that the mitochondria-enriched stem cells allow stabilization or amelioration of a disease or its symptoms, thereby increasing the survival rate of a subject whose survival rate is reduced or limited by the disease. For example, the mitochondria-enriched stem cells allow restoration of red blood cell (RBC), platelet (PLT) and white blood cell (WBC) levels, restoration of normal or normalized blood cell counts and mononuclear cell differentiation. Such restoration is part of the restoration of functional hematopoietic lineage. In certain embodiments, restoration of hematopoietic function or cell counts is characterized by improved cell differentiation, amelioration of anemia, reduction in the number of blasts, reduction in the number of ringed sideroblasts and / or reduction in the need for blood transfusions.

[0139] In one embodiment, the improved cell differentiation is achieved by improving CD34 + Includes erythroid differentiation.

[0140] In one embodiment, the invention provides a method of restoring hematopoietic lineage function in a subject in need thereof, comprising administering to the subject exogenous mitochondria-enriched stem and / or progenitor cells.

[0141] In one embodiment, the stem and / or progenitor cells are autologous.

[0142] As used herein, the term "stem cells are autologous to the subject" refers to the subject's own cells. Cells are isolated from the subject and undergo ex vivo modification (e.g., enrichment of healthy mitochondria). These mitochondrial-enriched stem cells are then expanded, selected as necessary, and injected into the subject. After transplantation, mitochondrial-enriched stem cells are expanded in peripheral blood.

[0143] In another embodiment, stem and / or progenitor cells enriched in exogenous mitochondria populate the bone marrow to establish new hematopoietic colonies.

[0144] In another embodiment, the method further comprises administering to the subject an MDS therapy selected from the group consisting of hypomethylating agents, erythropoietin stimulating agents (ESAs), erythroid maturation agents, immunosuppressive therapy (IST), growth factors, immunomodulatory agents, nucleoside analogs, blood transfusions, bone marrow transplants, or combinations thereof. In some embodiments, the MDS therapy is selected from the group consisting of azacitidine, decitabine, cedazuridine, luspatercept, granulocyte colony stimulating factor (G-CSF), lenalidomide, epoetin, and darbepoetin, or combinations thereof.

[0145] In one embodiment, the exogenous mitochondria constitute at least 1% of the total mitochondrial content in the mitochondrially-enriched stem and / or progenitor cells.

[0146] In one embodiment, at least 5×10 5 ~5×10 9 The mitochondrial-enriched stem and / or progenitor cells are administered to a subject.

[0147] Presented below are examples that discuss mitochondrial enrichment in stem and progenitor cells intended for the applications discussed. The following examples are provided to further illustrate embodiments of the invention, but are not intended to limit the scope of the invention. While they are typical of those that may be used, other procedures, methodologies, or techniques known to those of skill in the art may alternatively be used. EXAMPLES

[0148] Example 1 In vitro effects of mitochondrial enrichment in MDS hematopoietic stem / progenitor cells MDS is a disorder in which HSPC differentiation is inhibited before terminal differentiation. Ineffective hematopoiesis in MDS results from increased susceptibility of clonal bone marrow progenitors to apoptosis. This can be triggered by intrinsic factors such as mitochondrial polarization due to iron retention in ringed sideroblasts. Patient cells harbor mtDNA mutations and have lower O2 consumption rates. Preclinical mouse polg models (accumulating mitochondrial mutations / deletions) indicate that mitochondrial dysfunction in HSCs is responsible for the MDS phenotype and is cell intrinsic.

[0149] To evaluate the effect of mitochondrial enrichment in vitro, the proliferation, differentiation potential, and viability of MDS hematopoietic stem / progenitor cells were measured in vitro, as well as the mitochondrial function of enriched cells was assessed. Preclinical evidence suggested that MAT promotes the differentiation and proliferation of HSPCs into the B cell lineage. Therefore, according to the study design shown in Figure 1, we evaluated whether HSPCs from MDS patients could be differentiated into erythroid and / or platelet lineages by MAT to alleviate cytopenias. CD34+ cells were harvested from blood and enriched with mitochondria.

[0150] CD34 + Cells were isolated from bone marrow mononuclear cells (BM MNC) of MDS patients with early or late stage MDS.

[0151] Enrichment Process CD34 +Cells (1M cells / mL) were treated with or without mitochondria isolated from placenta or other sources at concentrations of 0.88, 4.4, 17.6, and 35 mU of CS activity per M cells.

[0152] (Table 1) Example of sample evaluation TIFF2024546826000002.tif108128

[0153] CD34 cells were incubated in vitro in liquid culture medium (StemSpan SFEM II medium) containing StemSpan™ Erythroid Expansion Supplement to induce erythroid lineage or StemSpan Megakaryocyte Expansion Supplement to induce megakaryocyte + platelet lineage. Medium was changed every 3 days.

[0154] Flow cytometric analysis was performed every 3 days in liquid cultures for approximately 14 days of culture. Flow cytometric analysis was performed to enumerate hematopoietic stem and progenitor cells using the CD34 marker. Megakaryocyte markers CD41a platelet and CD42b were used to enumerate myeloid progenitor cells. Glycophorin A (APC) and CD71 (erythroid progenitor) markers were used to enumerate erythroid progenitor cells.

[0155] In addition, to assess CFU formation, CD34 cells were incubated in vitro in semi-solid agar culture medium (MethoCult Medium for CFU Assay) which induces differentiation into CFU-GM, CFU-GEMM, BFU-E, and CFU-E colonies. Colonies were identified and quantified after 14+ / -2 days.

[0156] Bone marrow cells from MDS were either enhanced with 4.4 mU CS Hela-GFP mitochondria (Figure 2, right panel) or untreated (Figure 2, left panel) (untreated cells are cells that underwent the enhancement process without the addition of mitochondria). The percentage enhancement was assessed using flow analysis. Low-risk and intermediate-risk MDS-derived cells (top two rows) were CD34 + The NT and boosted groups were cultured for 14 days with a total of 20,000 cells each. The cells from high-risk MDS patients were CD34 + They did not undergo purification and were directly exposed to erythroid differentiation supplement for 14 days (100,000 cells each). Medium was replenished every 3 days and cells were counted at the end of culture using a nucleocounter and verified for CD71 / CD235 surface marker expression.

[0157] As shown in Figure 2, MAT favorably affected the erythroid differentiation capacity of MDS-HSC-derived bone marrow samples.

[0158] As shown in Figures 3A and 3B and Tables 2 and 3, MAT expresses MDS-CD34 + Improved erythroid differentiation, with higher levels of erythroid differentiation observed after augmentation of MDS patient-derived cells with GFP-mito (n=3).

[0159] (Table 2) TIFF2024546826000003.tif32130

[0160] (Table 3) TIFF2024546826000004.tif33130

[0161] Example 2 In vivo effects of mitochondrial enrichment in MDS hematopoietic stem / progenitor cells To assess the effects of mitochondrial enrichment in vivo, genetically engineered models of MDS are used.

[0162] Genetically engineered mouse models of MDS include mice with conditional deletion of Tet2 and / or Asxl1 and conditional expression of MDS-associated mutations in Sf3b1 (SF3B1K700E and K666N mutations), Srsf2 (SRSF2P95H), or Zrsr2 (Zrsr2 floxed mice). In particular, Sf3b1 and Srsf2 mutant mice have highly penetrant lymphopenia, macrocytosis, and reduced hematopoietic stem cell (HSC) reconstitution capacity, mimicking key aspects of human MDS. In the Sf3b1 model, there is a mutation in a spliceosome gene, a gene associated with most human MDS with BM ringed sideroblasts. However, the mouse model does not recapitulate ringed sideroblasts. It is associated with progressive anemia.

[0163] An additional mouse model used includes NUP98-HOXD13 (also known as NHD13). NHD13 faithfully contains all the key features of MDS including peripheral blood cytopenias, myelodysplasia, and apoptosis, and transformation to acute leukemia, inhibiting megakaryocytic differentiation and increasing apoptosis in the bone marrow. MDS that develops in NUP98-HOXD13 transgenic mice is uniformly fatal within 14 months.

[0164] Another mouse model involves NSG-humanized mice with enhanced patient-derived MDS cells.

[0165] Mitochondria are isolated from placenta or liver of wild-type (WT) control mice of a strain selected from Mx1-cre, C57, WSP, W8, CAST or NZB. Mitochondria are frozen in liquid nitrogen or at -80°C. HSPCs are isolated from 8-week-old CD45.2+ Mx1-cre Sf3b1 mutant or Srsf2 P95 / WTMitochondria are isolated from mutant or NHD13 mice. Mitochondria are thawed and cells are then incubated with or without 0.88, 4.4, 17.6, or 35 mU of CS activity per 1M cells for up to 24 hours. The medium is then removed and cells are washed and resuspended in 4.5% albumin in physiological cell suspension medium. Using sequence analysis, the enhancement is verified by identifying the presence of exogenous mitochondria in cells using sequencing methods.

[0166] Competitive bone marrow transplantation assays demonstrated that CD45.2 recipient mice were transplanted with or without mitochondrial enrichment. + Mx1-cre Sf3b1 mutant, Srsf2 P95 / WT This will be done using CD45.1 / CD45.2 double positive competitor cells mixed with an equal number of cells from NDH13 mutant or NDH13 mutant mice. Recipient mice will be bled monthly for 16 weeks after transplantation to assess the effect of mitochondrial enrichment on CD45.2 chimerism and differentiation of CD45.2 cells in peripheral blood. Specifically, time to recovery and final levels of red blood cells (RBCs), platelets (PLTs), and white blood cells (WBCs) will be monitored.

[0167] In addition, the effects of mitochondrial enrichment on subsets of hematopoietic stem and progenitor cells in bone marrow 16 weeks after transplantation were - Cells and Lin - SCA1 + c-KIT + The persistence of exogenous mitochondria is also monitored.

[0168] To measure the effect of MAT on long-term self-renewal, serial competitive transplants were performed using enriched or non-enriched linings. - LSK or lin from cell-treated animals - This is carried out by isolating cells and transplanting them into naive animals as described above.

[0169] In addition to the competitive transplantation assays described above, non-competitive transplantation of BM MNCs was also performed using 8-week-old CD45.2 + Mx1-cre Sf3b1 mutant or Srsf2 P95 / WT Transplantation of Mx1-cre Sf3b1 mutant or Srsf2 mutant mice into lethally irradiated CD45.1 wild-type recipient mice with and without mitochondrial enrichment was performed. Recipient mice were then bled monthly to assess the effect of mitochondrial enrichment on blood cell counts and MNC differentiation. These assays involve the transfer of Mx1-cre Sf3b1 mutant or Srsf2 mutant mice into lethally irradiated CD45.1 wild-type recipient mice. P95 / WT Mutant or NHD13 mutant cells have lymphopenia and macrocytic anemia, therefore the effect of mitochondrial enrichment on these parameters in particular is evaluated.

[0170] Example 3 Effect of mitochondrial enrichment in hematopoietic stem / progenitor cells for the treatment of MDS To evaluate the effect of mitochondrial enrichment in MDS patients, stem and / or progenitor cells are obtained from mobilized peripheral blood or directly from bone marrow of a subject with myelodysplastic syndrome (MDS) disease, disorder or symptoms. Exogenous mitochondria are obtained from a donor without MDS disease, disorder or symptoms. The stem and / or progenitor cells are contacted with exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the stem and / or progenitor cells, thereby producing a composition comprising mitochondrially enriched stem and / or progenitor cells.

[0171] The subject is administered mitochondrial-enriched stem and / or progenitor cells. Prevention of disease progression, improved survival, and alleviation of symptoms are assessed in the subject administered mitochondrial-enriched stem and / or progenitor cells compared to a control subject (e.g., a subject not administered mitochondrial-enriched stem and / or progenitor cells) by measuring changes in disease or disorder-related symptoms of MDS. For example, hematopoiesis, blood cytopenias, clonal instability, lymphopenia, erythrocytosis, hematopoietic stem cell (HSC) reconstitution, peripheral blood cytopenias, myelodysplasia, macrocytic anemia, apoptosis, frequency of transfusion requirements, and transformation to acute leukemia, as well as blood cell counts and MNC differentiation (e.g., recovery and end levels of red blood cells (RBCs), platelets (PLTs), and white blood cells (WBCs)). The subject may be administered subsequent doses of mitochondrial-enriched stem and / or progenitor cells. The subject may receive chemotherapy prior to administration of mitochondrial-enriched stem and / or progenitor cells.

[0172] Although the invention has been described in terms of the above examples, it will be understood that various modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the scope of the following claims.

Claims

1. 1. A pharmaceutical composition comprising stem and / or progenitor cells enriched with exogenous mitochondria and a pharmaceutically acceptable carrier, the stem and / or progenitor cells are obtained from a subject with a myelodysplastic syndrome (MDS) disease, disorder, or symptom thereof; The exogenous mitochondria are obtained from a donor who does not have MDS disease, disorder, or symptoms thereof, or a mitochondrial disease. The pharmaceutical composition.

2. The stem cells and / or progenitor cells enriched with exogenous mitochondria have the following characteristics compared to the stem cells and / or progenitor cells before mitochondria enrichment: (a) increased content of at least one mitochondrial protein; (b) Increased oxygen (O ２ ) consumption rate; (c) increased activity levels of citrate synthase, succinate, or tryptamine; (d) increased adenosine triphosphate (ATP) production rate; (e) increased mitochondrial DNA content; (f) increased colony-forming unit activity in liquid or solid media; (g) increased growth rate; (h) increased differentiation rate; and (i) any combination thereof 2. The pharmaceutical composition of claim 1, wherein

3. 2. The pharmaceutical composition of claim 1, wherein the exogenous mitochondria constitute at least 0.5% or at least 1% of the total mitochondria in the stem and / or progenitor cells enriched for exogenous mitochondria.

4. 2. The pharmaceutical composition of claim 1, wherein the exogenous mitochondria are isolated, derived, or partially purified human mitochondria.

5. The stem cells and / or progenitor cells include pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, hematopoietic progenitor cells, common myeloid progenitor cells, common lymphoid progenitor cells, CD34 ＋ cells, CD34 ＋ 2. The pharmaceutical composition of claim 1, wherein the cell is selected from the group consisting of a subset of cells and any combination thereof, and is optionally derived from whole blood, a blood fraction, peripheral blood, umbilical cord blood, bone marrow, or blood-mobilized bone marrow cells.

6. The stem and / or progenitor cells are CD34 ＋ cells or CD34 ＋ The pharmaceutical composition of claim 1, which is a subset of cells.

7. 10. The pharmaceutical composition of claim 1, wherein the exogenous mitochondria are isolated, derived, or purified from placenta, placental cells grown in culture, or blood cells.

8. The MDS disease or disorder is selected from the group consisting of myelodysplastic syndrome with single lineage dysplasia (MDS-SLD), myelodysplastic syndrome with multilineage dysplasia (MDS-MLD), myelodysplastic syndrome with ringed sideroblasts (MDS-RS), myelodysplastic syndrome with isolated del(5q), myelodysplastic syndrome with excess blasts (MDS-EB), unclassifiable myelodysplastic syndrome (MDS-U), and acute myeloid leukemia (AML); and / or 2. The pharmaceutical composition of claim 1, wherein the symptoms of MDS are selected from the group consisting of shortness of breath, weakness, fatigue, pallor, anemia, thrombocytopenia, leukopenia, bruising, hemorrhage, petechiae, ineffective hematopoiesis, blood cytopenias, clonal instability, and any combination thereof.

9. 10. The pharmaceutical composition of claim 1, wherein the subject is being treated or has been treated with an MDS treatment, and optionally the MDS treatment is a hypomethylating agent, an erythropoiesis-stimulating agent (ESA), a granulocyte colony-stimulating factor (G-CSF), azacitidine, decitabine, immunosuppressive therapy (1ST), luspatercept, or a combination thereof.

10. A pharmaceutical composition comprising stem and / or progenitor cells enriched with exogenous mitochondria according to any one of claims 1 to 9, for use in a method for treating or preventing one or more myelodysplastic syndrome (MDS) disease or disorder-related symptoms in a subject, comprising: The method comprises administering to the subject the pharmaceutical composition, thereby treating or preventing the one or more MDS disease or disorder-associated symptoms in the subject.

11. The pharmaceutical composition according to claim 10, wherein at least 5×10 5 to 5×10 9 mitochondria-enriched stem cells and / or progenitor cells are administered to the subject.

12. A method for producing stem cells and / or progenitor cells enriched with exogenous mitochondria as described in any one of claims 1 to 9, the method comprising a step of incubating the stem cells and / or progenitor cells with the exogenous mitochondria at a concentration of about 1 to 50 mU, about 4.4 mU, about 17.6 mU, or about 35 mU of citrate synthase (CS) activity per 10 6 cells.

13. The method of claim 12, wherein the step of incubating is performed at a ratio of about 1 million to 100 million mitochondrial particles per million cells, or at a ratio of about 10 million to 50 million mitochondrial particles per million cells.

14. The method of claim 12, wherein the process is incubated for a time ranging from about 0.5 to 30 hours at a temperature ranging from about 4 to 37°C.

15. A method according to claim 12, comprising a step of freezing / thawing the stem cells and / or progenitor cells and / or exogenous mitochondria prior to producing the enriched cells, and / or a step of subjecting the stem cells and / or progenitor cells to at least one freeze-thaw cycle after the exogenous mitochondria have been enriched.

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