Compositions and methods for treatment of amyotrophic lateral sclerosis (ALS) and other neurodegenerative diseases

Abstract

To provide a composition for use in a method for improving muscle function in a subject.SOLUTION: The present invention provides a composition comprising mitochondria isolated from cells of a donor other than the subject, the cells having been cultured without antibiotics, and a method comprising administering the composition comprising the mitochondria to the subject in an amount sufficient to improve muscle function.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS REFERENCE TO PENDING APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 958,592, filed January 8, 2020, entitled "Compositions And Methods For Treatment Of Amyotrophic Lateral Sclerosis (ALS) And Other Neurodegenerative Diseases, and Associated Methods for Preparing Said Compositions," which claims priority to and the benefit of U.S. Provisional Patent Application No. 16 / 937,388, filed July 23, 2020, entitled "Compositions And Methods For Treatment Of Amyotrophic Lateral Sclerosis (ALS) And Other Neurodegenerative Diseases, and Associated Methods for Preparing Said Compositions," the contents of each of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on August 14, 2020, is named 2013631-0004_SL.txt and is 6,469 bytes in size. [Background technology]

[0003] background Mitochondrial dysfunction is associated with neurodegenerative diseases (NDs), including amyotrophic lateral sclerosis (ALS). Researchers have tested the therapeutic efficacy of many mitochondrial-targeting agents, but the results have been disappointing without significant impact on disease survival. Several groups have demonstrated the introduction of isolated mitochondria into defective cells, but translation of this research into clinical applications has been limited.

[0004] ALS, known as Lou Gehrig's disease, involves both upper and lower motor neuron degeneration. ALS has sporadic and familial variants, with sporadic being more common. The well-known form is due to mutations in Cu, Zn superoxide dismutase (SOD1). Motor neuron degeneration leads to skeletal muscle atrophy, paralysis and death.

[0005] Mitochondrial dysfunction is involved in multiple NDs, such as Parkinson's, Alzheimer's and Huntington's diseases. Mitochondria are dynamic and important intracellular organelles involved in many essential cellular functions, such as energy production, regulation of reactive oxygen species (ROS), metabolism, cell signaling, apoptosis, autophagy and iron metabolism. Small defects in any of these functions can cause mitochondrial dysfunction. For example, excess ROS triggers free radicals that result in oxidative damage to mitochondrial DNA (mtDNA) and the decomposition of iron-sulfur clusters, which are accompanied by a collapse of the membrane potential and a significant decrease in energy production. This cascade leads to the death of motor neurons.

[0006] Currently, ND is incurable, and available treatments only manage symptoms or slow progression. Many NDs are extremely debilitating, and current treatments are inadequate to provide sufficient improvement in the symptoms of patients with these diseases. There is a need for treatments that improve the symptoms of patients with ND. Summary of the Invention [Means for solving the problem]

[0007] Abstract The present disclosure provides, inter alia, methods and compositions related to the treatment of neurodegenerative diseases (ND) and other mitochondrial disorders.Described herein are in vitro (cell culture) and in vivo (animal model) experimental examples that demonstrate mitochondrial organelle transplantation (MOT) for the treatment of ND, such as amyotrophic lateral sclerosis (ALS).Furthermore, as discussed herein, MOT has been performed in five human ALS patients, and positive results have been obtained, with measurable improvements in patient symptoms and no adverse events.

[0008] Evidence has been presented that confirms that the mitochondrial isolation and preservation protocols described herein sufficiently purify and maintain viable mitochondria for subsequent mitochondrial organelle transplantation in cell lines and patients. Surprisingly, it has been found that human fibroblast mitochondria do not express HLA antigens. Thus, human mitochondria can be used for allogeneic mitochondrial organelle transplantation without the need for a human leukocyte antigen (HLA)-matched donor. Furthermore, it has been discovered that human fibroblast mitochondria are more likely to enter cells with defective mitochondria than they are to enter healthy cells. Thus, diseased tissues and organs incorporate more fibroblast mitochondria than healthy tissues. Furthermore, it has been discovered that antibiotics (which are toxic to mammalian cell mitochondria) are not required to culture human fibroblast cells for the isolation and preservation of mitochondria to be used in the MOT treatment methods described herein. These discoveries, coupled with the details presented herein, have enabled the methods for the treatment of neurodegenerative diseases (ND) and other mitochondrial disorders, as well as compositions related to the methods, described herein.

[0009] In one aspect, the invention relates to a method for allogeneic transplantation of mitochondria in a human subject, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria isolated from a donor other than the subject.

[0010] In certain embodiments, the subject has a neurodegenerative disease or other condition related to mitochondrial dysfunction (e.g., the method is carried out to treat the disease or condition).In certain embodiments, the subject has a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), PD-related disorder, Alzheimer's disease (AD), Lewy body dementia (LBD), dementia, muscular dystrophy (MD), mitochondrial disorder, prion disease, motor neuron disease (MND), Huntington's disease (HD), multiple sclerosis (MS), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Batten disease and fatal familial insomnia.In certain embodiments, the subject has amyotrophic lateral sclerosis (ALS).

[0011] In certain embodiments, the composition further comprises a mitochondrial storage buffer having a potassium ion concentration that is safe for administration to a human (e.g., the mitochondrial storage buffer comprises a pharma- ceutically acceptable carrier).

[0012] In certain embodiments, administering comprises parenterally administering at least one unit dose of the composition to the subject (e.g., by intramuscular and intravenous injection). In certain embodiments, administering comprises both intramuscular and intravenous injection of the composition to the subject.

[0013] In certain embodiments, the method further comprises isolating the mitochondria from the donor. In certain embodiments, isolating the donor mitochondria comprises preparing a cell lysate from the tissue (or other biological sample) of the donor via tissue dissociation (e.g., using a bead tube shaking homogenizer). In certain embodiments, isolating the donor mitochondria comprises using a mitochondrial isolation buffer that includes a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMSF)) (e.g., to prevent or reduce damage to the donor mitochondria from digestive enzymes). In certain embodiments, the method comprises isolating the donor mitochondria without the use of antibiotics.

[0014] In certain embodiments, the donor and subject are not HLA (human leukocyte antigen) matched (e.g., not identically matched (e.g., based on matching of 8 or 10 tested HLA markers) and / or not haplotype matched (e.g., based on matching of 8 or 10 tested HLA markers), and / or of indeterminate match status (e.g., HLA markers not tested prior to the administering step)).

[0015] In certain embodiments, the composition administered to the subject does not include an antibiotic (eg, if the subject is not receiving an antibiotic).

[0016] In certain embodiments, the composition comprises mitochondria isolated from donor human primary fibroblasts.

[0017] In certain embodiments, the method further comprises isolating mitochondria (e.g., fibroblast mitochondria) from the donor tissue. In certain embodiments, the isolating is performed using a mitochondrial isolation buffer composition (e.g., a mitochondrial isolation buffer solution described herein). In certain embodiments, a mitochondrial isolation buffer composition (e.g., an aqueous solution) (e.g., for use in mitochondrial organelle transplantation) comprises a buffering agent [e.g., a zwitterionic sulfonic acid buffer, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, e.g., potassium HEPES, (K-HEPES)]; a chelating agent [e.g., ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) or a salt thereof, e.g., K-EGTA)]; a sugar (e.g., sucrose); an agent that acts as a membrane stabilizer and / or an oxygen radical scavenger and / or a binder of Ca2+ and / or a binder of free fatty acids (e.g., bovine serum albumin, BSA); and a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMSF), also known as phenylmethanesulfonyl fluoride) (e.g., when the composition further comprises isolated donor mitochondria, e.g., fibroblast mitochondria). In certain embodiments, the mitochondrial isolation buffer composition does not contain antibiotics. In certain embodiments, the method further comprises storing the isolated mitochondria at a temperature below -40°C (e.g., below -60°C, below -70°C, or below -80°C, e.g., using liquid nitrogen).

[0018] In certain embodiments, the methods include (eg, further include) administering to the subject an iron chelator (eg, desferrioxamine or deferasirox).

[0019] In certain embodiments, the methods include (e.g., further include) administering to the subject an antioxidant and / or a probiotic.

[0020] In certain embodiments, the composition is a pharmaceutical composition. In certain embodiments, the composition further comprises a pharma- ceutically acceptable carrier. In certain embodiments, the mitochondria is a therapeutic agent of the composition, present in a therapeutically effective amount.

[0021] In another aspect, the present invention relates to a method for improving muscle function in a subject, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function. In certain embodiments, the composition further comprises a pharma- ceutically acceptable carrier. In certain embodiments, the mitochondria are a therapeutic agent of the composition, present in a therapeutically effective amount.

[0022] In another aspect, when a subject has reduced muscle function due to administration of one or more members of the group consisting of an antibiotic, an antimalarial, an antiviral, or another treatment that causes or may cause mitochondrial damage, the invention relates to a method comprising administering to said subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function, e.g., where muscle is related to lung function.

[0023] In another aspect, when a subject has impaired muscle function due to one or more members of the group consisting of: (i) a lung-related disorder, such as viral pneumonia; (ii) a coronavirus infection, e.g., infection with a severe acute respiratory syndrome coronavirus, including SARS-Cov-1 or SARS-Cov-2 or related strains; (iii) influenza; (iv) a related genetic disorder; (v) chronic obstructive pulmonary disease (COPD); and (vi) another disease or condition in which mitochondrial damage occurs and / or is present, the invention relates to a method comprising administering to said subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function, e.g., where muscle is associated with lung function.

[0024] In another aspect, the present invention relates to a method for improving muscle function in a subject impaired by one or more members of the group consisting of: (i) a lung-related disorder, such as viral pneumonia; (ii) a coronavirus infection, e.g., infection with a severe acute respiratory syndrome coronavirus, including SARS-Cov-1 or SARS-Cov-2 and related strains, influenza, a related genetic disorder, chronic obstructive pulmonary disease (COPD), or another disease or condition in which mitochondrial damage occurs and / or is present, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function in the subject.

[0025] In another aspect, if a subject's iron levels, measured, for example, as serum ferritin in the range of 0.25 mg to 2.5 mg / mL, are higher than normal as understood by one of skill in the art, the invention relates to a method comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function, with or without (e.g., administered separately) administration of one or more iron chelating compounds, such as, for example, desferrioxamine (deferoxamine).

[0026] In another aspect, when a subject is being treated with a pharmaceutical agent, such as hydroxychloroquine and / or chloroquine, for an indication involving high reactive oxygen species (ROS), such as rheumatoid arthritis, the invention relates to a method comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function.

[0027] Those skilled in the art will understand that there are various known methods and techniques for assessing muscle function of a subject. In various embodiments, muscle function can refer to skeletal muscle function. In certain embodiments, muscle function can refer to cardiac muscle function and / or smooth muscle function. In various embodiments, muscle function can be tested in one or more muscles, including but not limited to upper limb muscles, lower limb muscles, leg muscles, tibialis (lower leg), foot muscles (e.g., heel), quadriceps, thigh flexors, upper arm, biceps, triceps, forearm, neck muscles, and / or gluteal muscles. Muscle function can include, for example, strength (e.g., force-generating capacity, e.g., maximum voluntary contraction, one-repetition maximum resistance, and / or grip strength measured by dynamometer), endurance (e.g., ability to maintain or repeat a particular task over time), fatigability (e.g., reversible decrease in force occurring during a given task), degree of atrophy, fiber type, oxidative capacity, and / or capillary formation. Strength tests include, for example, knee lifts, leg lifts, thigh flexors, and heel press tests.Other functional strength tests include, for example, the unsupported upper limb exercise test, stair running power test, abbreviated physical performance battery, 4-meter walking speed test, timed get-up-and-go test, and 5-repetition chair rise test.

[0028] In another aspect, the present invention relates to a method for treating a neurodegenerative disease or other condition associated with mitochondrial dysfunction in a subject in need of such treatment, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to treat the neurodegenerative disease or other condition. In certain embodiments, the composition further comprises a pharma- ceutical acceptable carrier. In certain embodiments, the mitochondria is the therapeutic agent of the composition, present in a therapeutically effective amount.

[0029] In another aspect, the present invention relates to a method for treating amyotrophic lateral sclerosis (ALS) in a subject in need of such treatment, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to treat the ALS. In certain embodiments, the composition further comprises a pharma- ceutically acceptable carrier. In certain embodiments, the mitochondria is the therapeutic agent of the composition, present in a therapeutically effective amount.

[0030] In another aspect, the invention provides a mitochondrial isolation buffer composition (e.g., an aqueous solution) (e.g., for use in practicing the methods described herein, e.g., for use in mitochondrial organelle transplantation), comprising a buffering agent [e.g., a zwitterionic sulfonic acid buffer, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, e.g., potassium HEPES, (K-HEPES)]; a chelating agent [e.g., ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof, e.g., K-EGTA)]; a sugar (e.g., sucrose); a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ and an agent that acts as a binder of free fatty acids (e.g., bovine serum albumin, BSA); and a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMSF), also known as phenylmethanesulfonyl fluoride). In certain embodiments, the mitochondrial isolation buffer composition does not include antibiotics.

[0031] In another aspect, the invention provides a mitochondrial storage buffer composition (e.g., an aqueous solution) (e.g., for use in practicing the methods described herein, e.g., for use in mitochondrial organelle transplantation), comprising one or more buffering agents [e.g., a zwitterionic sulfonic acid buffer, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, e.g., potassium HEPES salt, (K-HEPES)] [e.g., monopotassium phosphate (KH2PO4)]; a magnesium ion source [e.g., magnesium chloride (MgCl2)]; a chelating agent [e.g., ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof, e.g., K-EGTA)]; a sugar (e.g., sucrose); an antioxidant [e.g., taurine]; a cytoprotectant that binds calcium ions [e.g., lactobionate or a salt thereof, e.g., K-lactobionate]; and a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ In certain embodiments, the mitochondrial storage buffer composition comprises an agent that acts as a binder of free fatty acids (e.g., bovine serum albumin, BSA) (e.g., when the composition further comprises isolated donor mitochondria, e.g., fibroblast mitochondria). In certain embodiments, the mitochondrial storage buffer composition does not comprise an antibiotic.

[0032] In another aspect, the invention provides a kit comprising a donor mitochondrial composition (e.g., an aqueous composition) in a unit dosage amount effective to treat a neurodegenerative disease or other condition associated with mitochondrial dysfunction in a subject, said donor mitochondrial composition comprising mitochondria isolated from tissue of a donor (e.g., fibroblast mitochondria); one or more buffers [e.g., a zwitterionic sulfonic acid buffer, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, e.g., potassium salt of HEPES, (K -HEPES)] [e.g., monopotassium phosphate (KH2PO4)]; a magnesium ion source [e.g., magnesium chloride (MgCl2)]; a chelating agent [e.g., ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof, e.g., K-EGTA)]; a sugar (e.g., sucrose); an antioxidant [e.g., taurine]; a cytoprotectant that binds calcium ions [e.g., lactobionate or a salt thereof, e.g., K-lactobionate]; and a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ The present invention relates to a kit comprising an agent that acts as a binder for the IgG and / or a binder for free fatty acids (e.g., bovine serum albumin, BSA).

[0033] In certain embodiments, the donor and subject are not HLA (human leukocyte antigen) matched (e.g., not an identical match (e.g., based on matching of 8 or 10 tested HLA markers) and / or not a haplotype match (e.g., based on matching of 8 or 10 tested HLA markers), and / or of indeterminate match status (e.g., HLA markers not tested prior to administration)).

[0034] Humans have three major MHC class I loci, known as HLA-A, HLA-B and HLA-C, and each individual carries two alleles at each locus. Humans have six major MHC class II loci, known as HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1, and each individual carries two alleles at each locus. In general, donors and subjects will be matched based on the alleles present in the donor and subject at one or more HLA loci, such as HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 and / or HLA-DPB1. Various standards for HLA matching are known in the art. Matching of all eight alleles at the HLA-A, HLA-B, HLA-C and HLA-DRB1 loci can be referred to as an 8 / 8 match. A match of all ten alleles at the HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci can be referred to as a 10 / 10 match. For certain transplantation, various degrees of allele mismatch can be tolerated.Thus, for example, donor and subject can match for example in 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 alleles in HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1, or in 8, 7, 6, 5, 4, 3, 2, 1 or 0 alleles in 8 HLA-A, HLA-B, HLA-C and HLA-DRB1. As disclosed herein, it has been found that in various embodiments, the donor and subject for mitochondrial transplantation need not be HLA (human leukocyte antigen) matched (e.g., not identically matched (e.g., based on matching of eight or ten tested HLA markers) and / or not haplotype matched (e.g., based on matching of eight or ten tested HLA markers), and / or of indeterminate match status (e.g., HLA markers not tested prior to the administering step)).

[0035] In certain embodiments, the donor mitochondrial composition does not contain antibiotics.

[0036] In certain embodiments, the kit comprises instructions for administering the composition to a subject. In certain embodiments, the kit further comprises instructions for optimizing the dosage (e.g., unit dose) and / or frequency and / or route of administration of the composition.

[0037] In certain embodiments, the kit comprises a mitochondrial isolation buffer composition (e.g., an aqueous solution) comprising a buffering agent [e.g., a zwitterionic sulfonic acid buffer, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, e.g., potassium HEPES, (K-HEPES)]; a chelating agent [e.g., ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) or a salt thereof, e.g., K-EGTA)]; a sugar (e.g., sucrose); a membrane stabilizer and / or an oxygen radical scavenger and / or Ca 2+ and an agent that acts as a binder for free fatty acids (e.g., bovine serum albumin, BSA); and a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMSF), also known as phenylmethanesulfonyl fluoride) (e.g., when the composition further comprises isolated donor mitochondria, e.g., fibroblast mitochondria).

[0038] A limitation stated with respect to one aspect of the invention may, in certain embodiments, be implemented with respect to another aspect of the invention, for example, a limitation of a claim that depends directly or indirectly from a particular independent claim presented herein serves as support for a limitation presented in a further dependent claim of one or more other independent claims.

[0039] Throughout this specification, when a composition is described as having, including, or comprising particular components, or a method is described as having, including, or comprising particular steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are methods of the invention that consist essentially of or consist of the recited steps.

[0040] In certain embodiments, the compositions described herein are used to treat disease or condition.In certain embodiments, the compositions described herein are used to prevent the onset and / or progression of disease or condition (e.g., ND), for example, in subjects susceptible to said disease or condition.In certain embodiments, variants of one or more components of the compositions described herein are used instead of said one or more components.

[0041] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0042] The following description is merely illustrative and exemplary of the present disclosure and is not intended to limit the invention to the specific embodiments described.

[0043] The reference herein to any publication, for example in the Background section, is not an admission that that publication serves as prior art with respect to any of the claims presented herein. The Background section is presented for clarity and is not intended as a statement of prior art with respect to any claim. definition

[0044] A or An: The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" refers to one element or more than one element.

[0045] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system, for example to achieve delivery of the agent contained in or otherwise delivered by the composition.Non-limiting examples of administration include oral administration; parenteral administration (e.g., by subcutaneous, intramuscular, intravenous or epidural injection, for example, as a sterile solution or suspension or sustained release formulation); topical application (e.g., as a cream, ointment, patch or spray applied to, for example, the skin, lung or oral cavity); vaginal or rectal administration (e.g., as a pessary, suppository, cream or foam); ocular administration; nasal or pulmonary administration, etc.

[0046] Agent: As used herein, the term "agent" refers to an entity (e.g., a cell, a component of a cell such as, for example, a mitochondria or other organelle, a small molecule, a peptide, a polypeptide, a nucleic acid, a lipid, a polysaccharide, a complex, a combination, a mixture, a system, or a phenomenon such as heat, an electric current, an electric field, a magnetic force, a magnetic field, etc.).

[0047] Allogeneic: As used herein, the term "allogeneic" refers to any material derived from one subject (donor) and transplanted into another subject. Examples of allogeneic transplants include allogeneic T cell transplants, allogeneic stem cell transplants, and allogeneic mitochondrial transplants as discussed herein.

[0048] Amelioration: As used herein, the term "amelioration" refers to the prevention, alleviation, mitigation or improvement of a subject's condition. Amelioration includes, but does not require, complete recovery or complete prevention of a disease, disorder or condition.

[0049] Antibiotics: As used herein, the term "antibiotics" refers to antibacterial substances such as penicillin, gentamicin, streptomycin, cephalosporins, ciprofloxacin, etc., that are used to treat or prevent infection by killing or inhibiting the growth of bacteria in or on the body, are administered orally, topically or by injection, and are isolated from cultures of certain microorganisms (such as fungi) or are of semi-synthetic or synthetic origin.

[0050] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained from or derived from a biological source of interest (e.g., tissue or organism or cell culture) as described herein. In some embodiments, the biological source is or includes an organism, such as an animal or human, as described herein. In some embodiments, the biological sample is or includes a biological tissue or fluid, as described herein. In some embodiments, the biological sample can be or include a cell, tissue (e.g., skin tissue, muscle or other tissue), or bodily fluid, as described herein. In some embodiments, for example, as described herein, a biological sample can be or include blood, blood cells, cell-free DNA, free floating nucleic acid, ascites, biopsy sample, surgical specimen, cell-containing fluid, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural fluid, lymph, gynecological fluid, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as ductal washings or bronchoalveolar lavages, aspirates, scrapings, bone marrow. In some embodiments, for example, as described herein, a biological sample is or includes cells obtained from a single subject or from multiple subjects. A sample can be a "primary sample" obtained directly from a biological source, or a "processed sample". A biological sample can also be referred to as a "sample".

[0051] Improved, increased or decreased: as used herein, these terms or grammatically equivalent comparative terms refer to values ​​compared to comparable reference measurements. For example, in some embodiments, for example, as described herein, the assessment value achieved with an agent of interest can be "improved" compared to an assessment value obtained with an equivalent reference agent or without the agent. Alternatively or additionally, in some embodiments, for example, as described herein, the assessment value in a subject or system of interest can be "improved" compared to an assessment value obtained in the same subject or system under different conditions or at different times (e.g., before or after an event such as administration of the agent of interest), or in a different comparable subject (e.g., in a comparable subject or system that differs from the subject or system of interest in the presence of one or more indicators of a particular disease, disorder or condition of interest, or in prior exposure to a condition or agent, etc.). In some embodiments, for example, as described herein, the comparative term refers to a statistically valid difference (e.g., a difference in proportion and / or magnitude sufficient to achieve statistical validity). Those of ordinary skill in the art will know or be able to readily determine, in a given situation, the degree and / or percentage of difference necessary or sufficient to achieve such statistical significance.

[0052] Isolated: As used herein, "isolated" refers to a substance and / or entity (e.g., including one or more mitochondria) that is (a) separated from at least some of the components with which it was associated when originally produced (whether in nature, in a subject such as a donor, and / or in an experimental environment) and / or (b) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99% of the other components with which they were originally associated. In some embodiments, an isolated substance and / or entity is at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance and / or entity is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance and / or entity may still be considered "isolated" or "pure" after being combined with certain other components, such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.). In such embodiments, the percent isolation or purity of a substance and / or entity is calculated without including such carriers or excipients. Simply by way of example, in some embodiments, mitochondria present in nature can be referred to as "isolated" if (a) they are present in a composition that does not contain some or all of the components that naturally accompany them, for example in the donor from which they are derived, (b) they are substantially free of other organelles from the donor organism from which they are derived; (c) they are present in a cell or system that is different from the donor organism from which they are derived. Thus, for example, mitochondria removed from a donor for transplantation into a second, different subject can be referred to as "isolated".

[0053] Neurodegenerative disease: As used herein, the term "neurodegenerative disease" (also referred to as "degenerative neurological disease") is a collective term for conditions that primarily affect neurons in the human brain. In certain instances, neurodegenerative diseases are characterized by progressive loss of neurons accompanied by deposition of proteins that exhibit altered physicochemical properties in the brain and / or peripheral organs. Neurodegenerative diseases include, for example, amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD) and PD-related disorders, Alzheimer's disease (AD), Lewy body dementia (LBD), other forms of dementia, muscular dystrophies (MD), mitochondrial disorders, prion diseases, motor neuron diseases (MND), Huntington's disease (HD), multiple sclerosis (MS), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Batten disease, fatal familial insomnia, and the like.

[0054] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to a composition in which an active agent is provided together with one or more pharma- ceutically acceptable carriers. In some embodiments, for example, as described herein, the active agent is present in a unit dose suitable for administration to a subject, for example, in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to an appropriate population. In some embodiments, e.g., as described herein, a pharmaceutical composition may be formulated for administration in a particular form (e.g., in a solid or liquid form) and / or may be particularly adapted for, for example: oral administration (e.g., drenches (aqueous or non-aqueous solutions or suspensions), tablets, capsules, boluses, powders, granules, pastes, etc., which may be specifically formulated, e.g., for buccal, sublingual, or systemic absorption); parenteral administration (e.g., by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or a sustained release formulation); topical application (e.g., as a cream, ointment, patch, or spray applied, e.g., to the skin, lungs, or oral cavity); vaginal or rectal administration (e.g., as a pessary, suppository, cream, or foam); ocular administration; nasal or pulmonary administration, etc.

[0055] Pharmaceutically acceptable: As used herein, the term "pharmacologically acceptable" as applied to one or more or all ingredients for formulating a composition disclosed herein means that each ingredient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0056] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutical acceptable carrier" refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, that facilitates the formulation and / or modifies the bioavailability of an agent, e.g., a pharmaceutical agent. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose, as well as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; and its derivatives such as cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0057] Prevent or prophylaxis: The term "prevent" and "prevention" used herein in relation to the occurrence of disease, disorder or condition refers to reducing the risk of developing disease, disorder or condition; delaying the onset of disease, disorder or condition; delaying the onset of one or more characteristics or symptoms of disease, disorder or condition; and / or reducing the frequency and / or severity of one or more characteristics or symptoms of disease, disorder or condition. Prevention can refer to prevention in a specific subject or a statistical effect on a population of subjects. Prevention can be considered to be complete when the onset of disease, disorder or condition is delayed for a predetermined period of time.

[0058] Prognosis: As used herein, the term "prognosis" refers to determining the qualitative or quantitative probability of at least one possible future outcome or event.As used herein, prognosis can be determining the likely course of a disease, disorder or condition, such as cancer, in a subject, determining the subject's life expectancy, or determining the response to treatment, such as a particular treatment.

[0059] Reference: as used herein, describes a standard or control against which comparison is made. For example, in some embodiments, for example, as described herein, an agent, subject, animal, individual, population, sample, sequence or value of interest is compared to an agent, subject, animal, individual, population, sample, sequence or value of a reference or control. In some embodiments, for example, as described herein, the reference or its characteristics are tested and / or determined substantially simultaneously with the testing or determination of the characteristics in the sample of interest. In some embodiments, for example, as described herein, the reference is a past reference, optionally embodied in a tangible medium. Typically, as understood by those skilled in the art, the reference is determined or characterized under conditions or circumstances equivalent to those being evaluated, for example, with respect to the sample. Those skilled in the art will understand when there is sufficient similarity to justify reliance on and / or comparison to a particular possible reference or control.

[0060] Sample: As used herein, the term "sample" typically refers to an aliquot of material obtained from or derived from a source of interest. In some embodiments, for example, as described herein, the source of interest is a biological or environmental source. In some embodiments, for example, as described herein, the sample is a "primary sample" obtained directly from the source of interest. In some embodiments, for example, as described herein, as the context makes clear, the term "sample" refers to a preparation obtained by processing a primary sample (for example, by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample).

[0061] Susceptible to: An individual who is "susceptible to" a disease, disorder, or condition is at risk of developing said disease, disorder, or condition. In some embodiments, for example, as described herein, an individual who is susceptible to a disease, disorder, or condition does not show any symptoms of said disease, disorder, or condition. In some embodiments, for example, as described herein, an individual who is susceptible to a disease, disorder, or condition has never been diagnosed with said disease, disorder, and / or condition. In some embodiments, for example, as described herein, an individual who is susceptible to a disease, disorder, or condition is an individual who has been exposed to a condition associated with the development of the disease, disorder, or condition, or an individual who displays a biomarker status associated with the development of the disease, disorder, or condition. In some embodiments, for example, as described herein, the risk of developing a disease, disorder, and / or condition is a population-based risk (e.g., family members of an individual who suffers from a disease, disorder, or condition).

[0062] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, the subject is afflicted with a disease, disorder, or condition, e.g., as described herein. In some embodiments, the subject is susceptible to a disease, disorder, or condition, e.g., as described herein. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition, e.g., as described herein. In some embodiments, the subject is not afflicted with a disease, disorder, or condition, e.g., as described herein. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition, e.g., as described herein. In some embodiments, the subject has one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition, e.g., as described herein. In some embodiments, the subject is a patient, e.g., as described herein. In some embodiments, the subject is an individual to whom a diagnosis has been performed and / or a treatment has been administered, e.g., as described herein. In some cases, a human subject may be interchangeably referred to as an "individual," e.g., as described herein.

[0063] Therapeutic Agent, Pharmaceutical Product, and Active Agent: As used herein, the terms "therapeutic agent", "pharmaceutical product", and "active agent" are interchangeable and each refer to any agent that induces a desired pharmacological effect when administered to a subject. In some embodiments, for example, as described herein, an agent is considered to be a therapeutic agent if it shows a statistically significant effect across an appropriate population. In some embodiments, for example, as described herein, the appropriate population can be a population of model organisms or a human population. In some embodiments, for example, as described herein, the appropriate population can be defined by various criteria, such as a certain age group, sex, genetic background, pre-existing clinical symptoms, etc. In some embodiments, for example, as described herein, a therapeutic agent is a substance that can be used for the treatment of a disease, disorder, or condition. In some embodiments, for example, as described herein, a therapeutic agent is an agent that has been approved or needs to be approved by a government agency before it is commercially available for administration to humans. In some embodiments, for example, as described herein, a therapeutic agent is an agent that requires a medical prescription for administration to humans.

[0064] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount that produces a desired effect for which it is administered. In some embodiments, for example, as described herein, the term refers to an amount sufficient to treat a disease, disorder, or condition when administered to a population suffering from or susceptible to the disease, disorder, or condition according to a therapeutic dosing regimen. Those skilled in the art will understand that the term therapeutically effective amount does not actually require that successful treatment is achieved in a particular individual. Rather, a therapeutically effective amount may be an amount that, when administered to an individual in need of such treatment, provides a particular desired pharmacological response in a significant number of subjects. In some embodiments, for example, as described herein, reference to a therapeutically effective amount may be a reference to an amount measured in one or more particular tissues (e.g., tissues affected by a disease, disorder, or condition) or bodily fluids (e.g., blood, saliva, serum, sweat, tears, urine, etc.). Those skilled in the art will understand that in some embodiments, a therapeutically effective amount of a particular agent may be formulated and / or administered in a single dose. In some embodiments, e.g., as described herein, a therapeutically effective agent can be formulated and / or administered in multiple doses, e.g., as part of a multiple dose dosing regimen.

[0065] Treatment: As used herein, the term "treatment" (as well as "treat" or "treating") refers to the administration of a therapy administered for the purpose of partially or completely alleviating, ameliorating, alleviating, inhibiting, delaying the onset of, halting the progression of, delaying the progression of, reversing the progression of, reducing the severity of, and / or reducing the incidence of one or more symptoms, characteristics and / or causes of a particular disease, disorder or condition, or achieving any such result. In some embodiments, such treatment may be treatment of subjects who do not show signs of the relevant disease, disorder or condition and / or who show only early signs of the disease, disorder or condition, e.g., as described herein. Alternatively or additionally, such treatment may be treatment of subjects who show one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, such as, e.g., as described herein, treatment may be treatment of subjects who have been diagnosed as suffering from the relevant disease, disorder and / or condition. In some embodiments, for example, as described herein, the treatment can be a treatment of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing the relevant disease, disorder or condition. In various examples, the treatment is a treatment of cancer.

[0066] Unit dose: As used herein, the term "unit dose" refers to an amount administered as a single dose of a pharmaceutical composition and / or in a physically separate unit. In many embodiments, a unit dose contains a predetermined amount of an active agent, e.g., as described herein. In some embodiments, a unit dose contains a total single dose of an agent, e.g., as described herein. In some embodiments, more than one unit dose is administered to achieve a total single dose, e.g., as described herein. In some embodiments, administration of multiple unit doses is required or expected to be required to achieve an intended effect, e.g., as described herein. A unit dose can be, for example, a volume of liquid (e.g., an acceptable carrier) containing a predetermined amount of one or more therapeutic moieties, a predetermined amount of one or more therapeutic moieties in solid form, a sustained release formulation or drug delivery device containing a predetermined amount of one or more therapeutic moieties, and the like. It will be understood that a unit dose can be in a formulation that includes any of a variety of components in addition to a therapeutic agent. For example, an acceptable carrier (e.g., a pharma- ceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, and the like can be included. In many embodiments, for example, as described herein, the appropriate total daily dosage of a particular therapeutic agent can include a portion or a plurality of unit doses, and can be determined, for example, by a physician within the scope of sound medical judgment, as will be understood by those skilled in the art.In some embodiments, for example, as described herein, the specific effective dosage level for any particular subject or organism can depend on various factors, including the disorder being treated and the severity of the disorder; the activity of the specific active compound used; the specific composition used; the age, weight, general health, sex and diet of the subject; the time of administration and the rate of excretion of the specific active compound used; the duration of treatment; the drugs and / or additional treatments used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0067] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity, but is structurally different from the reference entity in the presence, absence, or level of one or more chemical moieties compared to the reference entity. In some embodiments, for example, as described herein, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a "variant" of a reference entity is based on its degree of structural identity with the reference entity. A variant can be a molecule that is equivalent to, but not identical to, the reference. For example, a variant peptide can differ from a reference peptide in one or more differences in amino acid sequence. In some embodiments, for example, as described herein, a variant peptide exhibits an overall sequence identity with a reference peptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 99%. In an embodiment of the present invention, for example, the following items are provided: (Item 1) 2. A method for allogeneic transplantation of mitochondria in a human subject, comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject. The method of claim 1, wherein the subject has a neurodegenerative disease or other condition associated with mitochondrial dysfunction. (Item 3) The method of claim 2, wherein the subject has a disease selected from the group consisting of amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), PD-related disorders, Alzheimer's disease (AD), dementia with Lewy bodies (LBD), dementia, muscular dystrophy (MD), mitochondrial disorders, prion diseases, motor neuron diseases (MND), Huntington's disease (HD), multiple sclerosis (MS), spinocerebellar ataxia (SCA), spinal muscular atrophy (SMA), Friedreich's ataxia, Batten disease, and fatal familial insomnia. (Item 4) The method of claim 2, wherein the subject has amyotrophic lateral sclerosis (ALS). (Item 5) 2. The method of any one of the preceding items, wherein the composition further comprises a mitochondrial storage buffer having a potassium ion concentration that is safe for administration to humans. (Item 6) The method of any one of the preceding items, wherein said administering comprises parenterally administering at least one unit dose of the composition to the subject. (Item 7) 7. The method of claim 6, wherein the administering comprises both intramuscular and intravenous injection of the composition into the subject. (Item 8) 2. The method of any one of the preceding items, further comprising isolating the mitochondria from the donor. (Item 9) 9. The method of claim 8, wherein isolating the donor mitochondria comprises preparing a cell lysate from the donor tissue via tissue dissociation. (Item 10) 10. The method of claim 8 or 9, wherein isolating the donor mitochondria comprises using a mitochondrial isolation buffer containing a serine protease inhibitor. (Item 11) 11. The method according to any one of items 8 to 10, comprising isolating the donor mitochondria without the use of antibiotics. (Item 12) 2. The method of any one of the preceding items, wherein the donor and the subject are not HLA (human leukocyte antigen) matched. (Item 13) Item 11. The method of any one of the preceding items, wherein the composition administered to the subject does not comprise an antibiotic. (Item 14) 11. The method of any one of the preceding items, wherein the composition comprises mitochondria isolated from human primary fibroblasts of the donor. (Item 15) 2. The method of any one of the preceding items, further comprising isolating said mitochondria from tissue of the donor. (Item 16) 16. The method of claim 15, wherein the isolating is performed using a mitochondrial isolation buffer composition. (Item 17) The mitochondrial isolation buffer composition comprises: A buffering agent; A chelating agent; Sugar and Membrane stabilizers and / or oxygen radical scavengers and / or Ca 2+ and / or an agent acting as a binder of free fatty acids; Serine protease inhibitors and 17. The method according to item 16, comprising: (Item 18) Item 19. The mitochondrial isolation buffer composition according to item 17, wherein the composition does not contain an antibiotic. 19. The method of any one of items 15 to 18, further comprising storing the isolated mitochondria at a temperature below -40°C (e.g., below -60°C, below -70°C, or below -80°C, e.g., using liquid nitrogen). (Item 20) The method of any one of the preceding items, comprising administering to the subject an iron chelator (e.g., desferrioxamine or deferasirox). (Item 21) The method of any one of the preceding items, comprising administering to the subject an antioxidant and / or a probiotic. (Item 22) 1. A method for improving muscle function in a subject, comprising administering to the subject a composition comprising mitochondria in an amount sufficient to improve muscle function. (Item 23) 1. A method for treating a neurodegenerative disease or other condition associated with mitochondrial dysfunction in a subject in need of such treatment, comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject in an amount sufficient to treat the neurodegenerative disease or other condition. (Item 24) 1. A method for treating amyotrophic lateral sclerosis (ALS) in a subject in need thereof, comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject in an amount sufficient to treat the ALS. (Item 25) A method for improving muscle function in a subject that has been impaired by administration of an agent that causes or can cause mitochondrial damage, the method comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject in an amount sufficient to improve muscle function in the subject. (Item 26) A method for treating a subject whose muscle function has been reduced by one or more members of the group consisting of: (i) a lung-related disorder, such as viral pneumonia; (ii) a coronavirus infection, e.g., an infection with a severe acute respiratory syndrome coronavirus, including SARS-Cov-1 or SARS-Cov-2 or related strains; (iii) influenza; (iv) an associated genetic disorder; (v) chronic obstructive pulmonary disease (COPD); and (vi) another disease or condition in which mitochondrial damage occurs and / or is present, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria (e.g., mitochondria isolated from a donor other than the subject) in an amount sufficient to improve muscle function in the subject. (Item 27) A method for improving muscle function in a subject that has been impaired by one or more members of the group consisting of: (i) a lung-related disorder, such as viral pneumonia; (ii) a coronavirus infection, e.g., infection with a severe acute respiratory syndrome coronavirus, including SARS-Cov-1 or SARS-Cov-2 and related strains, influenza, a related genetic disorder, chronic obstructive pulmonary disease (COPD), or another disease or condition in which mitochondrial damage occurs and / or is present, comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject in an amount sufficient to improve muscle function in the subject. (Item 28) 1. A method for treating a subject having high iron levels, comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject in an amount sufficient to improve muscle function in the subject. (Item 29) 1. A method for treating a subject being treated with a pharmaceutical agent, such as hydroxychloroquine and / or chloroquine, for an indication involving high reactive oxygen species (ROS), comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject. (Item 30) 1. A mitochondrial isolation buffer composition for use in mitochondrial organelle transplantation, comprising: A buffering agent; A chelating agent; Sugar and Membrane stabilizers and / or oxygen radical scavengers and / or Ca 2+ and / or an agent acting as a binder of free fatty acids; Serine protease inhibitors and 1. A mitochondrial isolation buffer composition comprising: (Item 31) 31. The mitochondrial isolation buffer composition of item 30, wherein the buffer is a zwitterionic sulfonic acid buffer. (Item 32) 32. The mitochondrial isolation buffer composition according to item 30 or 31, wherein the chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof. (Item 33) 33. The mitochondrial isolation buffer composition according to any one of items 30 to 32, wherein the sugar is sucrose. (Item 34) Membrane stabilizers and / or oxygen radical scavengers and / or Ca 2+ 34. The mitochondrial isolation buffer composition according to any one of items 30 to 33, wherein said agent acting as a binder of mitochondrial cleavage and / or free fatty acids is bovine serum albumin (BSA). (Item 35) 35. The mitochondrial isolation buffer composition according to any one of items 30 to 34, wherein the serine protease inhibitor is phenylmethylsulfonyl fluoride (PMSF). (Item 36) 36. The mitochondrial isolation buffer composition according to any one of items 30 to 35, wherein the composition further comprises isolated donor mitochondria. (Item 37) 37. The mitochondrial isolation buffer composition according to any one of items 30 to 36, wherein the composition does not contain an antibiotic. (Item 38) 1. A mitochondrial storage buffer composition for use in mitochondrial organelle transplantation, comprising: one or more buffering agents; a source of magnesium ions; A chelating agent; Sugar and Antioxidants, A cytoprotectant that binds to calcium ions; Membrane stabilizers and / or oxygen radical scavengers and / or Ca 2+ and / or an agent acting as a binder for free fatty acids. 1. A mitochondrial storage buffer composition comprising: (Item 39) 39. The mitochondrial storage buffer composition of item 38, wherein the one or more buffering agents comprise a zwitterionic sulfonic acid buffer and / or monopotassium phosphate (KH2PO4). (Item 40) 40. The mitochondrial storage buffer composition of claim 38 or 39, wherein the source of magnesium ions is magnesium chloride (MgCl2). (Item 41) 41. The mitochondrial storage buffer composition according to any one of items 38 to 40, wherein the chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof. (Item 42) 42. The mitochondrial storage buffer composition according to any one of items 38 to 41, wherein the sugar is sucrose. (Item 43) 43. The mitochondrial storage buffer composition according to any one of items 38 to 42, wherein the antioxidant is taurine. (Item 44) 44. The mitochondrial storage buffer composition according to any one of items 38 to 43, wherein the calcium ion-binding cytoprotectant is lactobionate or a salt thereof. (Item 45) Membrane stabilizers and / or oxygen radical scavengers and / or Ca 2+ 45. The mitochondrial storage buffer composition according to any one of items 38 to 44, wherein the agent acting as a binder of mitochondrial proteins and / or a binder of free fatty acids is bovine serum albumin (BSA). (Item 46) 46. ​​The mitochondrial storage buffer composition according to any one of items 38 to 45, wherein the composition further comprises isolated donor mitochondria. (Item 47) 47. The mitochondrial storage buffer composition according to any one of items 38 to 46, wherein the composition does not contain an antibiotic. (Item 48) 1. A kit comprising a donor mitochondrial composition in a unit dosage effective to treat a neurodegenerative disease or other condition associated with mitochondrial dysfunction in a subject, The donor mitochondrial composition comprises: Mitochondria isolated from donor tissue (e.g., fibroblast mitochondria); one or more buffering agents (e.g., a zwitterionic sulfonic acid buffer, e.g., 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) or a salt thereof, e.g., potassium HEPES, (K-HEPES) [e.g., monopotassium phosphate (KHPO)]; A source of magnesium ions [e.g., magnesium chloride (MgCl2)]; A chelating agent (e.g., ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof, e.g., K-EGTA); A sugar (e.g., sucrose); Antioxidants [e.g., taurine], A cytoprotectant that binds calcium ions (e.g., lactobionate or a salt thereof, e.g., K-lactobionate); Membrane stabilizers and / or oxygen radical scavengers and / or Ca 2+ and an agent that acts as a binder for free fatty acids (e.g., bovine serum albumin, BSA). Including the kit. (Item 49) 49. The kit of item 48, wherein the donor and the subject are not HLA (human leukocyte antigen) matched. (Item 50) 50. The kit of claim 48 or 49, wherein the donor mitochondrial composition does not contain an antibiotic. (Item 51) 51. The kit according to any one of items 48 to 50, further comprising instructions for optimizing the dosage and / or frequency and / or route of administration of the composition.

[0068] BRIEF DESCRIPTION OF THE DRAWINGS The foregoing and other objects, aspects, features and advantages of the present disclosure will become more apparent and be better understood by referring to the following description in conjunction with the accompanying drawings. [Brief description of the drawings]

[0069] [Figure 1] 1 is an image showing GFP-labeled mitochondria of fibroblasts according to an exemplary embodiment. There are 337±80 mitochondria per human fibroblast. Mitochondria were counted and analyzed by Olympus IX83 fluorescent microscope and Cellsense software.

[0070] [Diagram 2]2 is an image of live mitochondria isolated from fibroblasts, according to an exemplary embodiment. Mitochondria maintain a membrane potential gradient and actively concentrate JC-1, forming bright red fluorescent aggregates (J aggregates).

[0071] [Diagram 3] 3A-D are a series of images showing immunofluorescence staining of HLA-I antigens according to an exemplary embodiment. FIG. 3A shows a phase contrast image of a fibroblast. FIG. 3B shows green fluorescent staining on the cell surface of the fibroblast. FIG. 3C shows a phase contrast image of isolated mitochondria from a fibroblast. FIG. 3D shows no positive staining in isolated mitochondria from a fibroblast. FIG. 3A, 3C: phase contrast; FIG. 3B, 3D: fluorescence; FIG. 3A, 3B fibroblast; FIG. 3C, 3D: isolated mitochondria from a fibroblast.

[0072] [Figure 4] 4 is an image of fibroblasts introducing mitochondria into MCF-7 cancer cells, according to an exemplary embodiment. The cells indicated by the arrows have mitochondria from both fibroblasts and MCF-7. Thus, the mitochondria show orange fluorescence.

[0073] [Diagram 5] 5 is an image showing that NSC-34ρ0 cells lose mitochondrial DNA according to an exemplary embodiment. MT-CO1 and MT-ND1 genes are not amplified in NSC-34ρ0 cells. Genomic gene ACTB is the same as parental NSC-34 cells.

[0074] [Figure 6] 6A and 6B are a series of images showing mitochondria of NSC-34 and primary fibroblasts transplanted into NSC-34ρ0 cells according to an exemplary embodiment. NSC-34 and fibroblast mitochondria were labeled with MitoTracker Red, isolated, and co-cultured with NSC-34ρ0 cells for 24 hours. FIG. 6A transplanted NSC-34 mitochondria. FIG. 6B transplanted fibroblast mitochondria.

[0075] [Figure 7] 7 is an image showing that autologous NSC-34 mitochondria replenish mitochondrial DNA of NSC-34ρ0 cells according to an exemplary embodiment. MT-CO1 amplified bands are present 1, 3 and 6 days after mitochondrial transfer (MT).

[0076] [Figure 8] Figure 8 is an image showing that autologous NSC-34 mitochondria restores the MT-CO1 and MT-ND1 gene expression of NSC-34ρ0 cells according to an exemplary embodiment. NSC-34ρ0 cells are co-cultured with mitochondria isolated from NSC-34 cells (1 NSC-34ρ0 cell + mitochondria from 10 NSC-34 cells). Gene expression is measured by qPCR.

[0077] [Figure 9] 9 is an image showing that autologous NSC-34 mitochondria reverse the expression of glycolytic genes in NSC-34ρ0 cells according to an exemplary embodiment. NSC-34ρ0 cells are co-cultured with mitochondria isolated from NSC-34 cells (1 NSC-34ρ0 cell + mitochondria from 10 NSC-34 cells). Gene expression is measured by qPCR.

[0078] [Figure 10] 10 is an image demonstrating that, according to an exemplary embodiment, EpH4-Ev MitoTracker Orange-labeled mitochondria were successfully transplanted into 4T1 cells grown in mice. Orange fluorescence was observed in 4T1 cells on tumor smear slides 24 hours after injection of mitochondria.

[0079] [Figure 11]11 is an image of isolated mitochondria exhibiting red fluorescence (J aggregates) and retaining membrane electrochemical potential according to an exemplary embodiment. Mitochondria in fibroblasts are labeled with JC-1, isolated, and resuspended in mitochondrial storage buffer.

[0080] [Figure 12] Figures 12A and 12B are images showing immunofluorescence staining of HLA class I antigens according to an exemplary embodiment: Figure 12A: HLA-A / B / C, green fluorescence is seen mainly on the cell surface; Figure 12B: Mitochondria labeled with MitoTracker Red, red fluorescence is mainly in the cytoplasm.

[0081] [Figure 13-1] 13A-13D are images showing that isolated mitochondria from human fibroblasts labeled with MitoTracker Red are introduced into mouse motor neuron NSC-34 cells after 12 hours of co-culture according to an exemplary embodiment. Figures 13A, 13B: NSC-34, Figure 13A-phase contrast, Figure 13B-fluorescence; Figures 13C, 13D: NSC-34 treated with EtBr, Figure 13C-phase contrast, Figure 13D-fluorescence. [Figure 13-2] Same as above.

[0082] [Figure 14] FIG. 14 is a graph showing that injection of human fibroblast mitochondria improves quadriceps strength in ALS patients, according to an exemplary embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0083] Detailed Description The methods and compositions described herein demonstrate the use of human primary fibroblasts as a source of donor mitochondria for the treatment of other neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), Parkinson's disease and Alzheimer's disease, stroke, and other conditions and diseases that result in reduced muscle function due to mitochondrial damage.Fibroblasts share many characteristics with mesenchymal stem cells (MSCs), and it is found herein that allogeneic mitochondria from primary fibroblasts exhibit low or no immunogenicity and do not require HLA typing for mitochondrial transplantation.The mitochondrial respiration buffer (MRB) solution described herein (also referred to as mitochondrial storage buffer composition) maintains good mitochondrial viability and is safe for intravenous and intramuscular injection in human patients. In one embodiment, the MRB contains 240 mM sucrose at pH 7.2, 2 mM KH2PO4, 3 mM MgCl2, 10 mM K-HEPES, 1 mM K-EGTA, 20 mM taurine, 15 mM K-lactobionate, and 0.1% human serum albumin (HSA).

[0084] As described in more detail herein, the dosage of mitochondria can be determined for a specific subject.The number of fibroblasts can be used to estimate the number of mitochondria.In one embodiment, the starting dose of mitochondria isolated from 50 million primary fibroblasts can be used.

[0085] For example, according to the exemplary clinical administration protocol for ALS patients, in a given treatment, half of the mitochondria from 50 million primary fibroblasts is injected intravenously, and the other half is injected intramuscularly into multiple sites of affected muscles.For example, in some embodiments, when disease mainly affects the lower limbs, injection into the lower limbs is required, and when symptoms mainly occur in the upper limbs, together with developmental and swallowing muscles, etc., mitochondria is injected into the upper limbs and neck muscles.

[0086] In another example, according to an exemplary clinical dosing protocol for patients suffering from another neurodegenerative disease such as Parkinson's or Alzheimer's disease or patients who have suffered a stroke, all mitochondria from 50 million primary fibroblasts are injected intravenously.

[0087] As discussed in more detail herein, in addition to administration of mitochondria as part of mitochondrial organelle transplantation (MOT), the patient may be administered iron chelators (e.g., desferrioxamine or deferasirox), antioxidants, and / or probiotics. In some embodiments, administration of antibiotics is avoided due to potential adverse effects on transplanted mitochondria.

[0088] To prepare for mitochondrial transplantation studies in human neurodegenerative diseases, human fibroblasts were selected as mitochondrial donors because fibroblasts share many characteristics with mesenchymal stromal cells (MSCs). Human primary fibroblasts were isolated and generated mitochondrial DNA (mtDNA)-depleted mouse motor neuron NSC-34 cells (NSC-34ρ0 cells). Mitochondria from fibroblasts and NSC-34 cells were transferred to NSC-34ρ 0 Fibroblasts were co-cultured with NSC-34ρ cells. Mitochondrial transplantation was observed by fluorescence microscopy. Gene expression was determined by polymerase chain reaction (PCR) and real-time PCR (qPCR). Mitochondria were also injected into mice bearing breast adenocarcinoma 4T1 cells. The following results were found: 1) Abundant mitochondria are present in fibroblasts (337 ± 80 mitochondria per fibroblast). By using differential centrifugation, 42.4% live mitochondria were obtained. After co-culture, isolated mitochondria were transferred to NSC-34ρ cells. 02) Fibroblasts transfer mitochondria into human breast adenocarcinoma MCF-7 cells; 3) There is no expression of HLA-I antigens in fibroblast mitochondria, indicating that fibroblast mitochondria can be used for allogeneic mitochondrial transplantation without HLA antigen matching; 4) PCR and qPCR showed that NSC-34ρ 0 We show that cells lose mitochondrially encoded cytochrome c oxidase I (MT-CO1) and mitochondrially encoded NADH dehydrogenase 1 (MT-ND1) and upregulate the expression of glycolysis-related genes hexokinase (HK2), glucose transporter 1 (SLC2A1) and lactate dehydrogenase A (LDHA); 5) transplantation of NSC-34 mitochondria restores MT-CO1 and MT-ND1 and downregulates the gene expression of HK2, SLC2A1 and LDHA; 6) normal mammary epithelial mitochondria successfully enter 4T1 cells in mice. Subcutaneous injection of mitochondria is safe for mice. Mitochondrial transplantation replenishes mtDNA and rescues aerobic respiration of diseased cells with mitochondrial dysfunction. Human primary fibroblasts are suitable mitochondrial donors for mitochondrial transplantation studies in human neurodegenerative diseases.

[0089] In eukaryotic cells, mitochondria generate ATP by oxidative phosphorylation (OXPHOS) in the presence of oxygen. Mitochondria also play important roles in the synthesis of iron-sulfur (Fe-S) clusters, beta-oxidation of fatty acids, synthesis of heme prosthetic groups, the urea cycle, and homeostasis of calcium, iron and reactive oxygen species (ROS). Mitochondria are highly dynamic organelles that frequently fuse and fission. Mitochondrial fusion / fission allows for the separation of damaged mitochondria, mitophagy to remove damaged mitochondria and ultimately cell death if the damage is severe. In addition, mitochondria can be transferred between cells. Cells may be able to acquire functional mitochondria from other cells to meet their bioenergetic and biosynthetic needs. Without wishing to be bound by any particular theory, possible mechanisms include tunneling nanotubes, extracellular vesicles and partial or complete cell fusion.

[0090] Mitochondrial dysfunction contributes to many diseases, such as neurodegenerative diseases, heart disease and cancer.As known in the art, mitochondrial dysfunction broadly includes the condition that mitochondria of cell, tissue, organism or these samples are characterized by (1) reduced speed, amount or efficiency of ATP production; (2) reduced membrane potential of mitochondria; (3) reduced number or concentration of mitochondria; and / or (4) increased speed or amount of ROS production compared to reference.In some embodiments, the reference is a measurement or value representative of a healthy subject (e.g., a comparable subject with typical mitochondrial function and / or without diagnosed medical condition known to affect mitochondrial function).In some embodiments, the reference is a measurement or value representative of a population of healthy subjects.In some embodiments, the reference is a measurement or value representative of an earlier subject.Methods and techniques for measuring mitochondrial ATP production, mitochondrial membrane potential, mitochondrial number or concentration, and / or ROS production are known in the art.

[0091] Mitochondrial dysfunction has been reported in the literature, for example, in amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD) and Parkinson's disease (PD). Mitochondria are essential for neuronal function, as neurons are highly dependent on aerobic OXPHOS within mitochondria for their energy needs. Defects in mitochondrial respiration and ATP production in neurons result in neuronal dysfunction and degeneration. Mitochondria also produce ROS. If the oxidative stress of ROS overwhelms antioxidant defenses, mostly from superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX), ROS cause neuronal protein, lipid and DNA damage. Furthermore, mitochondrial calcium and iron excess impairs ATP production as well as mitochondrial and neuronal structure. Several proteins from genetic mutations have been associated with familial and sporadic ALS, including superoxide dismutase 1 (SOD1), TAR DNA binding protein (TARDBP; TDP-43), fused in sarcoma (FUS), chromosome 9 open reading frame 72 (C9orf72) and dipeptide repeat protein (DPR), which have been shown to interact with mitochondria in ALS mouse models and human patients.

[0092] Mitochondria are highly abundant in cardiac cells due to their increased energy demand. Mitochondrial dysfunction is associated with the development of numerous cardiac diseases, such as atherosclerosis, ischemia-reperfusion injury, hypertension, cardiac hypertrophy and heart failure.

[0093] Defects in mitochondrial function have also been linked to tumorigenesis. For example, cancer cells have been observed to have increased glycolysis and lactate production in the presence of oxygen without increased OXPHOS, known as the "Warburg effect." Many cancers have mitochondrial defects and dysfunction. Glycolysis inhibitors have been found to suppress tumor growth in animal models and clinical trials.

[0094] Experimental examples of mitochondrial transplantation (MT) into defective neurons and tumor cells in cell culture and mice, as well as the safety of mitochondrial injection in mice, are described below. Mitochondria were found to transfer between human fibroblasts and breast cancer cells after co-culture. Mitochondria isolated from human fibroblasts are candidates for transplantation into mouse mtDNA-depleted neurons. Transplantation of mouse motor neuron mitochondria replenishes mtDNA and rescues aerobic respiration of mouse mtDNA-depleted neurons. Furthermore, mitochondria isolated from mouse normal mammary epithelium are incorporated into mammary cancer cells in a mouse model. Injection of exogenous mitochondria is safe for experimental mice. EXAMPLES

[0095] Experimental Example 1 Isolation, primary culture and cryopreservation of human fibroblasts All protocols used aseptic technique in a class II, type A2 laminar flow hood. All culture plastic materials and scalpels were sterile and discarded. Human skin tissue was provided by young healthy males who signed informed consent. Skin tissue was surgically collected at an outpatient clinic and dropped into a 50 ml discarded sterile centrifuge tube containing 15 ml of sterile 0.9% sodium chloride injection. Skin (0.5 cm × 2 cm) was washed with cold sterile saline. Adipose tissue was trimmed off. Skin was cut into small pieces using a sterile discarded scalpel in a sterile discarded 100 mm culture dish. All tissue pieces and saline were transferred into a sterile glass test vessel with a lid and a stir bar. 4.5 ml of 3% collagenase type 3 (Worthington Biochemical Corporation, Lakewood, NJ, USA) was added to the test vessel. The final volume was 20 ml. For digestion, the test vessel was placed on a magnetic stirrer in a 37 °C oven. The tissue was digested for 5 hours with gentle agitation until tissue debris disappeared. The liquid was transferred into a sterile 50 ml centrifuge tube and centrifuged at 400 × g for 5 minutes. The supernatant was aspirated. The following was then added: 20 ml of pre-warmed (37 °C) complete alpha minimum essential medium (αMEM) (GIBCO, Carlsbad, CA, USA) containing 5% human platelet lysate (HPL) (Biological Industries, Cromwell, CT, USA) and 0.05 mg / ml gentamicin (GIBCO, Carlsbad, CA, USA). The cell pellet was then resuspended and the cell suspension was transferred to a 182 cm 2 The flasks were cultured overnight at 37°C in a 5% CO2 incubator. The next day, the medium and floating cells were aspirated. 20 ml of fresh medium was added to the flask. The flasks were returned to the incubator for culture, and the medium was replaced with fresh one every 3–4 days. When cell growth reached 80% confluency, the cells were subcultured into new flasks by 1:10 dilution. Primary fibroblasts of the second and third passages were collected, resuspended in Nutrifreez D10 cryopreservation medium (Biological Industries, Cromwell, CT, USA), and transferred to a cryopreservation vial at 1 × 10 6The cells were then aliquoted into 1 ml portions, frozen and stored in liquid nitrogen. cell culture

[0096] Human breast adenocarcinoma cell line MCF-7, mouse breast adenocarcinoma cell line 4T1 and mouse mammary epithelial cell line EpH4-Ev were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). MCF-7, 4T1 and human primary fibroblast cells were harvested from liquid nitrogen and cultured in alphaMEM (GIBCO, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS). EpH4-Ev cells were cultured in alphaMEM containing 10% FBS and 1.2 μg / mL puromycin (Sigma Aldrich, St. Louis, MO, USA). NSC-34 is a hybrid cell line produced by fusion of motor neuron-enriched embryonic mouse spinal cord cells with a mouse neuroblastoma. NSC-34 was purchased from Cedarlane Corporation (Ontario, Canada) and cultured in Dulbecco's modified Eagle's medium (DMEM) (GIBCO, Carlsbad, CA, USA) containing 10% FBS. When the cells grew to 80% full in the flask, they were digested with TrypLE expression solution (GIBCO, Carlsbad, CA, USA) and subcultured at 37°C and 5% CO2. Isolation of mitochondria

[0097] Mitochondria were isolated by differential centrifugation. All reagents were sterile. Cells were harvested with TrypLe expression solution and pelleted by centrifugation at 400 x g and 4 °C for 5 min. After aspiration of the solution and medium, the cell pellet was resuspended in ice-cold 300 mM sucrose mitochondrial isolation buffer (MIB) (Sigma Aldrich, St. Louis, MO, USA) and homogenized by bead beating (Bead Ruptor 12, Omni International Homogenizer, Kennesaw, GA, USA). The cell lysate was centrifuged at 700 × g and 4 °C for 10 min. The supernatant was then transferred to a new centrifuge tube and centrifuged at 9,000 × g and 4 °C for 10 min. The supernatant was removed. The mitochondrial pellet was resuspended in 240 mM sucrose mitochondrial respiration buffer (MRB) (Sigma Aldrich, St. Louis, MO, USA). To determine the number of isolated mitochondria, 10 μl of JC-1 stained mitochondria was placed on a glass slide and homogenized by bead beating (Bead Ruptor 12, Omni International Homogenizer, Kennesaw, GA, USA). 8 μm 2 ). Mitochondria were observed through a 20x objective lens, photographic images were taken in five fields, and the number of mitochondria was counted in each photograph by Cellsense software on an Olympus IX83 fluorescent microscope (Olympus, Tokyo, Japan). The object area of ​​each photograph was calculated by using the scale bar (μm) of the photograph. The number of mitochondria in 10 μl was calculated as follows: (coverslip area 4.84 × 10 8 μm 2 ÷μm per photo 2 The total number of isolated mitochondria was then calculated as the mean number of mitochondria per photograph (object area in units) × the number of mitochondria per photograph. Measurement of mitochondrial proteins

[0098] Mitochondrial protein content can be used to estimate the number of mitochondria. When mitochondria were used for protein content measurement, they were washed twice with protein-free MRB. Mitochondria were resuspended and lysed with RIPA buffer (Thermofisher Scientific, Waltham, MA, USA). 40 mg (≦5 × 10 6 For the wet pellet of 1000 ribosomal RNA (1000 ribosomal RNA) cells, 1 ml of RIPA buffer was used. Mitochondria were gently shaken on ice for 30 min. The mitochondrial lysate was then centrifuged at 14,000 × g for 10 min to pellet debris. The supernatant was transferred to a 1.5 ml vial for protein content measurement. Protein was quantified by Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). The procedure followed the protocol of the assay kit. Briefly, the instructions were to pipette 25 μl of standard or sample into duplicate wells, add 200 μl of BCR working reagent to each well, mix the plate thoroughly for 30 s, incubate the plate at 37 °C for 30 min, cool the plate to room temperature, and measure the absorbance at 562 nm in a plate reader. The protein concentration of the samples was calculated by a four-parameter logistic curve. Immunofluorescence staining of human leukocyte antigen I (HLA I) in primary fibroblasts and mitochondria

[0099] 20,000 primary fibroblast cells were cultured overnight on glass-bottom cell culture dishes. 5 × 10 6Mitochondria were isolated from fibroblasts. Fibroblasts in the dish and mitochondria in a 1.5 ml Eppendorf centrifuge tube were fixed with 4% paraformaldehyde solution for 15 min at room temperature. Rabbit anti-human HLA-ABC polyclonal antibody (Thermofisher Scientific, Waltham, MA, USA) reacts with all types of HLA-I antigens (HLA-A, -B and -C) (the abbreviation HLA stands for human leukocyte antigen). The brief procedure for immunofluorescence staining is as follows: wash twice with 1x phosphate-buffered saline (PBS), block with 5% goat serum in 1x PBS, wash once, incubate with 200 μl of 1:400 dilution of rabbit anti-human HLA-ABC polyclonal antibody at 4°C overnight, wash three times with 1x PBS, incubate with goat anti-rabbit IgG-Alexa488 at room temperature for 1 h, wash three times with 1x PBS, add 300 μl of 1x PBS to the dish and tube, resuspend mitochondria in a 1.5 ml Eppendorf tube, add 100 μl of mitochondria to the glass area of ​​a new glass-bottom dish, and observe the fluorescence under an Olympus IX83 fluorescence microscope. All pictures were taken with 1 second exposure. Staining of mitochondria with JC-1 or MitoTracker dyes

[0100] The mitochondrial membrane potential generated by proton pumps has been found to be a key component in the process of energy storage during OXPHOS. Membrane potential-dependent dyes such as JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) and MitoTracker dyes (rosamine or cyCarbocyanine-based probes) have been used to stain mitochondria and monitor mitochondrial potential. For JC-1 staining, mitochondria were stained with a mitochondrial staining kit (Sigma CS0390, St. Louis, MO, USA). The brief procedure is as follows: mix 25 μl of 200× JC-1 Stock Solution with 4 mL of ultrapure water in a test tube; close the tube and mix the solution by inverting or vortexing the tube briefly; incubate the tube at room temperature for 2 minutes to completely dissolve the JC-1; open the tube and add 1 ml of JC-1 Staining Buffer 5×; mix by inversion; then mix the staining mixture with an equal volume of complete medium for cell growth; aspirate the growth medium from the flask and overlay the cells with the above mixture; add 1 cm 2 Add 0.2-0.4 ml of the mixture per growth surface of 100 μl; Incubate the cells for 20 min at 37 °C in a humidified atmosphere containing 5% CO2; Aspirate the mixture; Then wash the cells twice with cold growth medium. Fluorescence was observed using an Olympus IX83 fluorescence microscope. In cells that maintained an electrochemical potential gradient, the dye was concentrated in the mitochondria, where it formed bright red fluorescent aggregates (J-aggregates). If the cells were unable to maintain a membrane potential, JC-1 dispersed throughout the cell, resulting in a shift from red to green fluorescence (JC-1 monomers).

[0101] For MitoTracker dye, the culture medium was aspirated. The cells were covered with fresh medium containing 150 nM MitoTracker Orange or Red dye (Thermofisher Scientific, Waltham, MA, USA) and incubated at 37 °C and 5% CO2 for 30 min. The staining solution was then removed. The cells were washed once, covered with fresh medium, and observed under a fluorescent microscope. Mitochondrial transfer between fibroblasts and cancer MCF-7 cells

[0102] CellLight Mitochondria-RFP or -GFP, BacMam2.0 was purchased from Invitrogen (Carlsbad, CA, USA). CellLight Mitochondria-RFP or -GFP, BacMam2.0 is a fluorescent, leader sequence of E1α pyruvate dehydrogenase fusion (in the mitochondrial matrix) that currently provides precise and specific targeting to mitochondria in live cell mitochondrial imaging. Cells were grown to 70% confluency on culture dishes. The appropriate volume of CellLight reagent for the number of cells was calculated according to the product protocol. The volume of CellLight Mitochondria-RFP or -GFP was added to the cells. Cells were incubated at 37°C for at least 16 hours and observed by Olympus IX83 fluorescence microscope. The average number of mitochondria per fibroblast was determined by Cellsense software. To test the transfer of mitochondria between adenocarcinoma cells MCF-7 and fibroblasts, mitochondria in MCF-7 and fibroblasts were labeled with CellLight mitochondrial-RFP or -GFP, respectively, for at least 16 h. The medium was removed. Cells were washed twice with pre-warmed fresh medium and detached with TrypLE express reagent. Equal numbers of MCF-7 and fibroblasts were mixed and co-cultured at 37°C and 5% CO2 for 24 h. Fluorescence in the cells was observed by an Olympus IX83 fluorescence microscope. Depleting mitochondrial DNA in NSC-34 cells with ethidium bromide

[0103] Long-term treatment of cells with low doses (0.1–2 μg / ml) of ethidium bromide (EtBr), an inhibitor of DNA / RNA synthesis, is thought to specifically suppress the replication and transcription of extrachromosomal genetic elements such as mtDNA, without affecting the replication and transcription of nuclear DNA. NSC-34 cells were maintained in complete DMEM medium containing 2 μg / ml EtBr and subcultured weekly with fresh medium changes every 3–4 days. After 3 months, total DNA of NSC-34 was isolated with a QIAamp DNA mini kit (Qiagen, Hilden, Germany). MT-CO1 and MT-ND1 were tested by PCR. PCR was performed in a total volume of 25 μl containing 2 μl of PCR Master mix (Thermofisher Scientific, Waltham, MA, USA), 0.5 μl of 100 μM forward and reverse primers, and 200 ng DNA template. The primers used for amplification of MT-CO1 were 5'-CTAGCCGCAGGCATTACTATAC-3' (SEQ ID NO: 1) and 5'-TGTCAAGGGATGAGTTGGATAAA-3' (SEQ ID NO: 2). The primers for MT-ND1 were 5'-GCCGTAGCCCAAACAATTTC-3' (SEQ ID NO: 3) and 5'-CGTAACGGAAGCGTGGATAA-3' (SEQ ID NO: 4). Mouse β-actin (mACTB) was used as a genomic gene control. The primers for amplification of mACTB were 5'-CTGAGTCTCCCTTGGATCTTTG-3' (SEQ ID NO: 5) and 5'-AGGGCAGGTGAAACTGTATG-3' (SEQ ID NO: 6). The amplification procedure included initial DNA denaturation at 95° C. for 3 min, followed by 35 cycles of denaturation at 95° C. for 30 s, primer annealing at 50° C. for 30 s and extension at 72° C. for 60 s, and a final extension at 72° C. for 10 min in a T100 Thermal Cycle (Bio-Rad, Hercules, CA, USA). PCR products were run on a 2% agarose gel and imaged by ethidium bromide fluorescence. mtDNA-depleted NSC-34 (NSC-34ρ 0 ) Mitochondrial transplantation

[0104] Mitochondria were isolated from mouse NSC-34 cells (1 × 10 6 1 × 10 mitochondria (from 10 cells) 5 NSC-34ρ 0 Mitochondria from 10 NSC-34 cells + 1 NSC-34ρ 0 Normal NSC-34 were used as a control. After 24 h of co-culture, the medium containing mitochondria was removed. The cells were washed twice, replenished with fresh complete DMEM, and continuously cultured. Cell DNA and RNA were isolated 1, 3, and 6 days after transplantation for PCR and real-time PCR examination, respectively. For imaging of mitochondrial transplantation, mitochondria of NSC-34 cells and primary fibroblasts were labeled with MitoTracker Red, isolated, and cultured in NSC-34ρ 0 Mitochondria from 10 NSC-34 cells or fibroblasts + 1 NSC-34ρ were co-cultured for 24 h with 0 After removal of the medium containing the labeled mitochondria, the cells were observed under a fluorescent microscope. Real-time PCR (qPCR)

[0105] Quantitative real-time polymerase chain reaction (qPCR) was used to measure mRNA levels of mitochondrial and glycolysis-related genes. The following were examined: mitochondrial OXPHOS genes, MT-CO1 and MT-ND1, and glycolysis-related genes HK2, SLC2A1 and LDHA, which catalyzes the reduction of pyruvate by NADH to form lactate. All gene expression quantification was performed using TaqMan Gene Expression Assay, a proven 5' nuclease-based real-time PCR chemistry. Primers and probes (PrimeTime Mini qPCR Assay) were synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa) (Table 1, SEQ ID NOs: 7-24). PCR was normalized for the amount of RNA added to the reverse transcription reaction using β-actin (ACTB) as an endogenous gene control. The probe contains FAM (6-carboxyfluorescein) as a fluorescent reporter dye at the 5' end and ZEN™ / Iowa Black FQ as a fluorescent dual quencher internally and at the 3' end. qPCR reactions were performed using a 7900HT Real-Time PCR System (Applied Biosystems, Grand Island, NY). Standard mode was run as 2 min at 50°C and 10 min at 95°C, with 40 cycles (15 s at 95°C, 1 min at 60°C). Target gene expression was determined by relative quantification, relating the signal of the target transcript in the experimental group to that of the control. Relative quantification was analyzed using RQ Manager 1.2.1 software (Applied Biosystems, Grand Island, NY, USA). Mitochondrial transplantation into mammary adenocarcinoma 4T1 cells in mice

[0106] An Institutional Animal Care and Use Committee (IACUC) protocol was submitted and approved by the LSU School of Veterinary Medicine. Female mice were 16-18 grams (mean 17.3 g) and 8 weeks old. 5Mouse mammary adenocarcinoma 4T1 cells were injected subcutaneously into each mouse. When the tumors grew to a diameter of 1 cm, 1.0 × 10 6 0.2 ml of isolated mitochondria from EpH4-Ev cells (5 x 10 7 1 × 10 mitochondria) were directly injected into the tumor. Mitochondria of normal epithelial EpH4-Ev were pre-labeled with MitoTracker orange dye, isolated, and stored in MRB on ice. After 24 hours, the tumor was biopsied and smeared on a glass slide. The tumor smear slide was observed under a fluorescent microscope to confirm whether the labeled mitochondria had entered into the 4T1 tumor cells. To examine the inhibition of normal mitochondria on tumor cells, 1 × 10 mitochondria were injected into 0.2 ml of MRB. 5 4T1 cells and mitochondria (5 × 10 7 Mice were injected subcutaneously in the dorsal neck region with a mixture of 1 × 10 mitochondria from 1 tumor 4T1 cell + 10 EpH4-Ev cells. Control mice received 1 × 10 mitochondria in 0.2 ml MRB per mouse. 5 Only 4T1 cells were injected. The control and mitochondria-treated groups contained 10 and 11 mice, respectively. 18 days after injection, the mice were sacrificed and autopsied. The tumor mass and organs, including lungs, liver, heart, spleen and kidneys, were removed and weighed. For histological studies, all tissues were fixed in 4% formalin solution. statistical analysis

[0107] Statistical significance was tested using Student's t-test. A p-value of less than 0.05 was considered statistically significant. result Number, protein content and viability of isolated mitochondria

[0108] The life span of human primary fibroblasts was found to be approximately 80 days. The doubling time of proliferation is 40 ± 6 hours. Fibroblast mitochondria were labeled with CellLight Mitochondria-GFP, and the number of mitochondria per fibroblast was counted in 30 different fields of view using Cellsense software on an Olympus IX83 fluorescent microscope. There were 337 ± 80 mitochondria (mean ± standard deviation) per human fibroblast. Figure 1 shows GFP-labeled mitochondria in fibroblasts. Mitochondrial viability was assessed using a mitochondrial fluorescent probe. Isolated mitochondria were found to maintain a membrane potential gradient, actively concentrate JC-1, and form bright red fluorescent aggregates (J-aggregates). Figure 2 shows viable mitochondria isolated from fibroblasts. This finding suggests that isolated mitochondria are viable and maintain an electrochemical potential gradient. A total of (3.37 ± 80) × 10 mitochondria were found in the cells. 9 1 x 10 mitochondria 7 A total of (1.43 ± 0.33) × 10 fibroblasts were 9 The mitochondrial isolation protocol yielded a yield of 42.4% [(1.41 × 10 9 )÷(3.37×10 9 ) × 100%] of fibroblast mitochondria can be obtained. Mitochondrial protein content is an alternative method to estimate the number of mitochondria. 7 Fibroblasts isolated from (1.43±0.33)×10 9 Each mitochondrion has a protein content of 190 ± 45 µg. Both the fibroblast number and the mitochondrial protein content can be used to estimate the number of isolated mitochondria. Fibroblast mitochondria do not express HLA-I antigens

[0109] Green fluorescence is seen on the cell surface of human fibroblasts. The distribution of green fluorescence is heterogeneous. This finding suggests that HLA class I antigens are mainly expressed on the cell surface of human fibroblasts. Isolated mitochondria have no green fluorescent staining. These results suggest that mitochondria of human fibroblasts do not express HLA antigens and can be used for allogeneic mitochondrial transplantation without HLA antigen matching. For illustration, Figures 3A-D show immunofluorescence staining of HLA-I antigens. Although green fluorescent staining is present on the cell surface of fibroblasts (Figure 3B), no positive staining is present in isolated mitochondria (Figure 3D). Figures 3A, 3C show phase contrast images; Figures 3B, 3D show fluorescence; Figures 3A, 3B show fibroblasts; Figures 3C, 3D show isolated mitochondria from fibroblasts. Mitochondrial transfer between human fibroblasts and breast adenocarcinoma MCF-7 cells

[0110] Fibroblasts labeled with CellLight Mitochondria-GFP were co-cultured with MCF-7 labeled with CellLight Mitochondria-RFP for 24 hours. Mitochondria transferring between fibroblasts (green) and MCF-7 cells (red) resulted in orange fluorescence in some cells (see arrows in Figure 4). Figure 4 shows that fibroblasts have transferred mitochondria to MCF-7 cancer cells. The cell indicated by the arrow has mitochondria from both fibroblasts and MCF-7 cancer cells. Thus, the mitochondria show orange fluorescence. NSC-34ρ 0 Transplantation of normal mitochondria into cells

[0111] After 3 months of treatment of NSC-34 with 2 μg / ml EtBr, PCR showed no amplification of the mitochondrial genes MT-CO1 and MT-ND1. Treatment had no effect on amplification of the genomic gene ACTB. For example, FIG. 5 shows that NSC-34ρ 0 The MT-CO1 and MT-ND1 genes were expressed in NSC-34ρ cells, indicating that the cells had lost mitochondrial DNA. 0The genomic gene ACTB was not amplified in the NSC-34 cells. The genomic gene ACTB is the same as in the parental NSC-34 cells. The results suggest that long-term EtBr treatment depleted the mtDNA in NSC-34 cells. 0 The cells proliferate much slower than the parental NSC-34. 0 The proliferation of the cells is approximately 10% of that of the parent NSC-34. MitoTracker Red-labeled mitochondria isolated from NSC-34 cells or primary fibroblasts were cultured in NSC-34ρ 0 After 24 hours of incubation, red fluorescent mitochondria were identified as NSC-34ρ cells. 0 6A and 6B show that mitochondria in NSC-34 and primary fibroblasts were downregulated by NSC-34ρ 0 Mitochondria from NSC-34 and fibroblast cells were labeled with MitoTracker Red, isolated, and transplanted into NSC-34ρ cells. 0 The cells were co-cultured with NSC-34ρ for 24 hours. Figure 6A shows that NSC-34 mitochondria were transplanted into the cells. Figure 6B shows that fibroblast mitochondria were transplanted into the cells. These results indicate that isolated autologous and heterologous mitochondria can effectively inhibit the defective NSC-34ρ 0 This suggests that the cells will be transplanted. NSC-34 mitochondria replenish mtDNA and NSC-34ρ 0 Rescuing cellular aerobic respiration

[0112] NSC-34ρ 0 After co-culturing cells with isolated NSC-34 mitochondria for 24 h, PCR confirmed that the MT-CO1 DNA gene was expressed in NSC-34ρ 0 MT-CO1 is amplified in cells. Furthermore, MT-CO1 persists after amplification 3 and 6 days after mitochondrial transplantation (FIG. 7). FIG. 7 shows that autologous NSC-34 mitochondria are amplified in NSC-34ρ cells. 0This indicates that the mitochondrial DNA of the cells is replenished. There is a clear MT-CO1 amplified band at 1, 3 and 6 days after mitochondrial transplantation (MT). The amplification of the genomic gene ACTB is not changed by mitochondrial transplantation. The gene expression level is calculated by the following formula: (NSC-34ρ 0 Gene expression in NSC-34 cells (gene expression in NSC-34 cells) × 100%. 0 The cells have very low levels of mRNA expression of the mitochondrial genes MT-CO1 and MT-ND1 (approximately 10% of parental NSC-34 cells), but demonstrate that mRNA expression is restored to normal NSC-34 levels 3 days after mitochondrial transplantation (Figure 8). 0 We show that the expression of MT-CO1 and MT-ND1 genes in NSC-34ρ cells is restored. 0 Cells were co-cultured with isolated mitochondria from NSC-34 cells (1 NSC-34ρ 0 cells + mitochondria from 10 NSC-34 cells). Gene expression was measured by qPCR. Expression of glycolytic genes HK2, LDHA and SLC2A1 was significantly higher in NSC-34ρ cells than in NSC-34ρ cells. 0 These are upregulated in NSC-34ρ cells that have defective mitochondria. 0 This suggests that cells increase aerobic glycolysis to produce cellular energy ATP. Mitochondrial transplantation downregulates the expression of glycolysis-related genes.

[0113] The levels of HK2, LDHA and SLC2A1 gene expression return to normal 3 days after transplantation of NSC-34 mitochondria (Figure 9). Figure 9 shows that autologous NSC-34 mitochondria increased the expression of NSC-34ρ 0 We show that it reverses the expression of glycolytic genes in cells. 0Cells were co-cultured with isolated mitochondria from NSC-34 cells (1 NSC-34ρ0 cell + mitochondria from 10 NSC-34 cells). Gene expression was measured by qPCR. These results indicate that isolated NSC-34 mitochondria effectively replenish mitochondrial DNA and NSC-34ρ 0 This suggests that aerobic respiration of cells can be rescued. In this study, mitochondria from 10 NSC-34 cells were compared with 1 NSC-34ρ, which has defective mitochondria. 0 The cells can be rescued.

[0114] Therefore, the above experiments demonstrated that heterologous mitochondria from human fibroblasts were 0 We demonstrate successful entry into cells (see, e.g., FIG. 6B and discussion above). Mitochondrial transplantation in the murine 4T1 tumor model

[0115] Orange fluorescence was observed in 4T1 cells 24 hours after injection of mitochondria labeled with MitoTracker orange from normal EpH4-Ev cells into the tumor mass (Figure 10). Figure 10 shows that EpH4-Ev mitochondria labeled with MitoTracker orange are successfully transplanted into 4T1 cells grown in mice. Orange fluorescence was observed in 4T1 cells on the tumor smear slide 24 hours after injection of mitochondria. This result suggests that mitochondria from EpH4-Ev cells are successfully transplanted into tumor 4T1 cells. On the 18th day of the experiment, the mice were autopsied. The tumor weight in the group receiving normal EpH4-Ev mitochondria is not different from that of the control group (p>0.05). Macroscopic examination shows that all mice have lung metastases in both groups. However, the lung weights of the group receiving mitochondria were smaller than those of the control group (see Table 2 in the Discussion subsection below), even though the difference was not significant (p=0.06). These results suggest that exogenous normal mitochondria can reduce metastatic burden in the lungs.

[0116] Mitochondria [5 × 10 per mouse 7 Subcutaneous injection of 10 mitochondria (6.6 μg protein) and MRB (0.2 ml / mouse) did not cause any side effects in mice. The dose in mice can be converted to a human dose based on body surface area as the formula: human dose per kilogram (kg) body weight = mouse dose per kg body weight × 0.081. In this study, the average mouse weight is 17.3 g. The mouse dose is 0.38 mg / kg [0.0066 mg (mitochondrial protein) ÷ 0.0173 kg mouse body weight], or 2.89 × 10 9 Mitochondria / kg (5×10 7 mitochondria ÷ 0.0173 kg mouse body weight). The equivalent human dose is 0.031 mg / kg protein (0.38 mg / kg in mice × 0.081) or 2.34 × 10 8 Mitochondria / kg (2.89 × 10 in mice) 9 × 0.081). These doses can be used as starting doses for clinical trials. Other doses may also be used. Subcutaneous injection of 0.2 ml of MRB is safe for mice. Conversion of MRB volumes to humans would be 2-5 ml intramuscular injection per site or <250 ml intravenous injection. Consideration

[0117] The exact pathogenesis of most neurodegenerative diseases (NDs) remains unclear. Current effective treatments for NDs are limited. NDs are often accompanied by mitochondrial dysfunction. Therefore, targeting mitochondrial-associated pathogenesis has attracted increasing attention for ND treatment. Traditional drugs or gene-targeting agents do not easily reach (or do not reach) specific subcompartments of mitochondria. Furthermore, the diversity of mitochondrial mutations among patients may make it impossible to develop one drug to cure one disease. In recent years, mitochondrial transplantation has shed new light on therapeutic interventions that benefit neuronal survival and regeneration for neurodegenerative diseases, stroke and CNS injury. As presented herein, exogenous healthy mitochondria are transplanted into defective neurons and found to promote neuronal viability, activity and neurite regrowth. In this study, we investigated the mechanism of action of mtDNA-depleted motor neuron NSC-34 (NSC-34ρ 0 ) cells grow much slower than the parental NSC-34 cells. NSC-34ρ 0 NSC-34ρ cells increase aerobic glycolysis, a pathway that is less efficient at generating ATP than OXPHOS (see Figure 9). 0 The cells can be a model of mitochondrial defects in motor neurons. In ALS patients, motor neurons in the brain and spinal cord are defective and do not function properly. NSC-34ρ 0 NSC-34 cells are a useful tool for studying mitochondrial pathogenesis in ALS. Transplantation of NSC-34 mitochondria resulted in the expression of NSC-34ρ, confirmed by PCR. 0 replenishes the mtDNA of cells (Figures 7-8). After mitochondrial transplantation, the expression of glycolytic genes HK2, LDHA and SLC2A1 is decreased at day 1 and reduced to normal levels in NSC-34 cells at day 3 (Figure 9).

[0118] A registered study on mitochondrial transplantation at ClinicalTrials.gov''Transplantation of Autologously Derived Mitochondria Following Ischemia'' (https: / / clinicaltrials.gov / ct2 / show / NCT02851758) describes that human autologous mitochondria of skeletal muscle are to be injected into ischemic myocardium. Like cardiac muscle, skeletal muscle is striated muscle. In addition, skeletal muscle is abundant in humans. Therefore, skeletal muscle is a reasonable mitochondrial donor tissue for mitochondrial transplantation after cardiac ischemia. However, since autologous repair and regeneration of the nervous system is limited, it is difficult to find allogeneic or autologous neurons as mitochondrial donors. In the study described herein, human fibroblasts were selected as donors of mitochondria in mitochondrial transplantation for human ND for the following reasons: 1) fibroblasts have abundant tissue sources and can regenerate in humans; 2) fibroblasts often exchange mitochondria with other cells, including neurons; 3) fibroblasts share many characteristics with mesenchymal stromal cells / stem cells (MSCs). MSC cell therapy has been used in trials against inflammatory, immune-mediated and degenerative diseases due to their immunomodulatory, immunosuppressive and regenerative abilities; 4) Human fibroblasts can be induced into functional neurons. In the study presented herein, human primary fibroblasts were found to be easily isolated and rapidly proliferated. Mitochondria are dynamic organelles with continuous fission and fusion. However, cells have a relatively stable number of mitochondria in normal physiological situations. The number of mitochondria in a cell can vary greatly between organs, tissues and cell types. For example, red blood cells have no mitochondria, while liver cells can have more than 2000 mitochondria each. Human fibroblasts have abundant mitochondria (an average of 337 mitochondria per fibroblast). In the study described herein, it was found that human fibroblasts transfer mitochondria to the human breast adenocarcinoma cell line MCF-7 (see Figure 4).Mitochondrial transfer between fibroblasts and cancer may be beneficial for cancer growth. However, fibroblast mitochondria transfer with normal cells may rescue the aerobic respiration of damaged cells and be beneficial for tissue repair and regeneration. The mitochondrial isolation protocol described herein resulted in 42.4% of intracellular mitochondria in fibroblasts. Due to the short life span of isolated mitochondria (less than a few hours on ice), fibroblast counts can be used as a rapid method to estimate mitochondrial number during mitochondrial transplantation studies. 1 x 10. 7 From 10 fibroblasts, the final number of mitochondria isolated was 1.43 x 10 9 (1×10 7 x 337 x 42.4% = 1.43 x 10 9 ). Mitochondrial protein content is an alternative method to determine the number of mitochondria, but this procedure often requires several hours. Therefore, mitochondrial protein content is less practical than cell number to estimate the number of mitochondria in mitochondrial transplantation studies. Isolated mitochondria maintain a membrane potential gradient. Mitochondrial membrane potential serves as an intermediate form of energy storage that is generated by proton pumps (complexes I, III and IV) and used by ATP synthase to make ATP. Membrane potential is also a factor that determines mitochondrial viability, part of which is the driving force for transporting charged compounds that are essential for mitochondrial viability. Loss of mitochondrial membrane potential induces mitophagy to eliminate damaged mitochondria. Thus, mitochondrial membrane potential is a marker of isolated mitochondria viability. Mitochondrial membrane potential can be determined by mitochondrial fluorescent probes such as JC-1, MitoTracker dyes, tetramethylrhodamine ethyl (TMRE) or methyl (TMRM) esters. The viability of isolated mitochondria can be determined quickly and easily by using mitochondrial fluorescent probes. Isolated fibroblast mitochondria express defective mouse motor neurons, NSC-34ρ 0The mitochondria were successfully transplanted into the cells (Figure 6B), indicating that heterologous mitochondria can be transplanted into recipient cells from different species. Taken together, human primary fibroblasts are found to be viable and potentially ideal mitochondrial donors for mitochondrial transplantation into NDs.

[0119] Allogeneic tissue donors require HLA type matching. The human leukocyte antigen (HLA) system or complex is a group of related proteins encoded by the major histocompatibility complex (MHC) gene complex in humans. HLAI molecules are one of two major classes of MHC molecules present on the cell surface of all nucleated cells and include HLA-A, HLA-B and HLA-C. HLAII is found only on professional antigen-presenting cells such as dendritic cells and B cells. The immune system uses HLA to distinguish autologous cells from non-autologous cells. Due to the similarity between fibroblasts and MSCs, fibroblasts may express low levels of HLAI antigens. The results described herein show that primary fibroblasts express HLA-I antigens on the cell surface, but mitochondria isolated from fibroblasts do not express any HLA-I antigens (Figure 3). These results suggest that allogeneic mitochondria exhibit low or no immunogenicity and do not require HLA type matching in mitochondrial transplantation. It is possible that fibroblast mitochondria can be used as a "ready-to-use" product for biotherapy.

[0120] Furthermore, mitochondrial transplantation may have potential for cancer therapy. Mitochondria isolated from normal mammary epithelium MCF-12A are transplanted into human breast carcinoma cell lines and are seen to downregulate gene expression of glycolytic enzymes and increase cancer drug sensitivity. In the study described herein, normal mitochondria are found to successfully enter tumor 4T1 cells in mice 24 hours after mitochondrial injection into the tumor mass (Figure 10). In another group of mice, normal mitochondria (mitochondria from 10 EpH4-Ev cells + 1 tumor 4T1 cell) are unable to inhibit tumor growth. Although normal mitochondria reduce lung weight, the difference is not as significant (p=0.06) compared to the control group. The inhibitory effect of normal mitochondria on tumor cells may depend on the dose of mitochondria. Subcutaneous injection of 0.2 ml of mitochondria and MRB was found to be safe for mice. The number of mitochondria and MRB volume in mice can be converted to human doses as an estimate of the starting dose for clinical trials.

[0121] In this experimental example, human primary fibroblasts are found to be suitable mitochondrial donors for mitochondrial transplantation for the treatment of human neurodegenerative diseases. Furthermore, it has been found that mitochondrial transplantation can replenish mtDNA and rescue aerobic respiration in cells with defective mitochondria. Table 1. List of primer and probe sequences for qPCR [Table 1] Table 1 contains sequences corresponding to SEQ ID NOs: 7 to 24 in the order shown in Table 1. Table 2. Tumor and lung weights in 4T1 mammary adenocarcinoma-bearing mice treated subcutaneously with EpH4-Ev mammary epithelial mitochondria. [Table 2] * Mean + standard deviation (number of mice) &Student's t-test was used, and a value of less than 0.05 was considered statistically significant. Experimental Example II

[0122] Fibroblasts were obtained from skin tissue of a young male adult. The donor was healthy and had no viral infections. The donor signed a release document for the use of his tissue. 250 mg of surgically removed skin tissue was minced with a sterile scalpel and resuspended in 6.25% fetal bovine serum (GIBCO). The cells were digested for 5 h at 37 °C in α-MEM containing 0.56% collagenase (GIBCO Invitrogen, Carlsbad, CA, USA), 0.56% collagenase (Worthington Biomedical, Lakewood, NJ), and 0.004% DNase (Sigma Aldrich, St. Louis, MO). The cell mixture was then centrifuged at 400 g for 5 min. The supernatant was removed. The cell pellet was resuspended in complete α-MEM supplemented with 10% fetal bovine serum and 1 mM glutamine (GIBCO Invitrogen, Carlsbad, CA, USA) and incubated at 37 °C with 5% CO2. After the appearance of large fibroblast colonies, the colonies were removed from the flask with a sterile cell scraper and transferred to a new flask containing fresh culture medium (same composition as above). When the fibroblasts had grown to 80% full in the flask, they were digested with 0.05% trypsin and 0.53 mM EDTA. Cells were grown in medium not supplemented with antibiotics such as gentamicin, penicillin, and streptomycin. A large number of primary fibroblasts were frozen and stored in liquid nitrogen for future use. Antibiotics in culture medium are toxic to mitochondria of mammalian cells. Therefore, for MOT, human fibroblasts were cultured in antibiotic-free complete α-MEM supplemented with 10% fetal bovine serum and 1 mM glutamine at 37°C with 5% CO2. Mitochondrial isolation and storage buffers

[0123] The mitochondrial isolation buffer in this example consists of 300 mM sucrose, 10 mM K-HEPES, 1 mM K-EGTA, 0.1% BSA, and 0.25 mM PMSF (Sigma Aldrich, St Louis, MO, USA). The osmolarity of the buffer is 325 mOsm. The concentration of potassium ions is 11 mM. Bovine serum albumin (BSA) is a membrane stabilizer, an oxygen radical scavenger, and a Ca 2+ and binds free fatty acids. Phenylmethylsulfonyl fluoride (PMSF), also called phenylmethanesulfonyl fluoride, is a serine protease inhibitor used in the preparation of cell lysates. Lysosomes are organelles that contain digestive enzymes that digest excess or weakened organelles. During the cell homogenization procedure, some lysosomes may be damaged and release digestive enzymes into the cell lysate. To prevent mitochondrial damage from digestive enzymes, we include PMSF in the isolation buffer.

[0124] The mitochondrial storage buffer in this experiment consists of 240 mM sucrose, 2 mM KH2PO4, 3 mM MgCl2, 10 mM K-HEPES, 20 mM taurine, 1 mM K-EGTA, 0.1% BSA and 15 mM K-lactobionate (Sigma Aldrich, St Louis, MO, USA). Taurine acts as an antioxidant that scavenges free radical species generated by mitochondria and is also involved in membrane stabilization, osmoregulation and ion channel regulation. Lactobionate has cytoprotective properties and prevents mitochondrial swelling. Lactobionate also binds calcium ions with high affinity and acts as a calcium chelator. The osmolarity of the mitochondrial storage buffer is 325 mOsm. The buffer contains 28 mM potassium ions. The buffer is sterilized by filtration through a 0.22 μm filter, aliquoted into small vials and stored at -80 °C.

[0125] In the clinical applications described below, fibroblast mitochondria in storage buffer are administered intramuscularly and intravenously to human patients. High potassium ion concentrations are dangerous for injection into humans (e.g., 91 mM potassium ion concentration from buffer B2 (PLOS One https: / / doi.org / 10.1371 / journal.pone.0187523)). In this experiment, all K + The salts used were 2 mM KH2PO4, 10 mM K-HEPES and 15 mM K-lactobionate. + The final concentration of was 28 mEq, similar to clinical intravenous solutions containing potassium chloride (20 mEq and 40 mEq). The concentration of sucrose was increased to keep the osmolality similar. Isolation of mitochondria

[0126] In a preferred embodiment, it is observed that the use of cooling and rapid isolation is important. All materials and reagents for mitochondrial isolation are pre-chilled on ice. All steps are performed at 0°C-4°C. The brief procedure is as follows: fibroblasts are digested from flasks and pelleted by centrifugation. The fibroblast pellet is resuspended in mitochondrial isolation buffer and transferred to a 2 ml sterile tube containing 1.0 mm beads. The bead tube is shaken at 4000 rpm for 2 minutes to homogenize the fibroblasts. The cell homogenate is poured onto a 40 μm cell strainer and filtered into a 50 ml collection tube on ice, washed with 5 ml of mitochondrial isolation buffer, and the process is repeated once. The homogenate is loaded onto a 10 μm cell strainer and filtered into a 50 ml collection tube on ice. The homogenate is centrifuged at 9000 g for 10 minutes at 4°C. The mitochondrial pellet is resuspended in mitochondrial storage buffer and stored on ice or used immediately. Immunofluorescence staining of HLA class I in human fibroblasts and mitochondria

[0127] Human leukocyte antigen (HLA) complexes include class I (HLA-A, B, C) and class II (HLA-DPA1, DPB1, DQA1, DQB1, DRA and DRB1). HLA class I antigens are present on the surface of almost all cells. HLA class II antigens are present almost exclusively on the surface of certain immune system cells. Mismatches of HLA antigens between donors and recipients often result in cell and organ rejection after cell therapy and organ transplantation. For the staining process, 20,000 fibroblasts are cultured overnight on 35 mm glass-bottom dishes. The mitochondria of fibroblasts are stained with MitoTracker Red (Molecular Probes, Eugene, OR). MitoTracker Red labels mitochondria in living cells by accumulation dependent on the mitochondrial membrane potential. MitoTracker is chemically reactive and binds to thiol groups in mitochondria. The dye becomes permanently bound to mitochondria and therefore remains after cells are killed or fixed. The cells are fixed with 4% paraformaldehyde solution at room temperature for 15 min and washed with phosphate buffered saline (PBS). The cells are incubated with protein blocker at room temperature for 1 h, washed three times with PBS, and incubated with rabbit anti-human HLA-ABC polyclonal antibody (Invitrogen, Carlsbad, CA) overnight at 4°C. The cells in the blank control dish are incubated with PBS without HLA antibody. The dish is washed four times with PBS, incubated with goat anti-rabbit IgG-Alexa488 at room temperature for 1 h, and washed four times again. The cells are covered with 0.4 ml of PBS and observed for fluorescence under an Olympus IX83 fluorescent microscope (Tokyo, Japan). Furthermore, isolated mitochondria are fixed and incubated with antibodies as for fibroblasts, and immunofluorescence staining is observed. Viability of isolated mitochondria

[0128] To determine the viability of mitochondria isolated from human fibroblasts, a technique involving the use of 5,5,6,6'-tetrachloro-1,1',3,3'tetraethylbenzimidazoylcarbocyanine iodide (JC-1) dye (Sigma Aldrich, St Louis, MO, USA) was used to measure the mitochondrial membrane potential (ΔΨ M ) is detected. M In the case of cells with mitochondrion, JC-1 dye enters and accumulates in the negatively charged mitochondria, which generate voltage, and spontaneously forms red fluorescent J-aggregates. In contrast, in unhealthy or apoptotic cells, the inside of the mitochondria is less negative due to increased membrane permeability, resulting in a loss of electrochemical potential, so JC-1 dye also enters the mitochondria, but to a lesser extent. Under this condition, JC-1 retains its original green fluorescence, since it does not reach a sufficient concentration to cause the formation of J-aggregates. JC-1 can be used to assess ΔΨM in both intact isolated mitochondria and tissues. Stain fibroblasts with JC-1 (Sigma Aldrich, St Louis, MO). Isolate mitochondria stained with JC-1 by the protocol described above. Observe the fluorescence of the isolated mitochondria under a fluorescent microscope. Red fluorescence suggests that the isolated mitochondria maintain their membrane electrochemical potential and are alive. Mitochondrial organelle transplantation (MOT) in mouse neuromotor cell line NSC-34

[0129] NSC-34 is a hybrid cell line produced by fusion of motor neuron-rich embryonic mouse spinal cord cells with mouse neuroblastoma (Cellutions Biosystems Inc., Burlington, Ontario, L7L 5R2, Canada). NSC-34 cells are cultured in complete DMEM medium containing 10% fetal bovine serum. Human fibroblast mitochondria are labeled with MitoTracker Red, isolated, and co-cultured with NSC-34 cells for 12 h at 37°C with 5% CO2. The culture medium is aspirated. The cells are washed three times with pre-warmed DMEM and covered with 0.5 ml of fresh DMEM medium. The fluorescence of NSC-34 cells is observed under an Olympus IX83 fluorescence microscope (Tokyo, Japan). Additionally, NSC-34 cells are cultured in DMEM medium containing 2,000 ng / ml ethidium bromide (EtBr). EtBr intercalates (i.e., inserts itself between the strands) into double-stranded DNA, deforming the DNA and affecting DNA biological processes, including DNA replication and transcription. EtBr causes mitochondrial DNA defects and ultimately depletes mitochondrial DNA in NSC-34 cells. Fibroblast mitochondrial MOT is examined in diseased NSC-34 cells and healthy NSC-34 treated with EtBr. Preparation of MOT in amyotrophic lateral sclerosis (ALS)

[0130] 80-100×10 mitochondria 6The chimeric mitochondrial permeabilizing peptide was isolated from 11 human fibroblasts. Mitochondria were resuspended in 11 ml of mitochondrial storage buffer. 4 mg of the chimeric mitochondrial permeabilizing peptide was dissolved in 2 ml of mitochondrial storage buffer and sterilized by filtering through a 0.22 μm filter. The peptide consisted of the leader peptide of mitochondrial aldehyde dehydrogenase (ALDH2) with an arginine-rich peptide. The amino acid sequence of the peptide was MLRAAARFGPRLGRRLLSARKKRRQRRR. (SEQ ID NO: 25). The leader peptide can bind to the mitochondrial outer membrane via the protein translocase outer membrane (Tom), and the arginine-rich sequence can insert into the cell membrane. Thus, the chimeric peptide may increase the possibility of exogenous mitochondria entering the cell. The peptide solution was added into 11 ml of fibroblast mitochondria and incubated on ice for 30 minutes. Mitochondria were drawn into eight 1 ml syringes and one 5 ml syringe. The syringes were placed on ice and used for intramuscular and intravenous injections within 30 minutes. result Isolated mitochondria maintain their membrane electrochemical potential and viability.

[0131] Isolated mitochondria can concentrate the JC-1 dye and form J aggregates. Thus, red fluorescence is found in mitochondrial colonies (Figure 11). This finding confirms that the mitochondrial isolation and preservation protocol is capable of purifying and maintaining live mitochondria for the following MOT in cell lines and patients. Human mitochondria do not express HLA antigens

[0132] Green fluorescence is seen on the cell surface of human fibroblasts. The distribution of green fluorescence is heterogeneous (Figure 12A). This finding suggests that HLA class I antigens are mainly expressed on the cell surface of human fibroblasts. The red fluorescence of mitochondria labeled by MitoTracker Red is seen mainly in the cytoplasm (Figure 12B). These findings indicate that mitochondria have no or extremely low expression of HLA class I antigens. Furthermore, using the same immunofluorescence technique, the HLA class I antigens of isolated mitochondria of fibroblasts were examined. There was no positive green fluorescence in mitochondria. These results suggest that mitochondria of human fibroblasts do not express HLA antigens and can be used for allogeneic MOT without the need for HLA antigen matching.

[0133] Human leukocyte antigens (HLA) are associated with the rejection or acceptance of organ or cell transplants. HLA mismatch between donor and recipient often results in cell and organ rejection after stem cell therapy and organ transplantation. In the experiments described herein, it was found that there is no expression of HLA in mitochondria. Therefore, it is possible to transplant mitochondria from any healthy donor into a recipient without HLA matching. There is no problem of donor source. This is a significant advantage over stem cell therapy and organ transplantation, which require HLA matching between donor and recipient. Human fibroblast mitochondria enter mitochondrial DNA-deficient NSC-34 more easily than healthy NSC-34

[0134] The NSC-34 cell line provides a motor neuron model for in vitro studies. After co-culturing NSC-34 cells with isolated mitochondria labeled by MitoTracker Red for 12 hours, red fluorescence is found in NSC-34 cells (Figure 13A, 13B). This suggests that human fibroblast mitochondria enter into mouse motor neuron NSC-34. Furthermore, higher intensity fluorescence was found in NSC-34 cells treated with EtBr (Figure 13C, 13D). This indicates that fibroblast mitochondria transfer more easily into mitochondrial DNA-deficient NSC-34 cells. These results reveal that diseased tissues and organs incorporate more fibroblast mitochondria than healthy tissues.

[0135] The preferential renewal of mitochondria by defective cells compared to healthy cells can be quantified as follows: the fluorescence intensity of pre-labeled mitochondria can be measured in defective and healthy cells by fluorescence microscopy after co-culture of these cells with pre-labeled isolated mitochondria. The intensity can then be compared between defective and healthy cells. Clinical Trial I Early clinical results of mitochondrial organelle transplantation (MOT) in two human patients with amyotrophic lateral sclerosis (ALS)

[0136] We first performed mitochondrial organelle transplantation (MOT) in two ALS patients, one female and one male. The female patient is a 58-year-old Caucasian woman who had progressive ALS for over 3 years (at the start of treatment). She had failed all previous treatments. After several lengthy discussions about the procedure and proof-of-principle research evidence for possible efficacy, she signed informed consent for this experimental treatment. The male is a 48-year-old Caucasian man who had progressive ALS for 3 years (at the start of treatment). He was fully informed, wanted to try MOT, and signed a detailed consent form.

[0137] This series of initial clinical results for this female patient (Clinical Procedure A) describes six MOT procedures beginning in June 2019. These procedures were performed approximately 5-6 weeks apart. Prior to the procedures, she underwent strength testing of the leg muscles, quadriceps and thigh flexors. The first four implantations were performed only in the leg and upper arm muscles. Multiple injections of live isolated allogeneic fibroblast mitochondria in our buffered preservation solution were given into the muscle in multiple areas. In the fifth and sixth procedures, she received an intramuscular injection and was also given 5ml of isolated mitochondria in preservation solution intravenously.

[0138] The patient had no adverse events and no signs of rejection, autoimmunity or infection were present. Muscle strength (Figure 14) and mobility improved, and hope and mood were greatly improved. The greatest improvement was the improvement in her sensory sensation. At presentation, she had almost no sensation in her arms and legs and did not require local anesthesia for intramuscular injections. After the third transplant, sensation and sensory stimulation were restored and remained normal. It is believed that the restoration of sensation helped the motor neuron function. The great result of MOT is that the progression of the disease was halted and her sensation, strength and mobility improved. Her treatment is an ongoing study and MOT is complemented by nutritional supplements for sensory stimulation and mitochondrial health. Due to high serum ferritin, an indicator of iron overload in motor neurons, she is given iron chelators weekly. Her next MOT is planned for mid-March 2020 after a 3-month interval since her last MOT. Her results reveal that MOT can alleviate, stabilize and possibly reverse motor neuron degeneration in ALS. To our knowledge, this woman is the first person to undergo allogeneic fibroblast mitochondrial MOT for ALS.

[0139] The male patient is a 48 year old Caucasian male with 3+ years of progressive ALS (at the start of the procedure). He has severe generalized muscle weakness with bulbar ALS variant. He speaks very slowly and it is very difficult to understand him when he speaks. He had his first MOT procedure on December 11, 2019. He was given multiple injections of isolated fibroblast mitochondria in the leg, upper arm and posterior neck muscles. He was also given 2ml of isolated mitochondria in a preservative solution intravenously. He had no adverse events and within 2 hours of the procedure he was speaking faster, stronger and could be understood. After 5 days he was able to get out of bed without assistance and walk to the bathroom without assistance. He went deer hunting with his son but using a special chair. His mood, mental state and attitude have improved and he is more positive and eager to continue the MOT. He states that his energy has increased extremely rapidly. He was always unable to hold his head up straight and drooping, but now he no longer droops.

[0140] In the seven mitochondrial transplant procedures in the two patients above (clinical procedure A), the first four injections were intramuscular. The last three injections were a combination of intramuscular (2 / 3 mitochondria from approximately 100 million fibroblasts) and intravenous (1 / 3 mitochondria from approximately 100 million fibroblasts). The last three injections show better response (strength, sensation, energy).

[0141] In summary, the experience with MOT in these two ALS patients in clinical procedure A was beneficial in terms of treating their disease and / or halting their disease progression. Some of the key findings are: (1) allogeneic mitochondria can be safely transplanted into humans without HLA antigen matching; (2) allogeneic mitochondria do not cause autoimmunity; (3) ALS symptoms are improved; (4) MOT can extend life or remission and possibly cure ALS; and (5) MOT can be performed for the treatment of other neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, muscular dystrophies and other mitochondrial disorders. Clinical Trial II Long-term clinical outcomes of MOT in five human patients with ALS

[0142] A total of five patients have undergone the Mitochondrial Organelle Transplantation (MOT) procedure, for a total of 21 procedures to date. (Clinical Trial II described here encompasses Clinical Trial I, the first study involving two of the five patients.) Patient code number A5 underwent the procedure approximately one year ago and has had no adverse effects to date. All patients have reported no adverse effects, with some pain reported during the injection procedure.

[0143] In all cases, patients report an increased ability to perform multiple tasks through improved strength, control and movement. In addition, they all report higher energy levels and improved mood following the MOT procedure. These reports are verified through video, exams, and consultations during clinical visits and are further supported by the clinician's clinical notes.

[0144] In some cases, patients reported that overall disease progression had stopped, and this was observed and documented with observations from the clinical team. In three of the five patients, videotaped sessions with the clinical team on various measures of improved health and functionality indicated that disease progression had stopped. Patient code number A5

[0145] A 58-year-old woman from the South, previously in good health with no known pre-existing conditions, was given a definitive diagnosis of ALS in June 2019. She was diagnosed in 2016, 3 years prior to beginning treatment with mitochondrial organelle transplantation (MOT). No significant signs were present on routine physical exam. She presented with predominant symptoms in the lower body, to a lesser extent in the right arm, and even less in the left arm. She had little to no sensory or motor control below the waist, but had significant use of her arms and hands, and her voice was good.

[0146] June 3, 2019 - Patient admission inspection

[0147] Upon admission, a physical examination was completed and she appeared to be in good condition except for ALS. Baseline muscle testing was completed.

[0148] June 4, 2019 - First treatment

[0149] To our knowledge, this is the first patient to receive isolated allergenic mitochondria from fibroblasts.

[0150] Prior to the procedure, we filmed a video with her permission, completed a muscle test, and identified 18 areas for injection. Each location was marked and prepared, and the injection sites were anesthetized using 1% xylocaine. Each of the 18 sites was administered 3 / 10cc of mitochondrial solution. There was minimal bleeding from the puncture wounds, which were washed with Betadine and dressed with a sterile dressing. The patient was observed for 3 hours with no adverse events. There was no sign of allergy, and these mitochondria were allogeneic from a fibroblast human cell line.

[0151] During the observation period, she began to notice warmth and heat in her legs and over her right lower leg that she had not felt before. She stated that she began to have some sensation before leaving the office and felt it more strongly in her left leg, and she was able to lift, flex, and extend her left leg without difficulty. No toxicity or adverse events were observed. The patient was scheduled for follow-up on each of the next 2 days.

[0152] June 5, 2019 - Day 1 after treatment

[0153] The patient arrived in the morning for follow-up. Initial signs were favorable and she reported feeling better. The patient reported tingling and warmth in both legs. She was able to get out of bed on her own in the morning, something she had not been able to do before, and she was standing. Muscle strength was tested and improved as shown in Table 3a - Treatment 1. She had no adverse effects, toxicity, or fever. We have achieved objective proof that MOT injected into the quadriceps improved muscle strength in ALS patients.

[0154] June 6, 2019 - Day 2 after treatment

[0155] The patient arrived and reported no adverse effects, toxicity, or fever. We tested and recorded muscle strength, discussed self-monitoring guidelines for when she returned home, and scheduled her next procedure for June 27. After discharge, we called the patient several times before returning for the procedure, and no adverse effects were reported.

[0156] June 27, 2019 - 2nd treatment

[0157] Serum ferritin levels were recorded as 228 ng / mL pretreatment, and transferrin was 293 mg / dL (reference range 200-360). Upon arrival, we assessed her physical condition and again performed muscle strength testing. Results showed a slightly better pretreatment baseline than the initial baseline on June 4th.

[0158] When examined during follow-up on June 28, the scores were overall slightly better than they were before treatment on June 27. The areas of improvement were not as dramatic as the day after the first treatment, but there was an overall improvement as shown in Table 3a - Treatment 2.

[0159] There were no adverse effects, toxicity or fever experienced, and on the night of the procedure the patient sent a text message indicating he was able to get out of bed and walk much more easily.

[0160] As previously observed, she experienced some tingling and burning in the legs, but nothing dramatic.

[0161] We performed a CBC and iron workup on her. The CBC was good. Interestingly, in the iron workup, the iron binding capacity, saturation serum, and total IBC in unsaturated iron binding capacity (IBC) and calculated iron saturation were all within normal levels (WNL). However, her serum ferritin was high.

[0162] Subsequent procedures and patients are monitored for iron metabolism. Chelation therapy with deferoxamine or similar may be considered. The patient was informed of all test results and the next treatment procedure was scheduled for the first week of August.

[0163] The patient was followed up with regular follow-up appointments to track her progress and symptoms. A phone call on June 30th indicated that she and her family had noticed improvement since the second treatment.

[0164] July 7, 2019 - Special note in the file

[0165] A detailed review of the strength tests was completed by the team and we added these charts to her file. The good news is that even though there may be some variability at certain points, the overall trend is that strength increases in all areas injected during treatment.

[0166] August 8, 2019 - 3rd treatment

[0167] Prior to treatment, muscle strength was tested as shown in Table 3a - Treatment 3. The patient and her family noted that the patient was noticeably better than before treatment began in early June. The patient observed that she was now able to get out of bed more easily and turn in bed without assistance.

[0168] For this implantation, there were several areas selected in the quadriceps of both legs, some thigh flexors and buttocks, and both arms and forearms. There were no adverse events or toxicities experienced after the procedure. During the evening after the procedure, she was checked and was doing well. She was able to hold her bottle with her left hand, which was a further improvement. There were no calls or problems during the night.

[0169] During follow-up the day after treatment, muscle strength is tested and shows a slight decrease, but is still higher than the original baseline.

[0170] No adverse effects or problems were reported during the treatment visits, but there was some continued improvement in daily abilities.

[0171] October 2, 2019 - 4th treatment

[0172] Her most recent lab work CBC showed eosinophil WNL present. For this treatment she was given 30 injections in one area of ​​the quadriceps, thigh flexors, buttocks and forearm as well as the right biceps region. We utilized a hep-lock placed for adverse events for the first time to slowly administer 5cc through an IV port. No adverse effects were reported and she returned for follow-up very satisfied with the overall progress of the treatment and her improvement. Muscle strength was tested pre-treatment and the day after follow-up and showed slight improvement overall as shown in Table 3a - Treatment 4.

[0173] During the inter-procedure check-ups, she reported no adverse events or problems. By early November, she still reported good sensation in both feet, but not in her hands or arms. She maintained strength in her legs. Some percentage of eosinophils were elevated, but absolute counts were normal. She was scheduled to return on the 6th for her next procedure.

[0174] November 6, 2019 - 5th treatment

[0175] October 30, 2019 Serum ferritin level was recorded as 362ng / mL prior to treatment.

[0176] She came in for her 5th MOT today. She is doing well. Disease is fairly stable and some areas have improved significantly especially perception and sensation. During the first MOT treatment she did not have enough sensation to feel the intramuscular injection but as the treatment continued she confirmed that she felt the injection at an increasing level. Prior to the treatment, muscle testing was completed. For this treatment we started with 5cc of cold mitochondria in isolation medium administered slowly over 6 minutes. It was very cold and she developed an immediate headache and felt a little uncomfortable but no serious adverse reactions. We laid her down and her vital signs were good. We elevated her legs and symptoms subsided for approximately 30 minutes. For the intramuscular injection we injected into the quadriceps but not as much in either leg as last time and we injected into the femoral flexors of both legs and this time we injected downward towards the tibialis of both legs. We also injected into the biceps. She was doing well and was observed for 3.5 hours before being discharged without any adverse events or issues. During the follow-up visit on November 7th, we completed muscle testing for the comparisons shown in Table 3a - Treatment 5. Some areas had improved, some were stable, and some had slightly worsened. We scheduled the next treatment for December 11th, followed by a 3-4 month break in treatment while monitoring symptoms and signs. It now seems time to add another patient to our protocol.

[0177] Over the course of three days, November 8-10, we spoke several times and she reported feeling extremely well, although she did experience some lightheadedness and dizziness on a few occasions. She reported that she had more strength in her legs than ever before. As of November 11, she reported no dizziness and felt fine. In an additional call before her sixth treatment, she reported no side effects or adverse events, but continued to have strength, especially in her legs and arms.

[0178] December 11, 2019 - 6th treatment

[0179] Dec 4, 2019 Serum ferritin levels were recorded as 290ng / mL pre-procedure and transferrin was 271mg / dL.

[0180] She came in today for her 6th implant and biotherapy. She reported feeling much better and has much more sensation back than she has in the past month, especially in her legs. She reported increased strength and stamina seemed good. Injections were given in the quadriceps, thigh flexors and upper arms, slowly through an IV giving 5 cc. Patient experienced another episode of headache and dizziness, but as in November, they were transient. No adverse effects were noted during post-procedure observation and she was dismissed for the evening. During the evening call she stated she felt much better and had taken a nap.

[0181] When she arrived for follow-up on December 12th, the day after the procedure, the patient reported feeling good, energetic, and better than the past few weeks. We also discussed taking an iron chelator. We are investigating the role of iron metabolism in these neurons, which may be contributing to mitochondrial dysfunction. We suggested giving her 500 mg of deferoxamine, administered by injection from her local practitioner, once a week for 4-6 weeks, which she completed.

[0182] Interacting with the patient during the procedure

[0183] We spoke with the patient and possibly her family about her symptoms and progression. No adverse events or episodes of severe dizziness or headaches were reported. She was originally scheduled to return on March 11, 2020, but due to the COVID-19 pandemic, this was postponed until mid-April.

[0184] An extended telephone consultation was conducted on March 3. She reported that her disease progression has stabilized and she is pleased with her progress since last June, but there are still periods of inactivity. She reported that she is still doing physical therapy and receiving muscle stimulation.

[0185] A serum ferritin test was performed on April 10th and came back in the normal range. The patient was called to share the good news.

[0186] Repeat blood tests were ordered and received later in the week. Serum ferritin had fallen to the normal range of 181.8. She reported some delayed improvement, but overall, she was doing well.

[0187] April 15, 2020 - 7th treatment

[0188] She came in on April 15th for her 7th transplant, 4 months after her last procedure. She reported feeling a little more fatigue recently. We believe we have halted her rapid progression, but she has felt some increasing weakness and she thought she may be regressing slightly. She was anxious to receive the transplant. We started the IV, marked all areas selected for injection in the muscle, and placed approximately 8cc of mitochondrial isolation solution in a syringe to give slowly through the IV over a period of time. We injected 3-4 / 10cc of mitochondrial isolation solution into each injection site, 2 injections on each side of the right and left posterior of the area. We administered 3 injections into the forearms on both sides and 5 injections into the quadriceps on both sides, i.e., 1 centrally and 1 anteriorly, 2 laterally and 2 medially on both sides. The treatment was well tolerated.

[0189] At follow-up the next morning, she reported feeling more energetic and stated that her left side felt much better and stronger. Things were moving in the right direction for her and we believe that this treatment has halted the rapid progression of ALS in this patient.

[0190] She sent an email saying she felt great after returning home and felt like her last transplant on April 15th was one of her better transplants. She reported that everything is going well for her and that she has increased energy. We have observed significant improvement and believe that the progression of her disease has stopped. She reported a desire to continue this protocol and we suggested that at least two further treatments at 6 week intervals followed by an additional 3-4 months of rest may be an excellent protocol for this patient.

[0191] June 17, 2020 - 8th treatment

[0192] On June 17, 2020, the patient arrived for her 8th implantation procedure, one year after the first procedure, which was performed on June 4, 2019. During this year, we accomplished a lot to help this patient, including halting the progression. For this procedure, we started an IV, planned out the areas we planned to inject, and created a diagram in her chart showing the areas of injection. We injected two in the upper neck on both sides slightly posteriorly. We gave several additional injections in the upper arm, lower arm and hand and thumb area as well as the quadriceps and thigh flexor area and calf. She tolerated the procedure well when we gave the IV infusion, even though the IV infusion was cold. We gave the IV infusion very slowly and she did not develop any of the headaches or dizziness as before. This was the first IV given without headaches and dizziness. During post-procedure observation, she began to notice warmth and tingling in her legs (a buzzing sensation as she called it). This observation was considered a good sign in this patient.

[0193] We took blood for a new ferritin reading before removing the IV. There were no reported adverse effects or events. During follow-up the next day, she reported feeling stronger and appeared to be in good shape. We had a lengthy discussion with her and her husband about her progress over the past year. They agreed to video document our discussion to be used in conjunction with her study results.

[0194] The patient's progress will be monitored by telephone until she returns for her next treatment in approximately 6 weeks.

[0195] Muscle strength testing was performed pre- and post-injection for the first five treatments using an ActivForce Digital Dynamometer System, as recorded in Table 3a below. [Table 3-1] [Table 3-2]

[0196] Table 3b shows complete blood counts (CBCs) with differentials (complete blood cell panel) performed on the same or approximately the same day as ferritin, also from June 2019 to April 2020, for patient A5. [Table 3-3] Patient code number A4

[0197] A 48-year-old man from the Southeast, previously in excellent health, served with honor in the United States Army, worked for a large transportation delivery company, and coached recreational league baseball locally. He presented in mid-December 2019 for treatment with a confirmed diagnosis of ALS. He had been symptomatic since 2016 and was diagnosed in 2018. He has been prescribed riluzole and edaravone for ALS, but has not seen any noticeable benefit. He also takes alprazolam, naproxen, fluoxetine HCL, tylenol, and melatonin. Testing on December 9th showed his serum ferritin level was 319 ng / mL and transferrin was 264 mg / dL.

[0198] A full evaluation was conducted on December 17, 2019. He has significant physical disabilities, has great difficulty speaking, virtually no use of either his arms or hands, cannot hold his head up, and has significant weakness in his legs and lower body.

[0199] For the procedure, the patient was started on an IV in his left hand. Finding a suitable vein was difficult, but we were successful in securing one on the left side and suggested that we consider placing a Med port on him for his safety in case of an emergency and to make the procedure easier. We injected into the deltoid and triceps areas of both arms, the forearms, the posterior neck area on both sides, and the quadriceps and calves. He received approximately 30 total intramuscular injections and 2 cc of MOT via hep-lock IV. The patient had no adverse events or effects. He reported feeling much better at follow-up and believed he had more strength. Post-procedure follow-up, primarily by phone with the caregiver, indicated this patient was doing better. He had a great increase in energy, was walking better, could move his arms better, and could turn the shower knob to turn the water on. His Med port was placed on January 10th. Follow-up calls indicated he was doing well. Five weeks after the first MOT treatment, the patient arrived for the second treatment. In observing the patient, we noted how much better he appeared to be and that the first treatment had lasted.

[0200] The patient reported improved mobility, was able to get out of bed on his own, went to the bathroom on his own, had increased energy, and his head and neck were no longer tilted forward as they were before the first procedure. He still had significant problems with speech, but his speech had improved, and although still very slow and slurred, he could be understood better and his speech was slightly more forceful. For this procedure, injections were given in the quadriceps on both sides, the inner calf on both sides, the biceps on both sides of the forearm, and the posterior neck muscles on both sides. He had a Med port, which allowed for easier access, and he received a slow IV push of 2cc of MOT for 5-6 minutes. He tolerated the procedure well and observations showed no adverse events or side effects. He reported the next morning for follow-up and he was not feeling well. Upon examination, we determined that he had eaten two large meals with lots of carbohydrates and drank a large amount of beer. He was hydrated with Gatorade and we instructed him to rest, not eat a lot, and to return tomorrow. He reported feeling much better the second morning after the MOT than the previous day. He reported feeling extremely positive about his improvement. In fact, last night he felt so good that the patient made a short video of himself dancing, something he had not done in a long time. The patient was asked to stay hydrated at all times.

[0201] During the treatment call, the patient reported no adverse events or side effects, and continued to see various levels of improvement in his condition. He reported attending a local meet last weekend and was able to do more than he had before. He reported feeling excited about his next transplant, and lithium orotate has been added to his protocol.

[0202] On March 18th the patient returned for his 4th MOT implant and we had some difficulty accessing his med port. We managed to get a good result and stabilized it. We gave him approximately 5cc of isolated liquid MOT through the IV port. For intramuscular injections, we injected into the right and left side of his neck, both deltoid and triceps areas and muscles, biceps and forearm muscles, we injected more on the left side which is his weakest area, on both sides in several areas of the quadriceps, and into both calves. He tolerated the procedure well. Follow-ups, as well as between-procedure phone consultations, did not result in any adverse events.

[0203] On April 22nd, the patient reported for his 5th MOT implantation. They had extreme difficulty traveling due to the COVID-19 pandemic. The patient had stayed at the airport the night before the procedure and was tired but feeling fine on the day of the procedure. All the usual procedure preparation was done. As part of the procedure, diagrams will be included on the chart to show the injection sites for this procedure including the neck, upper arm, forearm, calf and thigh which were the primary injection locations. We administered approximately 8cc of MOT through the IV Med port over a period of 8-10 minutes. No adverse effects were evident in any of the given locations. The patient received an intramuscular injection and was then observed for approximately 1.5 hours while receiving IV fluids and water for hydration. We discharged him without incident and no adverse events or effects were observed. However, as with the previous procedure, during the IV delivery of the procedure, this patient had pain that appeared in the lower back approximately halfway through the IV treatment delivery. There is no specific explanation for this pain in this area occurring simultaneously during each procedure. One possible explanation is that the mitochondria are immediately directed to this area of ​​his body due to a problem he has in his spine. I checked on him during the evening and he felt fine. Due to a 6am flight leaving, he did not come to the office for his usual next day follow up. I instructed him to call if he had any problems.

[0204] During multiple telephone consultations between procedures, the patient reported no adverse events, side effects, or fevers, but continues to experience improvement.

[0205] He reported for his 6th MOT treatment on May 27th. Since starting treatment in mid-December, he has made a lot of progress and has improved in many ways. He still has difficulty speaking at times and is difficult to understand reliably, but his speech is much improved than it was 6 months ago. The patient reported that his VA physical therapy exam, done at home prior to any MOT treatment, had a score of 12. He was retested approximately 2 weeks ago and had a score of 28. This is a significant improvement in approximately 6 months.

[0206] For this procedure, he received 34 intramuscular injections of MOT, 3-4 / 10cc each and 8cc via slow IV push over approximately 10 minutes. As with the previous procedure via IV (approximately 4cc intravenously), he again experienced intense pain in the lumbar spinal region, but this time the pain seemed more intense. We stopped the injections for a while, moved to intramuscular, and then terminated the IV administration at the end of the procedure. No adverse events were experienced or reported during observation. He was doing well when checked in the late afternoon and at night. He reported the absence of adverse reactions the following morning. He felt well, strong, and now had a comfortable neck position. He was instructed to stay hydrated and to report any problems immediately. Telephone consultations with this patient continue to be constructive. He has maintained the energy and his mobility he gained through MOT. Although he still has significant problems, his overall quality of life and control over his daily activities have changed dramatically in his favor since he first reported to us last December. We will maintain follow-up by telephone and will hear from him shortly as to our thoughts on when he should be scheduled for his next MOT. Patient code number A3

[0207] A 68-year-old man from the South, previously in fairly good health, presented with a confirmed diagnosis of ALS made in December 2019. Prior to retirement, he was a police officer for over 20 years. He has type 2 diabetes, but not uncontrolled, and is treated for high blood pressure, also not uncontrolled. He experienced symptoms for 2 years prior to diagnosis, primarily in his left arm and hand. He dismissed the onset of symptoms as possibly developing arthritis. As the pre-diagnosis period well into its second year, he began to have other symptoms, primarily muscle spasms or fasciculations. He has been examined and seen by several physicians in Huntsville, Alabama, and Atlanta, Georgia, who confirmed the diagnosis and confirmed that no meaningful treatment exists to improve his symptoms. He has been prescribed riluzole, but, like other patients, has seen little to no substantial benefit. He also takes amlodipine, alprazolam, hydrochlorothiazide, metformin, and Toprol XL for other existing conditions. His transferrin was recorded as 241 ng / mL.

[0208] His greatest difficulty is use of his left arm and hand, and to a lesser extent his right arm and leg, so the inventors identified injection sites and had them all ready to go to address his current symptoms. He was given 2.5cc via slow IV push and 3 / 10cc intramuscular injections in the shoulder, arm, hand and leg. Sites were focused on the left arm and hand, with more injections in the right arm with an emphasis on the triceps and biceps. The inventors injected in mirror image positions in the quadriceps and calves of both legs. He was monitored and hydrated for over 2 hours after the procedure with no adverse events or reactions.

[0209] A follow-up appointment the next day revealed no side effects or adverse events. In a telephone consultation the following week, he stated he was still feeling well and had much improved gripping ability with his left hand. Subsequent follow-up calls showed slow, continuing improvement.

[0210] He was scheduled for his second MOT on April 1st but was forced to delay due to the current COVID-19 pandemic. He returned on May 20th, over 3 1 / 2 months after his first MOT. He had lost some of the early improvements but was still better than before his first treatment. We completed the standard preparation and started a hep-lock IV in preparation for the treatment. All of the previously treated areas were used, as well as additional positions on the left hand including the leg, arm and between the thumb and index finger. A total of 30 injections were given, 8cc given via IV over 9 minutes. The treatment was well tolerated with no adverse events or side effects. When he returned the next day he was very much improved and was happy to report that after yesterday and this morning's treatment he was able to open his car door with his left hand, something he had not been able to do for over a year.

[0211] His third MOT treatment was on June 24th. The patient reported feeling that the gains from his last MOT were maintained, feeling stronger, and experiencing infrequent fasciculations. He reported having very good use of his left arm and hand. Patient observations indicated he was breathing well, walking well, and sleeping better than previously reported. The second treatment injection and slow IV push was repeated, which was tolerated without adverse events or reactions. His follow-up appointment confirmed that he is feeling even better overall, and that the strength of his arms, hands, and legs continues to improve. He reports being pleased with his progress and wishes to continue this treatment protocol. He plans to return in 4-6 weeks. Patient code number A2

[0212] A 73-year-old man from the South, previously in good health, presented on April 8, 2020 with a confirmed diagnosis of ALS. He has been prescribed Riluzole, a drug currently available for its potential to slow symptom progression in ALS patients. He has not experienced a significant delay in symptom progression with the use of this drug. Prior to the procedure, his symptomology was reviewed and he completed the ALS Functional Assessment Scale. He scored 37 on this scale, with 6 of 12 criteria affected. His greatest impairment is his right arm and hand, which have the greatest degree of weakness and are unable to use them. He has problems with his other limbs, but to a lesser extent. The first procedure focused on the right arm and hand as well as the quadriceps and thighs of both legs. Standard site preparation and identification was completed and a hep lock IV was started. Approximately 20 intramuscular injections of 3 / 10cc and a slow push delivery of 5cc in the IV were administered. The procedure was well tolerated and no adverse effects were experienced during the observation period in the office or after discharge. As he left the office after the first treatment, approximately 2 hours after we began, his wife observed that he was walking more easily and steadily. A follow-up visit and between treatments with this patient showed positive results, with some improvement in both the strength and functionality of his legs and his right arm and hand. Serum ferritin was reported at 190 ng / mL, and transferrin was 259 mg / dL.

[0213] The patient underwent additional treatments on May 6th and June 10th, both consisting of 8cc slow push IV and 30ml intramuscular injections. Injection areas included each side of the posterior neck, both upper arms, hands and thumbs, thigh area of ​​both legs, quadriceps, thigh flexors and calves. There were no adverse events or side effects. Treatment was well tolerated and continued incremental improvement in strength and functionality was noted by the patient. The patient expressed a desire to continue the treatment protocol and we will work to schedule him for every 4-6 weeks. Patient code number A1

[0214] A 30-year-old male from the West Coast presented for treatment in May 2020 with a diagnosis of ALS. He experienced symptoms in March 2019 and was diagnosed in August 2019 after testing including an MRI, blood work and electromyography (EMG). He had been prescribed riluzole but experienced no reduction in symptom progression. He had also undergone stem cell treatment with little to no positive results. His serum ferritin level was recorded as 381ng / mL prior to treatment and transferrin was recorded as 239mg / dL. On routine physical exam, no notable signs were present. He had a score of 44 on the ALS Functional Assessment Scale with predominant symptoms being the left hand and arm, to a lesser extent the right hand and arm, and to a lesser extent both legs. He was able to walk well. Muscle strength testing was performed using the ActivForce Digital Dynamometer System before and after treatment with mitochondrial organelle transplantation (MOT), as recorded in Table 4 below. [Table 4-1] [Table 4-2]

[0215] At this patient's first procedure, an IV was started and 8 cc of mitochondrial organelle transfer (MOT) was administered by IV over 9 minutes. The areas identified for intramuscular injection of MT were marked and prepared (including administration of xylocaine to numb the injection site). In this patient's case, injection sites included the back of the neck, shoulder, upper arm around the wrist, hand and forearm, thigh and calf. Approximately 29 injections of 3 / 10 cc were administered into these muscle areas. No issues were observed. He was given an additional 250 cc of fluid push IV. The hep-lock intravenous (IV) catheter was left in place for approximately 1.5 hours during post-procedure observation. During observation, he was encouraged to drink water, no adverse events were observed, and the hep-lock was removed and wrapped in a pressure wrap for later removal that evening. At his scheduled 24-hour follow-up examination, muscle strength was retested and showed signs of overall improvement. His walking improved and he reported that he was able to extend or "thumbs up" his thumb during the day after follow-up, whereas he had not been able to do so the previous two days, and had regained the ability to "thumbs up" with his left thumb. His grip with his left hand was also significantly improved from the previous two days.

[0216] Post-treatment telephone follow-up with this patient was good and improvement from the first treatment continued to be stable. His second MOT is scheduled for late July. SEQ ID NO: (nucleotide sequence) [ka] [ka] [ka] [ka] Sequence (amino acid sequence) [ka] Other Aspects

[0217] While we have described several embodiments, it is clear that our basic disclosure and examples may provide other embodiments that utilize or are encompassed by the compositions and methods described herein, and therefore, it will be understood that the scope should be defined not by the specific embodiments presented as examples, but by what can be understood from this disclosure and the appended claims.

[0218] All references cited herein are hereby incorporated by reference.

Claims

1. A composition for use in a method for improving muscle function in a subject, the composition comprising mitochondria isolated from cells of a donor other than the subject, cultured without antibiotics, the method comprising administering to the subject the composition comprising the mitochondria in an amount sufficient to improve muscle function.

2. A composition for use in a method for improving muscle function in a subject that has been impaired by administration of an agent that causes or may cause mitochondrial damage, said composition comprising mitochondria isolated from cells of a donor other than the subject, cultured without antibiotics, said method comprising administering to the subject the composition comprising the mitochondria in an amount sufficient to improve muscle function in the subject.

3. A composition for use in a method for treating a subject whose muscle function has been reduced by one or more members of the group consisting of: (i) a lung-related disorder such as viral pneumonia, (ii) a coronavirus infection, e.g., an infection with a severe acute respiratory syndrome coronavirus including SARS-Cov-1 or SARS-Cov-2 or related strains, (iii) influenza, (iv) an associated genetic disorder, (v) chronic obstructive pulmonary disease (COPD), and (vi) another disease or condition in which mitochondrial damage occurs and / or is present, said composition comprising mitochondria isolated from cells of a donor other than the subject, cultured without antibiotics, said method comprising administering to the subject the composition comprising the mitochondria in an amount sufficient to improve muscle function in the subject.

4. A composition for use in a method for improving muscle function in a subject impaired by one or more members of the group consisting of (i) a lung-related disorder such as viral pneumonia, (ii) a coronavirus infection, e.g., infection with a severe acute respiratory syndrome coronavirus including SARS-Cov-1 or SARS-Cov-2 and related strains, influenza, a related genetic disorder, chronic obstructive pulmonary disease (COPD), or another disease or condition in which mitochondrial damage occurs and / or is present, said composition comprising mitochondria isolated from cells of a donor other than the subject, cultured without antibiotics, said method comprising administering to the subject the composition comprising the mitochondria in an amount sufficient to improve muscle function in the subject.

5. A composition for use in a method for treating a subject having high iron levels, the composition comprising mitochondria isolated from cells of a donor other than the subject, cultured without antibiotics, the method comprising administering to the subject the composition comprising the mitochondria in an amount sufficient to improve muscle function in the subject.

6. A composition for use in a method for treating a subject being treated with a pharmaceutical agent such as hydroxychloroquine and / or chloroquine for an indication involving high reactive oxygen species (ROS), the composition comprising mitochondria isolated from cells of a donor other than the subject and cultured without antibiotics, the method comprising administering the composition comprising the mitochondria to the subject.

7. A mitochondrial isolation buffer composition for use in mitochondrial organelle transplantation, comprising: A buffering agent; A chelating agent; Sugar and an agent that acts as a membrane stabilizer and / or an oxygen radical scavenger and / or a binder of Ca2+ and / or a binder of free fatty acids; Serine protease inhibitors and 1. A mitochondrial isolation buffer composition comprising:

8. The mitochondrial isolation buffer composition of claim 7, wherein the buffer is a zwitterionic sulfonic acid buffer.

9. The mitochondrial isolation buffer composition of claim 7, wherein the chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof.

10. The mitochondrial isolation buffer composition of claim 7, wherein the sugar is sucrose.

11. A mitochondrial isolation buffer composition described in any one of claims 7 to 10, wherein the agent acting as a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca2+ binder and / or a free fatty acid binder is bovine serum albumin (BSA).

12. The mitochondrial isolation buffer composition described in any one of claims 7 to 11, wherein the serine protease inhibitor is phenylmethylsulfonyl fluoride (PMSF).

13. The mitochondrial isolation buffer composition of any one of claims 7 to 12, wherein the composition further comprises isolated donor mitochondria.

14. The mitochondrial isolation buffer composition of any one of claims 7 to 13, wherein the composition does not contain an antibiotic.

15. A mitochondrial storage buffer composition for use in mitochondrial organelle transplantation, comprising: one or more buffering agents; a source of magnesium ions; A chelating agent; Sugar and Antioxidants, A cytoprotectant that binds to calcium ions; an agent that acts as a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ binder and / or a free fatty acid binder; 1. A mitochondrial storage buffer composition comprising:

16. The mitochondrial storage buffer composition of claim 15, wherein the one or more buffering agents include a zwitterionic sulfonic acid buffer and / or monopotassium phosphate (KH2PO4).

17. The mitochondrial storage buffer composition of claim 15 or claim 16, wherein the source of magnesium ions is magnesium chloride (MgCl 2 ).

18. The mitochondrial storage buffer composition of claim 15, wherein the chelating agent is ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or a salt thereof.

19. A mitochondrial storage buffer composition described in any one of claims 15 to 18, wherein the sugar is sucrose.

20. A mitochondrial storage buffer composition described in any one of claims 15 to 19, wherein the antioxidant is taurine.

21. A mitochondrial storage buffer composition described in any one of claims 15 to 20, wherein the cell protective agent that binds to the calcium ions is lactobionate or a salt thereof.

22. A mitochondrial storage buffer composition described in any one of claims 15 to 21, wherein the agent acting as a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca2+ binder and / or a free fatty acid binder is bovine serum albumin (BSA).

23. A mitochondrial storage buffer composition described in any one of claims 15 to 22, wherein the composition further comprises isolated donor mitochondria.

24. A mitochondrial storage buffer composition according to any one of claims 15 to 23, wherein the composition does not contain an antibiotic.

25. A kit comprising a donor mitochondrial composition in a unit dosage effective to treat a neurodegenerative disease or other condition associated with mitochondrial dysfunction in a subject, comprising: The donor mitochondrial composition comprises: Mitochondria isolated from donor tissue; one or more buffering agents; a source of magnesium ions; A chelating agent; Sugar and Antioxidants, A cytoprotectant that binds to calcium ions; an agent that acts as a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ binder and / or a free fatty acid binder; Including the kit.

26. The kit described in claim 25, wherein the donor and the subject are not HLA (human leukocyte antigen) matched.

27. ​​The kit described in claim 25 or 26, wherein the donor mitochondrial composition does not contain an antibiotic.

28. A kit described in any one of claims 25 to 27, further comprising instructions for optimizing the dosage and / or frequency and / or route of administration of the composition.

29. The composition of any one of claims 2 to 6, wherein the composition comprises mitochondria isolated from mesenchymal stromal cells (MSCs) of the donor.

30. The composition described in any one of claims 2 to 6, wherein the composition comprises mitochondria isolated from human primary fibroblasts of the donor.

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