Compositions and methods for the treatment of traumatic brain injury (TBI), e.g., mild traumatic brain injury (mTBI)

Abstract

Presented herein are compositions and methods involving mitochondrial organelle transplantation for use in treating traumatic brain injury (TBI), e.g., mild traumatic brain injury (mTBI), in a subject. Compositions and methods for treating traumatic brain injury (tbi), e.g., mild traumatic brain injury (mtbi). The present invention provides a method for allogeneic transplantation of mitochondria in a subject, e.g., for the treatment of traumatic brain injury (TBI), the method comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject, wherein the subject has mTBI.

Description

[Technical Field]

[0001] Cross-reference to pending applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 405,336, filed September 9, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Field The subject matter described herein relates to compositions and methods for treating patients. In certain embodiments, described herein are compositions and methods involving mitochondrial organelle transplantation for use in treating traumatic brain injury (TBI), such as mild traumatic brain injury (mTBI), in a subject. [Background technology]

[0003] Over the 10-year period from October 2001 to 2011, 1.6 million military personnel deployed to Iraq and Afghanistan to fight the war on terror. The number of combatants suffering from concussions is estimated to be between 5 and 35%. Concussive injuries, also known as mild traumatic brain injury (mTBI), have been associated with cognitive impairment in 14% of study participants, even one year later. Another recent report by Cole et al. showed that over 50% of soldiers in their study reported "abnormally high" symptom levels upon "return to duty," and over 50% reported at least one significant symptom after "return to duty."

[0004] Therefore, new approaches are needed to alleviate post-concussion symptoms in combatants who may be in austere environments. Summary of the Invention [Means for solving the problem]

[0005] Described herein are compositions and methods involving mitochondrial organelle transplantation for use in treating traumatic brain injury (TBI), such as mild traumatic brain injury (mTBI), in a subject.

[0006] Injuries resulting in traumatic brain injury (TBI) are a part of everyday life. While some individuals may experience a TBI through a fall or car accident, others work in occupations that constantly expose them to TBI. For example, military and sports occupations (e.g., soccer) are constantly exposed to scenarios that can result in TBI. As described herein, TBI (e.g., mTBI) is often the result of mechanical injury to an individual's head. TBI also includes minor TBI (mTBI) (e.g., concussion). After an individual experiences a TBI, they may experience many symptoms associated with TBI depending on its severity. Symptoms such as nausea, blurred vision, and persistent headaches are common in those who suffer from TBI. While not wishing to be bound by any particular theory, the applicant believes that many symptoms associated with TBI are the result of mitochondrial damage and dysfunction. Mitochondrial damage and dysfunction can lead to secondary damage to the brain, thus causing many of the symptoms associated with TBI.

[0007] Applicant believes that many symptoms associated with TBI can be treated by administering mitochondria to a subject.As disclosed herein, Applicant has developed techniques, methods and compositions for administering mitochondria to a subject.Based on Applicant's experience in administering mitochondria to a subject suffering from neurodegenerative disease, administering healthy allogeneic mitochondria replenishes the mitochondria lost in TBI and repairs damaged mitochondria.Furthermore, in certain embodiments, repairing and replacing the mitochondria of a subject can help alleviate the symptoms associated with TBI.

[0008] Described herein are methods for treating TBI in a subject, including mild traumatic brain injury (mTBI). In certain embodiments, described herein are compositions and methods involving mitochondrial organelle transplantation for use in treating TBI, such as mild traumatic brain injury (mTBI), in a subject.

[0009] In one aspect, the present invention is directed to a method for allogeneic transplantation of mitochondria in a subject (e.g., a human subject) for the treatment of traumatic brain injury (TBI) (e.g., mild traumatic brain injury (mTBI)), the method comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising mitochondria isolated from a donor other than the subject, wherein the subject has mTBI (e.g., has been diagnosed with mTBI).

[0010] In certain embodiments, the composition further comprises a mitochondrial storage buffer having a potassium ion concentration that is safe for human administration (e.g., the mitochondrial storage buffer comprises a pharmaceutically acceptable carrier).

[0011] In certain embodiments, the administering step comprises parenterally administering (e.g., by subcutaneous, intramuscular, or intravenous injection) at least one unit dose of the composition to the subject. In certain embodiments, the administering step comprises both intramuscular and intravenous injection of the composition to the subject.

[0012] 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 donor tissue via tissue dissociation (e.g., using a bead tube shaking homogenizer). In certain embodiments, isolating the donor mitochondria comprises using a mitochondrial isolation buffer containing a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMFS)) (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 using antibiotics.

[0013] 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 an indeterminate match (e.g., HLA markers not tested prior to the administering step)).

[0014] In certain embodiments, the composition administered to the subject does not include an antibiotic (eg, if the subject is not receiving an antibiotic).

[0015] In certain embodiments, the composition comprises mitochondria isolated from donor human primary fibroblasts.

[0016] In certain embodiments, the method further comprises isolating mitochondria (e.g., fibroblast mitochondria) from the donor tissue. In certain embodiments, the isolating step is performed using a mitochondrial isolation buffer composition (e.g., a mitochondrial isolation buffer described herein). In certain embodiments, the mitochondrial isolation buffer composition comprises a buffering agent, a chelating agent, a sugar, a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ and a serine protease inhibitor. In certain embodiments, the composition does not include an antibiotic.

[0017] 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 administering to the subject an iron chelator (e.g., desferrioxamine or deferasirox).

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

[0020] In another aspect, the invention provides a mitochondrial isolation buffer composition (e.g., an aqueous solution) for use in performing any of the methods described herein (e.g., for use in mitochondrial organelle transplantation), the composition 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 a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMFS), also known as phenylmethanesulfonyl fluoride).

[0021] In certain embodiments, the composition does not include an antibiotic.

[0022] In another aspect, the invention provides a mitochondrial storage buffer composition (e.g., an aqueous solution) for use in performing any of the methods described herein (e.g., for use in mitochondrial organelle transplantation), the composition 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., HEPES potassium 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 cytoprotective agent that binds calcium ions [e.g., lactobionate or a salt thereof, e.g., K-lactobionate]; a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ The present invention is directed to a mitochondrial storage buffer composition comprising an agent that acts as a binder of free fatty acids and / or a binder of free fatty acids (e.g., bovine serum albumin, BSA) (e.g., the composition further comprises isolated donor mitochondria, e.g., fibroblast mitochondria).

[0023] In certain embodiments, the composition does not include an antibiotic.

[0024] In another aspect, the present invention provides a kit comprising a unit dose of a donor mitochondrial composition (e.g., an aqueous composition) effective to treat traumatic brain injury (TBI) (e.g., mild traumatic brain injury (mTBI)) in a subject, wherein the donor mitochondrial composition comprises 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., HEPES potassium salt ( a magnesium ion source [e.g., magnesium chloride (MgCl)]; 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 cytoprotective agent that binds calcium ions [e.g., lactobionate or a salt thereof, e.g., K-lactobionate]; a membrane stabilizer and / or an oxygen radical scavenger and / or Ca 2+ The present invention relates to a kit comprising an agent that acts as a binder for the free fatty acids and / or a binder for the free fatty acids (e.g., bovine serum albumin, BSA).

[0025] 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 an indeterminate match (e.g., HLA markers not tested prior to administration)).

[0026] In certain embodiments, the donor mitochondrial composition does not include antibiotics.

[0027] In certain embodiments, the kit further comprises instructions for optimizing the dosage and / or frequency and / or route of administration of the composition.

[0028] The systems, devices, methods, and processes of the claimed invention are intended to encompass variations and adaptations developed using information from the embodiments described herein. Adaptations and / or modifications of the systems, devices, methods, and processes described herein can be implemented as contemplated by the present specification.

[0029] In another aspect, the invention relates to a method of treating traumatic brain injury (TBI), comprising administering to a subject a composition comprising allogeneic mitochondria.

[0030] In certain embodiments, the subject has experienced a TBI (eg, a fall, explosion, car accident, shock wave, or other mechanical force).

[0031] In certain embodiments, the subject is suffering from TBI symptoms (e.g., headache (e.g., that gradually worsens and / or does not go away), recurrent vomiting, nausea, convulsions (e.g., seizures), inability to awaken from sleep, dilated pupils in one or both eyes, slurred speech, weakness and / or numbness in the limbs, loss of coordination, increased confusion, restlessness, and agitation).

[0032] In certain embodiments, the subject has experienced a mild traumatic brain injury (mTBI) (eg, a concussion).

[0033] In certain embodiments, the subject is suffering from mTBI symptoms (e.g., confusion, dizziness, blurred vision, eye strain, tinnitus, unpleasant taste in the mouth, fatigue, lethargy, changes in sleep patterns, behavioral and / or mood changes, and difficulty with memory, concentration, attention, and / or thinking).

[0034] In certain embodiments, the subject is administered the composition at the time of injury that may result in a TBI (eg, mTBI).

[0035] In certain embodiments, the subject is administered the composition a period of time (e.g., 1 hour, 1 day, 1 week, 1 month, 1 year, 5 years) after a TBI (e.g., mTBI) or suspected TBI.

[0036] In certain embodiments, the TBI (eg, mTBI) results in mitochondrial loss in the subject.

[0037] In certain embodiments, TBI (eg, mTBI) results in mitochondrial damage dysfunction.

[0038] In certain embodiments, the method further comprises isolating the mitochondria.

[0039] In certain embodiments, the methods involve isolating mitochondria from a donor other than the subject (e.g., a healthy donor).

[0040] In certain embodiments, the methods include isolating mitochondria from a subject.

[0041] In certain embodiments, the method further comprises creating a cell bank from a donor other than the subject (e.g., a healthy donor).

[0042] In certain embodiments, the cell bank comprises fibroblasts (eg, primary fibroblasts).

[0043] In certain embodiments, the cell bank comprises mesenchymal stromal cells (MSCs).

[0044] In certain embodiments, the method includes obtaining a tissue biopsy (eg, a skin tissue biopsy) from the donor.

[0045] In certain embodiments, the method includes creating a tissue bank (eg, from a biopsy, eg, a skin tissue biopsy) (eg, a frozen tissue bank) from a donor.

[0046] In certain embodiments, the composition further comprises a mitochondrial storage buffer having a potassium ion concentration that is safe for human administration.

[0047] In certain embodiments, the administering step comprises parenterally administering at least one unit dose of said composition to the subject.

[0048] In certain embodiments, the administering step comprises both intramuscular and intravenous injection of the composition into the subject.

[0049] In certain embodiments, isolating mitochondria comprises preparing a cell lysate from the tissue via tissue dissociation.

[0050] In certain embodiments, isolating mitochondria comprises using a mitochondrial isolation buffer that includes a serine protease inhibitor.

[0051] In certain embodiments, the method further comprises isolating the mitochondria without the use of antibiotics.

[0052] In certain embodiments, the subject and donor from whom the mitochondria are obtained are not HLA (human leukocyte antigen) matched.

[0053] In certain embodiments, the composition administered to the subject does not include an antibiotic.

[0054] In certain embodiments, the isolating step is carried out using a mitochondrial isolation buffer composition.

[0055] In certain embodiments, the mitochondrial isolation buffer composition comprises a buffering agent, a chelating agent, a sugar, a membrane stabilizer and / or an oxygen radical scavenger and / or Ca 2+ and a serine protease inhibitor.

[0056] In certain embodiments, the mitochondrial isolation buffer composition does not include antibiotics.

[0057] In certain embodiments, the method further comprises storing the isolated mitochondria at a temperature below -40°C.

[0058] In certain embodiments, the method further comprises administering to the subject an iron chelator.

[0059] In certain embodiments, the method further comprises administering an antioxidant and / or a probiotic to the subject.

[0060] In certain embodiments, the method further comprises a mitochondrial isolation buffer composition for use, said composition comprising a buffering agent, a chelating agent, a sugar, a membrane stabilizer and / or an oxygen radical scavenger and / or Ca 2+ and a serine protease inhibitor.

[0061] In certain embodiments, the mitochondrial isolation buffer composition does not include antibiotics.

[0062] In certain embodiments, the mitochondrial storage buffer composition comprises one or more buffering agents, a magnesium ion source, a chelating agent, a sugar, an antioxidant, a cytoprotective agent that binds calcium ions, a membrane stabilizer and / or an oxygen radical scavenger and / or a Ca 2+ and an agent that acts as a binder for the free fatty acids and / or a binder for the free fatty acids.

[0063] In certain embodiments, the mitochondrial storage buffer composition does not include antibiotics.

[0064] In another aspect, the present invention is directed to the use of allogeneic mitochondria (e.g., in the methods described herein) (e.g., using the compositions described herein) for the treatment of traumatic brain injury (TBI) (e.g., mTBI).

[0065] In another aspect, the present invention is directed to the use of allogeneic mitochondria (e.g., in methods described herein) (e.g., using compositions described herein) for the treatment of traumatic brain injury (TBI) (e.g., mTBI), wherein the allogeneic mitochondria are administered to a subject suffering from traumatic brain injury (TBI) (e.g., mTBI)-associated mitochondrial damage.

[0066] In certain embodiments, the subject has experienced a TBI (eg, mTBI) (eg, a fall, explosion, car accident, shock wave, or other mechanical force).

[0067] In certain embodiments, the subject is suffering from TBI symptoms (e.g., headache (e.g., that gradually worsens and / or does not go away), recurrent vomiting, nausea, convulsions (e.g., seizures), inability to awaken from sleep, dilated pupils in one or both eyes, slurred speech, weakness and / or numbness in the limbs, loss of coordination, increased confusion, restlessness, and agitation).

[0068] In certain embodiments, the subject has experienced a mild traumatic brain injury (mTBI) (eg, a concussion).

[0069] In certain embodiments, the subject is suffering from mTBI symptoms (e.g., confusion, dizziness, blurred vision, eye strain, tinnitus, unpleasant taste in the mouth, fatigue, lethargy, changes in sleep patterns, behavioral and / or mood changes, and difficulty with memory, concentration, attention, and / or thinking).

[0070] In certain embodiments, the use involves administering (e.g., intravenously, intramuscularly, subcutaneously) (e.g., both intravenously and intramuscularly) a composition (e.g., a composition described herein) comprising allogeneic mitochondria at the time of injury that may result in TBI (e.g., mTBI).

[0071] In certain embodiments, the use involves administering a composition comprising allogeneic mitochondria (e.g., a composition described herein) a period of time (e.g., 1 hour, 1 day, 1 week, 1 month, 1 year, 5 years) after a TBI (e.g., mTBI) or suspected TBI.

[0072] In certain embodiments, the subject is suffering from mitochondrial loss.

[0073] In certain embodiments, the subject is suffering from a mitochondrial impairment dysfunction. definition

[0074] 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 to more than one element.

[0075] Administration: As used herein, the term "administration" typically refers to the administration of a composition to a subject or system, for example, to achieve the delivery of the agent contained in the composition 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 that is applied to 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.

[0076] 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.).

[0077] Allogeneic: As used herein, the term "allogeneic" refers to any material derived from one subject (donor) and transplanted into another. Examples of allogeneic transplants include allogeneic T cell transplants, allogeneic stem cell transplants, and allogeneic mitochondrial transplants as discussed herein.

[0078] Amelioration: As used herein, the term "amelioration" refers to the prevention, alleviation, relief, or improvement of a subject's condition. Amelioration includes, but does not require, complete reversal or complete prevention of a disease, disorder, or symptom.

[0079] Antibiotics: As used herein, the term "antibiotics" refers to antibacterial substances such as penicillin, gentamicin, streptomycin, cephalosporins, ciprofloxacin, and the like, which 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.

[0080] Autologous: As used herein, the term "autologous" refers to any material derived from one subject (donor) and transplanted back into that same subject. As used herein, "autologous" and "autogenous" are used interchangeably. Examples of autologous transplants include autologous stem cell transplants and autologous stem cell transplants, as well as autologous mitochondrial transplants as discussed herein.

[0081] 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., a tissue or organism or cell culture), as described herein. In some embodiments, for example, as described herein, the biological source is or includes an organism, such as an animal or human. In some embodiments, for example, as described herein, the biological sample is or includes a biological tissue or fluid. In some embodiments, for example, as described herein, the biological sample can be or include a cell, tissue (e.g., skin tissue, muscle or other tissue), or bodily fluid. In some embodiments, for example, as described herein, a biological sample can be or include blood, blood cells, cell-free DNA, free-floating nucleic acids, ascites, biopsy samples, surgical specimens, cell-containing bodily fluids, sputum, saliva, feces, urine, cerebrospinal fluid, peritoneal fluid, pleural effusion, lymph, gynecological fluids, secretions, excretions, skin swabs, vaginal swabs, oral swabs, nasal swabs, washings or lavages such as ductal washings or bronchoalveolar lavage, aspirates, scrapings, or bone marrow. In some embodiments, for example, as described herein, a biological sample is or includes cells obtained from a single subject or multiple subjects. A sample can be a "primary sample" obtained directly from a biological source, or can be a "processed sample." A biological sample can also be referred to as a "sample."

[0082] Improved, increased, or decreased: As used herein, these terms or grammatically equivalent comparative terms refer to a value compared to an equivalent reference measurement.For example, in some embodiments, for example, as described herein, the assessment value achieved with an agent of interest can be "improved" compared to the 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 the 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 the administration of an 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 symptom of interest, or in prior exposure to a symptom, 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 skilled 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.

[0083] 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 setting) and / or (b) designed, produced, prepared, and / or manufactured by the hand of man. Isolated substances and / or entities can 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 more 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. By way of example only, in some embodiments, naturally occurring mitochondria can be referred to as "isolated" if (a) the mitochondria 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 were derived, (b) the mitochondria are substantially free of other organelles from the donor organism from which they were derived; or (c) the mitochondria are present in a cell or system that is different from the donor organism from which they were derived. Thus, for example, mitochondria removed from a donor for transplantation into a second, different subject can be referred to as "isolated."

[0084] Neurodegenerative disease: As used herein, the term "neurodegenerative disease" (also referred to as "degenerative neurological disease") refers to a general term for conditions that primarily affect neurons in the human brain. In certain instances, neurodegenerative diseases are characterized by the progressive loss of neurons accompanied by the deposition of proteins with 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), dementia with Lewy bodies (LBD), other forms of dementia, muscular dystrophy (MD), mitochondrial disorders, prion diseases, motor neuron disease (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.

[0085] 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 pharmaceutically acceptable carriers. In some embodiments, e.g., as described herein, the active agent is present in a unit dose suitable for administration to a subject, e.g., 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 can be formulated for administration in a particular form (e.g., in a solid or liquid form) and / or can be particularly adapted for, for example: oral administration (e.g., a drench (aqueous or non-aqueous solution or suspension), tablet, capsule, bolus, powder, granule, paste, etc., which can 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 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.

[0086] Pharmaceutically acceptable: As used herein, the term "pharmaceutically acceptable" as applied to one or more or all ingredients for formulating the compositions disclosed herein means that each ingredient must be compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

[0087] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically 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 which can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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 materials used in pharmaceutical formulations.

[0088] Prevent or prevention: As used herein, the term "prevent" and "prevention" in relation to the occurrence of disease, disorder or symptom refers to reducing the risk of developing disease, disorder or symptom; delaying the onset of disease, disorder or symptom; delaying the onset of one or more characteristics or symptoms of disease, disorder or symptom; and / or reducing the frequency and / or severity of one or more characteristics or symptoms of disease, disorder or symptom.Prevention can refer to the prevention in a specific subject or the statistical effect on a population of subjects.If the onset of disease, disorder or symptom is delayed for a predetermined period of time, prevention can be considered complete.

[0089] 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 a treatment, for example, a particular treatment.

[0090] Reference: As used herein, it describes a standard or control to 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 with 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, a reference is determined or characterized under conditions or circumstances equivalent to those being evaluated, for example, for a 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.

[0091] 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 is clear from the context, the term "sample" refers to a preparation obtained by processing the primary sample (e.g., by removing one or more components of the primary sample and / or by adding one or more agents to the primary sample).

[0092] 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, e.g., as described herein, an individual who is susceptible to a disease, disorder, or condition does not exhibit any symptoms of said disease, disorder, or condition. In some embodiments, e.g., as described herein, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with said disease, disorder, and / or condition. In some embodiments, e.g., 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 exhibits a biomarker status associated with the development of the disease, disorder, or condition. In some embodiments, e.g., 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 suffering from the disease, disorder, or condition).

[0093] Subject: As used herein, the term "subject" refers to an organism, typically a mammal (e.g., a human). In some embodiments, e.g., as described herein, the subject is afflicted with a disease, disorder, or condition. In some embodiments, e.g., as described herein, the subject is susceptible to a disease, disorder, or condition. In some embodiments, e.g., as described herein, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, e.g., as described herein, the subject does not suffer from a disease, disorder, or condition. In some embodiments, e.g., as described herein, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, e.g., as described herein, the subject possesses one or more characteristics characteristic of susceptibility to or risk for a disease, disorder, or condition. In some embodiments, e.g., as described herein, the subject is a patient. In some embodiments, e.g., as described herein, the subject is an individual for whom a diagnosis has been performed and / or a treatment has been administered. In some cases, e.g., as described herein, a human subject may be referred to interchangeably as an "individual."

[0094] Syngeneic: As used herein, the term "syngeneic" refers to any material derived from one subject (donor) and transplanted into another subject where the subject and donor are genetically identical. Examples of syngeneic transplants include syngeneic T cell transplants, syngeneic bone marrow transplants, and syngeneic mitochondrial transplants as discussed herein.

[0095] 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, e.g., as described herein, an agent is considered to be a therapeutic agent if it exhibits a statistically significant effect across an appropriate population. In some embodiments, e.g., as described herein, the appropriate population may be a population of model organisms or a human population. In some embodiments, e.g., as described herein, the appropriate population may be defined by various criteria, such as a particular age group, sex, genetic background, pre-existing clinical symptoms, etc. In some embodiments, e.g., 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, e.g., as described herein, a therapeutic agent is an agent that has been or needs to be approved by a government agency before being commercially available for administration to humans. In some embodiments, e.g., as described herein, a therapeutic agent is an agent that requires a medical prescription for administration to humans.

[0096] 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 be achieved in specific individuals. Rather, a therapeutically effective amount may be an amount that, when administered to individuals in need of such treatment, provides a specific, 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 the amount measured in one or more specific 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 can 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.

[0097] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to the administration of a therapy administered with the purpose of partially or completely alleviating, ameliorating, alleviating, inhibiting, delaying the onset of, halting the progression of, slowing 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, e.g., as described herein, such treatment can be treatment of a subject who does not exhibit signs of the relevant disease, disorder, or condition and / or who exhibits only early signs of the disease, disorder, or condition. Alternatively or additionally, such treatment can be treatment of a subject who exhibits one or more established signs of the relevant disease, disorder, and / or condition. In some embodiments, e.g., as described herein, treatment can be treatment of a subject who has been diagnosed with the relevant disease, disorder, and / or condition. In some embodiments, for example, as described herein, the treatment can be treatment of a subject known to have one or more susceptibility factors that statistically correlate with an increased risk of developing the associated disease, disorder, or condition. In various examples, the treatment is treatment of cancer.

[0098] 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 discrete unit. In many embodiments, e.g., as described herein, a unit dose contains a predetermined amount of an active agent. In some embodiments, e.g., as described herein, a unit dose contains a whole single dose of an agent. In some embodiments, e.g., as described herein, more than one unit dose is administered to achieve a total single dose. In some embodiments, e.g., as described herein, administration of multiple unit doses is required or expected to be required to achieve the 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, etc. It will be understood that a unit dose can be present in a formulation containing any of a variety of ingredients in addition to a therapeutic agent. For example, an acceptable carrier (e.g., a pharmaceutically acceptable carrier), a diluent, a stabilizer, a buffer, a preservative, etc. may be included. In many embodiments, for example, as described herein, the appropriate total daily dosage of specific therapeutic agent can comprise a portion or a plurality of unit doses, and can be determined 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 specific 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 excretion rate of the specific active compound used; the duration of treatment; the drug and / or additional treatment used in combination with or simultaneously with the specific compound used, and similar factors well known in the medical field.

[0099] Variant: As used herein, the term "variant" refers to an entity that exhibits significant structural identity with a reference entity, but that structurally differs 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 the reference entity. Generally, whether a particular entity is properly considered 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 the 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 the reference peptide that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99%.

[0100] Xenogeneic: As used herein, the term "xenogeneic" refers to any material derived from one subject (donor) and transplanted into a subject where the subject and donor are of different species. Examples of xenogeneic transplants include xenogeneic skin transplants, xenogeneic heart valve transplants, and, as discussed herein, xenogeneic mitochondrial transplants. DETAILED DESCRIPTION OF THE INVENTION

[0101] Described herein are methods for treating traumatic brain injury (TBI), including mild traumatic brain injury (mTBI), by applying the methods and compositions described below.

[0102] International (PCT) Patent Application No. PCT / US2020 / 047359, filed August 21, 2020, and published as WO 2021 / 141637, describes compositions and methods for treating amyotrophic lateral sclerosis (ALS), other neurodegenerative diseases (NDs), and other mitochondrial disorders. In particular, experimental examples demonstrating Mitochondrial Organelle Transplantation™ (MOT™) for the treatment of NDs, such as ALS, are described therein. This application incorporates by reference U.S. Patent Application No. 16 / 937,388, filed July 23, 2020, and U.S. Provisional Patent Application No. 62 / 958,592, filed January 8, 2020. The contents of each of the above-referenced patent applications are incorporated herein by reference in their entirety. Additionally, all publications mentioned herein are incorporated herein by reference in their entirety.

[0103] Hubbard et al. investigated the effects of mitochondrial bioenergetics in a mouse model of mild closed head injury (CHI). This study established that mTBI results in mitochondrial dysfunction. Targeting this dysfunction through the injection of healthy allogeneic mitochondria is important for alleviating mTBI symptoms resulting from mitochondrial damage. With several reports of intercellular mitochondrial transfer as a means of damaged tissue repair, mitochondrial replacement offers a viable means for rescuing injured cells after mTBI.

[0104] Using data and knowledge gained from ongoing research to treat neurodegenerative diseases, particularly ALS and Parkinson's disease, it can be extrapolated that MOT™ may reduce the severity of mTBI symptoms and the length of time required for recovery, facilitating a more rapid and more complete "return to duty" (e.g., for military professionals).

[0105] As of the filing of this application, MOT™ for the treatment of ALS is undergoing IND submission for a Phase I clinical trial. MOT™ material is currently at Technology Readiness Level 4 (TRL4). Applicant has previously published on traumatic brain injury (TBI) in mice with controlled cortical injury (CCI), closed head TBI, and mild repetitive TBI (rmTBI) models. In addition, Applicant has published extensively on stroke, spinal cord injury (SCI), rodent models of ALS, and other models of injury in rodents. Furthermore, Applicant has reported that TBI reduces mitochondrial respiration, enhances ROS production, and induces apoptotic cell death, suggesting a prominent role of mitochondria in the pathophysiology of TBI. A primary cause of TBI-related brain injury is secondary damage, including mitochondrial dysfunction. Mitochondrial dysfunction impairs hippocampal development, resulting in the loss of NSCs and adult neurogenesis. Genetic diseases that result in mitochondrial abnormalities often also present with progressive clinical signs of cognitive impairment. Complex mitochondrial dysfunction after TBI requires treatment that specifically addresses the secondary injury(ies). Once future studies are completed, the MOT™ for mTBI recovery index will be TRL5. A. Mitochondria

[0106] In eukaryotic cells, mitochondria are known as powerhouses for generating adenosine triphosphate (ATP) through oxidative phosphorylation (OXPHOS). They also play important roles in the synthesis of iron-sulfur clusters and heme, the beta-oxidation of fatty acids, and calcium, iron, and reactive oxygen species (ROS) homeostasis. Mitochondria are particularly important in neurons. Neurons have high metabolic demands, and the brain, despite accounting for only 2% of the body's mass, consumes 20% of the body's resting ATP production. Furthermore, mitochondria are essential calcium-buffering organelles in neurons that modulate local calcium dynamics, for example, regulating neurotransmitter release. Neurons are long-lived cells that persist throughout an individual's lifespan and are therefore susceptible to the cumulative damage that results from mitochondrial dysfunction. Severe mitochondrial dysfunction can take many forms, including defective OXPHOS, excessive ROS, impaired calcium buffering capacity, and defective mitochondrial dynamics.

[0107] In eukaryotic cells, mitochondria generate ATP through oxidative phosphorylation (OXPHOS) in the presence of oxygen. Mitochondria also play important roles in iron-sulfur (Fe-S) cluster synthesis, fatty acid β-oxidation, heme prosthetic group synthesis, the urea cycle, and calcium, iron, and reactive oxygen species (ROS) homeostasis. 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 damage is severe. Furthermore, mitochondria can move 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.

[0108] Mitochondrial dysfunction contributes to many diseases, such as neurodegenerative diseases, heart diseases, and cancer. Mitochondrial dysfunction broadly includes a condition in which the mitochondria of a cell, tissue, organism, or sample thereof are characterized by (1) a decrease in the rate, amount, or efficiency of ATP production; (2) a decrease in mitochondrial membrane potential; (3) a decrease in the number or concentration of mitochondria; and / or (4) an increase in the rate or amount of ROS production compared to a reference. In some embodiments, the reference is a measurement or value representing a healthy subject (e.g., a comparable subject with typical mitochondrial function and / or without a diagnosed medical condition known to affect mitochondrial function). In some embodiments, the reference is a measurement or value representing a population of healthy subjects. In some embodiments, the reference is a measurement or value representing a subject at an earlier time point. Methods and techniques for measuring mitochondrial ATP production, mitochondrial membrane potential, the number or concentration of mitochondria, and / or ROS production are known in the art.

[0109] Mitochondrial dysfunction has been demonstrated in, for example, amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Parkinson's disease (PD). Because neurons are highly dependent on aerobic OXPHOS within mitochondria for their energetic needs, mitochondria are essential for neuronal function. Defects in mitochondrial respiration and ATP production in neurons result in neuronal dysfunction and degeneration. Mitochondria also produce ROS. When ROS oxidative stress overwhelms antioxidant defenses from superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX), ROS cause protein, lipid, and DNA damage in neurons. Furthermore, excess mitochondrial calcium and iron impair ATP production and mitochondrial and neuronal structure.

[0110] Mitochondria are highly abundant in cardiac cells due to their increased energy demands. Mitochondrial dysfunction is associated with the development of numerous cardiac diseases, including atherosclerosis, ischemia-reperfusion injury, hypertension, cardiac hypertrophy, and heart failure.

[0111] 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. B. Traumatic brain injury

[0112] In some embodiments, the methods and compositions described herein are used for treating the subject suffering from traumatic brain injury (TBI) or symptoms related to traumatic brain injury (TBI).Without wishing to be bound by any particular theory, TBI can cause mitochondrial damage and / or loss, which can lead to secondary damage to the brain.The secondary damage caused by mitochondrial damage (for example, dysfunction) or loss can cause symptoms related to TBI.

[0113] In certain embodiments, the TBI is caused by mechanical injury to the head from an external force (e.g., a fall, an explosion, a car accident, a shock wave, or other mechanical force). In certain embodiments, the mechanical injury to the head includes a bump, blow, and / or impact to the head, and / or a penetrating head injury.

[0114] In certain embodiments, a TBI may result in one or more symptoms including, but not limited to, headache (e.g., that gradually worsens and / or does not go away), recurrent vomiting, nausea, convulsions (e.g., seizures), inability to awaken from sleep, dilated pupils in one or both eyes, slurred speech, weakness and / or numbness in the extremities, loss of coordination, increased confusion, restlessness, and agitation.

[0115] In certain embodiments, the TBI is a mild traumatic brain injury (mTBI) (e.g., a concussion). In certain embodiments, an mTBI can result in one or more symptoms including, but not limited to, lightheadedness, dizziness, blurred vision, eye fatigue, tinnitus, a bad taste in the mouth, fatigue, lethargy, changes in sleep patterns, changes in behavior and / or mood, and difficulty with memory, concentration, attention, and / or thinking.

[0116] In certain embodiments, the severity of the TBI is determined at or near the time of injury, hi certain embodiments, the severity of the TBI is based on one or more factors, including, but not limited to, the length of loss of consciousness, the length of any memory loss or disorientation, and the responsiveness of the individual following the injury.

[0117] In certain embodiments, treatment for TBI may be administered at the time of the initial injury or a period of time thereafter (e.g., immediately after injury, 1 hour after injury, 1 day after injury, 1 week after injury, 1 month after injury, 1 year after injury, 5 years after injury). C. Tissue Donor Selection

[0118] When allogeneic mitochondria are used (e.g., from a donor other than the subject receiving the mitochondria), the donor is screened before providing tissue (e.g., skin) for use in creating a cell bank (e.g., a fibroblast cell bank). In certain embodiments, screening the donor ensures that the donor is suitable for providing the tissue (e.g., skin tissue) from which the cells are obtained. Mitochondria are isolated from the cells obtained from the donor.

[0119] In certain embodiments, the donor is selected as required by FDA 21 CFR Part 1271, which is incorporated by reference in its entirety. In certain embodiments, the donor is screened for one or more diseases (e.g., a communicable disease, e.g., a virus). In certain embodiments, the donor is free of risk factors and clinical evidence of infection with relevant communicable disease pathogens and / or diseases. In certain embodiments, the donor is free of communicable disease risks associated with xenotransplantation. In certain embodiments, the donor is tested for communicable disease pathogens and determined to be negative or non-reactive. In certain embodiments, the donor is free of diseases including, but not limited to, human immunodeficiency virus (HIV) (e.g., HIV1, HIV2, HBV, HCV), hepatitis B virus, hepatitis C virus, human transmissible spongiform encephalopathies (e.g., Creutzfeldt-Jakob disease), Treponema pallidum, and infectious diseases of the genitourinary tract (e.g., Chlamydia trachomatis, Neisseria gonorrhea).

[0120] In certain embodiments, the donor does not have a neurodegenerative disease and / or other condition associated with mitochondrial dysfunction. In certain embodiments, the donor does not have a disease, including but not limited to, 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.

[0121] In certain embodiments, the donor has no history of cancer, diabetes, or genetic mitochondrial disease. In certain embodiments, the donor is not pregnant or is a minor or adult individual lacking the capacity to consent.

[0122] In certain embodiments, the donor is between 18 and 40 years of age. In certain embodiments, the donor is older than 18 years of age. In certain embodiments, the donor is under 40 years of age (e.g., under 30 years of age, under 25 years of age, under 20 years of age).

[0123] Mitochondria derived from primary fibroblasts exhibit low or no immunogenicity, and therefore do not require HLA matching for mitochondrial transplantation. Humans have three major MHC class I loci known as HLA-A, HLA-B, and HLAC, with each individual possessing 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, with each individual possessing two alleles at each locus. Generally, 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. The match of all eight alleles in HLA-A, HLA-B, HLA-C and HLA-DRB1 loci can be called 8 / 8 match.The match of all ten alleles in HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1 loci can be called 10 / 10 match.In certain transplants, various degrees of allele mismatch can be tolerated.Therefore, for example, donor and subject can be matched at 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 of the 10 alleles of HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1, or at 8, 7, 6, 5, 4, 3, 2, 1 or 0 of the 8 alleles of HLA-A, HLA-B, HLA-C and HLA-DRB1.

[0124] As disclosed herein, in various embodiments, the donor and subject for mitochondrial transplantation need not be HLA (human leukocyte antigen) matched (e.g., not an identical match (e.g., based on a match of 8 or 10 tested HLA markers), and / or not a haploidentical match (e.g., based on a match of 8 or 10 tested HLA markers), and / or an indeterminate match (e.g., no HLA markers tested prior to the administering step). This is a significant advantage over stem cell therapy and organ transplantation, which require HLA matching of donor and recipient. D. Obtaining Cells from Donor Tissue

[0125] In some embodiments, the cell bank is generated from cells of a donor (e.g., a donor different from the subject receiving the mitochondrial composition). In some embodiments, the cell bank is a frozen cell bank. In some aspects, the cells are preserved in the cell bank by freezing. In certain embodiments, the cells are frozen at a temperature below -40°C (e.g., below -60°C, below -70°C, below -80°C, e.g., using liquid nitrogen). In certain embodiments, the medium used to freeze the cells to generate the cell bank does not contain antibiotics.

[0126] In some embodiments, skin tissue is used as a source material for creating a cell bank. In certain embodiments, fibroblasts from skin tissue are obtained to create a cell bank. In certain embodiments, fibroblasts from skin tissue are expanded before freezing. In certain embodiments, fibroblasts from skin tissue are expanded in culture medium that is not supplemented with antibiotics (e.g., gentamicin, penicillin, streptomycin). Antibiotics in culture medium can be toxic to mitochondria in mammalian cells. In certain embodiments, fibroblasts from the first, second, or third passage are frozen to create a cell bank.

[0127] In certain embodiments, mesenchymal stem cells (MSCs) (eg, primary MSCs) are obtained from a donor (eg, to create a cell bank).

[0128] Those skilled in the art will be able to select appropriate freezing protocols for the tissues and cell types described herein based on the information provided in this disclosure. E. Mitochondrial Isolation and Storage Buffer

[0129] In certain embodiments, the methods and compositions described herein utilize mitochondrial isolation and storage buffers. In certain embodiments, the mitochondrial isolation and storage buffers used are described in International (PCT) Patent Application No. PCT / US2020 / 047359, filed August 21, 2020, and incorporated by reference in its entirety.

[0130] In certain embodiments, the mitochondrial isolation buffer (e.g., for isolating mitochondria) 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); a membrane stabilizer and / or an oxygen radical scavenger and / or Ca 2+ and a serine protease inhibitor (e.g., phenylmethylsulfonyl fluoride (PMFS), also known as phenylmethanesulfonyl fluoride). In certain embodiments, the composition further comprises isolated donor mitochondria, e.g., fibroblast mitochondria.

[0131] In certain embodiments, the mitochondrial isolation buffer 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). In certain embodiments, the osmolality of the buffer is about 325 mOsm. In certain embodiments, 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 to free fatty acids. Phenylmethylsulfonyl fluoride (PMSF), also known as 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 worn-out organelles. During the cell homogenization procedure (e.g., to obtain mitochondria), some lysosomes may be damaged and release digestive enzymes into the cell lysate. In certain embodiments, PMSF can be included in the isolation buffer to prevent mitochondrial damage from digestive enzymes. In certain embodiments, the isolation buffer does not contain antibiotics.

[0132] In certain embodiments, a mitochondrial storage buffer is administered to a subject and / or used to stably maintain isolated mitochondria in solution. In certain embodiments, the storage buffer comprises 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 (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 calcium ion-binding cytoprotective agent (e.g., lactobionate or a salt thereof, e.g., K-lactobionate); and a membrane stabilizer and / or an oxygen radical scavenger and / or Ca 2+In certain embodiments, the composition further comprises isolated donor mitochondria (e.g., fibroblast mitochondria). In certain embodiments, the storage buffer does not contain antibiotics.

[0133] In certain embodiments, the mitochondrial storage buffer 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. In certain embodiments, the osmolality of the mitochondrial storage buffer is about 325 mOsm. In certain embodiments, the mitochondrial storage buffer contains about 28 mM potassium ions. In certain embodiments, the buffer is sterilized by filtration (eg, through a 0.22 μm filter), aliquoted into small vials, and stored at -80°C.

[0134] In certain embodiments (e.g., clinical uses discussed herein), mitochondria in a storage buffer are administered to a human subject (e.g., intramuscularly, intravenously, subcutaneously) (e.g., both intramuscularly and intravenously). High potassium ion concentrations are dangerous for injection into humans (e.g., 91 mM potassium ion concentration). In certain embodiments, reduced concentrations of all K + The salts used in the mitochondrial storage buffer (e.g., 2 mM KH2PO4, 10 mM K-HEPES, and 15 mM K-lactobionate) are: +The final concentration of is similar to that of a clinical intravenous solution (e.g., 20 mEq to 40 mEq) containing potassium chloride (e.g., about 28 mEq). To maintain the osmolality of the storage buffer at a desired level, the concentration of sucrose in the storage buffer may be increased. In certain embodiments, the storage buffer is not administered with an antibiotic. F. Explanation of Future Research, Scientific Rationale, and Initial Data

[0135] The scope of this effort by the VA partners is to test the efficacy of MOT™ administered by infusion to shorten the time to recovery and reduce symptom severity in animal models of mTBI. Using key findings from neurodegeneration in ALS and Parkinson's disease, as well as knowledge from brain injury models from the VA, applicants hypothesize that mitochondrial dysfunction is responsible for many of the symptoms associated with mTBI. Therefore, the infusion of healthy allogeneic mitochondria via transplantation will supplement mitochondria lost during injury and repair damaged mitochondria, resulting in objective improvement of mTBI in the model. Over the course of these studies, information will be obtained confirming safety in the specific context of mTBI, and manufacturing improvements will be investigated, focusing on delivery and use in austere environments. Successful completion of these studies will allow for the submission of an Investigational New Drug (IND) application to the FDA, paving the way for the therapeutic use of MOT™ to shorten the time required for military personnel to return to duty and reduce the severity of their symptoms after their return to duty.

[0136] In recent years, mitochondrial transplantation (MT) has emerged as a promising therapeutic intervention benefiting neuronal survival and regeneration for neurodegenerative diseases, stroke, and central nervous system (CNS) injuries. McCully's group has extensively studied the role of injected isolated mitochondria for cardioprotection during ischemia-reperfusion. Exogenous mitochondria entered cardiomyocytes 2 hours after injection, maintained viability and function, and produced sufficient ATP levels. We also demonstrated that exogenous mitochondria provide cardioprotection both extracellularly and intracellularly. Shi et al. intravenously injected heterologous mitochondria derived from human hepatoma into experimental Parkinson's disease (PD) rat models. MT prevented the progression of experimental PD by increasing electron transport chain activity, reducing ROS levels, and preventing cell apoptosis and necrosis. In 2016, Huang's group intracerebrally or intra-arterially injected heterologous mitochondria from hamster cells into a rat model of cerebral ischemia. Their results confirmed that hamster mitochondria protected neuronal function and promoted functional recovery in rats with cerebral ischemia. They found that mitochondrial internalization into neurons could not fully explain the high rescue of neuronal injury.

[0137] Extracellular exogenous mitochondria can be a source of ATP and ROS scavengers to protect cells from free radical damage. Applicant has demonstrated that human mitochondria from primary fibroblasts enhance the mitochondrial DNA (mtDNA) depletion (ρ) of rat motor neurons NSC-34. 0 reported that MTs replenish mtDNA, restore mitochondrial-encoded cytochrome c oxidase 1 (MT-CO1) and mitochondrial-encoded NADH dehydrogenase 1 (MT-ND1), and promote the growth of NSC-34ρ. 0 Furthermore, Applicants found that isolated human mitochondria contained no detectable human leukocyte antigen I (HLA-I), suggesting low or no immunogenicity and allogeneic mitochondria that do not require HLA binding matching in MTs.

[0138] Mesenchymal stem cell / stromal cell (MSC) therapy has also received significant attention.

[0139] Bidirectional mitochondrial transfer has been observed between donor and recipient cells, affecting both. Mitochondrial transfer improves cell viability, promotes anti-inflammatory responses, and promotes stem cell differentiation. Adding isolated, healthy mitochondria to induced pluripotent stem cells (iPSCs) allowed them to enter the stem cells within minutes and promote neuronal differentiation by increasing the expression of neuronal and glutamatergic markers β3-tubulin and synapsin 1, as well as activating the glutamate-glutamine cycle. MOT™ was able to replenish mitochondria and mtDNA and restore mitochondrial function in neurons damaged by mTBI. Gollihue et al. reported that transplantation of exogenous mitochondria concentration-dependently maintained acute bioenergetic changes in the injured spinal cord. Elliott et al. reported the case of an ALS patient treated with MOT™ under the Compassionate Care Act. The patient participated in two previous clinical trials, but his disease continued to progress and his condition declined. After four implantation procedures at six-week intervals, the patient had no adverse reactions or complications, but had significant improvement in leg muscle strength according to the tests performed and clinical function measured, and the patient regained full sensation in both legs.

[0140] The MOT™ protocol uses fibroblasts from healthy donors. Cells were stored in liquid nitrogen at the Elliott MRC Laboratory. Fibroblasts were recovered from liquid nitrogen and expanded in a GLP cell culture facility. Mitochondria were then isolated from the fibroblasts in a GLP cell culture facility. The isolation protocol is simple and rapid, maintaining good mitochondrial yield, viability, and quality. Mitochondria can be injected intramuscularly and intravenously.

[0141] Applicant has conducted a pilot study of repeated intramuscular and intravenous administration of MOT™ in a series of five patients with ALS over the past 2.5 years, with no observed adverse events across a total of 47 doses. These patients were followed for a period of 45-90 days between treatment cycles. While this small sample size was not powered to detect efficacy, four patients reported symptomatic improvement observed over several weeks following administration. This real-world data provided the basis for the Human Experience component of the IND submission to the FDA. Applicant is in communication with FDA leadership (Dr. Peter Marks), who confirmed that MOT™ will be appropriately regulated via the IND pathway within the Center for Biologics Evaluation and Research (CBER).

[0142] Supporting the above preliminary clinical data, the applicant successfully manufactured clinical-grade batches of MOT™, which were used to provide doses for the case series. The MOT™ preparation demonstrated consistency in terms of mitochondrial wet weight. For this project, the team will provide GLP-grade batches of MOT™. MOT™ material is being prepared to achieve GMP production development for use in the treatment of ALS. Methods for MOT™ are available to the team to enable progression from TRL4 to TRL5. GMP processing of MOT™ material will be overseen by KRS Global Biotechnology, an industry-leading, customized, FDA-registered and inspected 503b Human Outsourcing Facility that provides sterile and non-sterile compounding services to patients, surgery centers, ophthalmology clinics, hospitals, and universities. In collaboration with KRS, the applicant will evaluate the shipment and use of MOT™ in a challenging environment. Following successful completion of this study, clinical trials can begin. G. Constructive Examples

[0143] Healthy human mitochondria improve motor function and post-concussion recovery in animal mTBI models. First, human primary fibroblasts are selected from healthy donors for use in MOT™. A primary fibroblast bank is developed. Skin tissue is provided by healthy volunteers aged 18-40 years. Donors with a history of infectious diseases (HIV1, HIV2, HBV, HCV, and Treponema pallidum), cancer, diabetes, or genetic mitochondrial disorders, pregnant women, and minors or adults who lack the capacity to consent for themselves are excluded from donation. The tissue collection protocol and consent form are approved by the Institutional Review Board (WCG IRB [formerly Western] for Elliott MRC at SABBF). The isolation, culture, and cryopreservation of human primary fibroblasts were previously reported in Jiang et al. (Jiang XP, Baucom CC, Elliott RL. Mitochondria dynamically transplant into cells in vitro and in mice and rescue aerobic respiration of mitochondrial DNA-depleted motor neurons NSC-34. J. Biomedical Sci. Engineering. 2020;13:203-221. doi:10.4236 / jbise.2020.139019). Second, primary fibroblasts were harvested and expanded from a human fibroblast bank. Mitochondria were isolated from the fibroblasts by differential centrifugation (e.g., according to Jiang et al.). Third, assays for determining mitochondrial number, integrity, viability, and potency were further developed. Data from the assays were used to refine mitochondrial isolation and preservation protocols and quality control parameters for MOT™ in animal mTBI models.

[0144] Data from the assay will be used to refine mitochondrial administration of MOT™ in animal mTBI models. Fourth, the safety of mitochondria and buffer will be tested via different administration routes, such as subcutaneous or intravenous injection. Fifth, mitochondrial distribution in tissues, including the spinal cord and brain, will be examined by injection of fluorescently labeled mitochondria. Sixth, efficacy will be tested in mice and rats, including motor function and neurological behavior, using previously reported biomarkers and assays (e.g., those described in References 27 and 28). These biomarkers may enable predictions about the potential effectiveness of MOT™ treatment for mTBI treatment. Seventh, pathological and ultrastructural studies will be completed on mouse and rat tissues, including the spinal cord, brain, and muscle, to demonstrate the mechanism of MOT™ at the endpoint. These studies will be repeated in a pig model following analysis of the mouse and rat studies to confirm findings in a larger animal model. Finally, the data will be statistically analyzed, reported, and published in an appropriate peer-reviewed journal.

[0145] The proposed study will demonstrate 1) the impact of MOT™ on recovery time in three animal mTBI models (mouse, rat, and pig), and 2) reducing the severity of mTBI symptoms following injury in the animal models. Success would validate MOT™ as a viable treatment for mTBI in reducing "return to duty" time and, if approved by the FDA, would be a powerful boost to the Department of Defense and Ready Forces. [ka] [ka] [ka] [ka] [ka]

[0146] The systems, compositions, methods, and processes of the claimed inventions are intended to encompass variations and adaptations developed using information from the embodiments described herein. Adaptations and / or modifications of the systems, compositions, methods, and processes described herein can be implemented as contemplated by the present specification.

[0147] Throughout this specification, when articles, compositions and systems are described as having, including, or comprising particular components, or when processes and methods are described as having, including, or comprising particular steps, it is further contemplated that there are articles, compositions and systems of the invention that consist essentially of, or consist of, the recited components, and there are processes and methods of the invention that consist essentially of, or consist of, the recited processing steps.

[0148] 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.

[0149] The citation of any publication herein is not an admission that the publication serves as prior art with respect to any of the claims presented herein.

[0150] The documents are incorporated herein by reference as if set forth. In the event of a discrepancy in the meaning of a particular term, the meaning provided in this document shall control.

[0151] Headers are provided for the convenience of the reader, and the presence and / or placement of headers is not intended to limit the scope of the subject matter described herein.

[0152] While the present invention has been particularly shown and described with reference to certain preferred embodiments, it should be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. 1. A method for allogeneic transplantation of mitochondria in a subject for the treatment of traumatic brain injury (TBI), the method comprising administering to the subject a composition comprising mitochondria isolated from a donor other than the subject, the subject having mTBI.

2. 10. The method of claim 1, wherein the composition further comprises a mitochondrial storage buffer having a potassium ion concentration that is safe for human administration.

3. 10. The method of claim 1, wherein the administering step comprises parenterally administering at least one unit dose of the composition to the subject.

4. 4. The method of claim 3, wherein the administering step comprises both intramuscular and intravenous injection of the composition into the subject.

5. 10. The method of claim 1, further comprising isolating the mitochondria from the donor.

6. 6. The method of claim 5, wherein isolating the donor mitochondria comprises preparing a cell lysate from the donor tissue via tissue dissociation.

7. 6. The method of claim 5, wherein isolating the donor mitochondria comprises using a mitochondrial isolation buffer containing a serine protease inhibitor.

8. 6. The method of claim 5, comprising isolating the donor mitochondria without the use of antibiotics.

9. The method of claim 1 , wherein the donor and the subject are not HLA (human leukocyte antigen) matched.

10. 10. The method of claim 1, wherein the composition administered to the subject does not contain an antibiotic.

11. 10. The method of claim 1, wherein the composition comprises mitochondria isolated from human primary fibroblasts of the donor.

12. 10. The method of claim 1, further comprising isolating the mitochondria from the donor tissue.

13. 13. The method of claim 12, wherein the isolating step is carried out using a mitochondrial isolation buffer composition.

14. The mitochondrial isolation buffer composition comprises: A buffering agent; A chelating agent; Sugar and Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; a serine protease inhibitor; 14. The method of claim 13, comprising:

15. 15. The mitochondrial isolation buffer composition of claim 14, wherein the composition is antibiotic-free.

16. 13. The method of claim 12, further comprising storing the isolated mitochondria at a temperature below -40°C.

17. 10. The method of claim 1, comprising administering to the subject an iron chelator.

18. 10. The method of claim 1, comprising administering to the subject an antioxidant and / or a probiotic.

19. 10. A mitochondrial isolation buffer composition for use in practicing the method of claim 1, said composition comprising: A buffering agent; A chelating agent; Sugar and Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; a serine protease inhibitor; 1. A mitochondrial isolation buffer composition comprising:

20. 20. The mitochondrial isolation buffer composition of claim 19, wherein the composition is antibiotic-free.

21. 10. A mitochondrial storage buffer composition for use in practicing the method of claim 1, said composition comprising: one or more buffering agents; a source of magnesium ions; A chelating agent; Sugar and Antioxidants and a cytoprotective agent that binds to calcium ions; Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; 1. A mitochondrial storage buffer composition comprising:

22. 22. The mitochondrial storage buffer composition of claim 21, wherein the composition is antibiotic-free.

23. 1. A kit comprising a unit dose of a donor mitochondrial composition effective to treat traumatic brain injury (TBI) in a subject, the donor mitochondrial composition comprising: Mitochondria isolated from donor tissue; one or more buffering agents; a source of magnesium ions; A chelating agent; Sugar and Antioxidants and a cytoprotective agent that binds to calcium ions; Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; Kit including:

24. 24. The kit of claim 23, wherein the donor and the subject are not HLA (human leukocyte antigen) matched.

25. 24. The kit of claim 23, wherein the donor mitochondrial composition does not contain antibiotics.

26. 24. The kit of claim 23, further comprising instructions for optimizing the dosage and / or frequency and / or route of administration of the composition.

27. 1. A method of treating traumatic brain injury (TBI), said method comprising: A method comprising administering to a subject a composition comprising allogeneic mitochondria.

28. 28. The method of claim 27, wherein the subject has experienced a TBI.

29. 28. The method of claim 27, wherein the subject is suffering from TBI symptoms.

30. 28. The method of claim 27, wherein the subject has experienced mild traumatic brain injury (mTBI).

31. 28. The method of claim 27, wherein the subject is suffering from mTBI symptoms.

32. 28. The method of claim 27, wherein the subject is administered the composition at the time of an injury that may result in a TBI.

33. 28. The method of claim 27, wherein the subject is administered the composition a period of time after TBI or suspected TBI.

34. 29. The method of claim 28, wherein the TBI results in mitochondrial loss in the subject.

35. 29. The method of claim 28, wherein the TBI results in mitochondrial damage dysfunction.

36. 28. The method of claim 27, wherein the method further comprises isolating mitochondria.

37. 37. The method of claim 36, wherein the method comprises isolating mitochondria from a donor other than the subject.

38. 37. The method of claim 36, wherein the method comprises isolating mitochondria from the subject.

39. 28. The method of claim 27, wherein the method further comprises creating a cell bank from a donor other than the subject.

40. 40. The method of claim 39, wherein the cell bank comprises fibroblasts.

41. 40. The method of claim 39, wherein the cell bank comprises mesenchymal stromal cells (MSCs).

42. 28. The method of claim 27, wherein the method comprises obtaining a tissue biopsy from a donor.

43. 28. The method of claim 27, wherein the method comprises creating a tissue bank from a donor.

44. 28. The method of claim 27, wherein the composition further comprises a mitochondrial storage buffer having a potassium ion concentration that is safe for administration to humans.

45. 28. The method of claim 27, wherein the administering step comprises parenterally administering at least one unit dose of the composition to the subject.

46. 46. ​​The method of claim 45, wherein the administering step comprises both intramuscular and intravenous injection of the composition into the subject.

47. 37. The method of claim 36, wherein isolating the mitochondria comprises preparing a cell lysate from the tissue via tissue dissociation.

48. 37. The method of claim 36, wherein isolating the mitochondria comprises using a mitochondrial isolation buffer containing a serine protease inhibitor.

49. 37. The method of claim 36, comprising isolating the mitochondria without the use of antibiotics.

50. 28. The method of claim 27, wherein the subject and the donor from whom the mitochondria are obtained are not HLA (human leukocyte antigen) matched.

51. 28. The method of claim 27, wherein the composition administered to the subject does not include an antibiotic.

52. 37. The method of claim 36, wherein the isolating step is carried out using a mitochondrial isolation buffer composition.

53. The mitochondrial isolation buffer composition comprises: A buffering agent; A chelating agent; Sugar and Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; a serine protease inhibitor; 53. The method of claim 52, comprising:

54. 54. The mitochondrial isolation buffer composition of claim 53, wherein the composition is antibiotic-free.

55. 37. The method of claim 36, further comprising storing the isolated mitochondria at a temperature below -40°C.

56. 28. The method of claim 27, comprising administering to the subject an iron chelator.

57. 28. The method of claim 27, comprising administering to the subject an antioxidant and / or a probiotic.

58. 28. A mitochondrial isolation buffer composition for use in practicing the method of claim 27, said composition comprising: A buffering agent; A chelating agent; Sugar and Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; a serine protease inhibitor; 1. A mitochondrial isolation buffer composition comprising:

59. 59. The mitochondrial isolation buffer composition of claim 58, wherein the composition does not contain an antibiotic.

60. 28. A mitochondrial storage buffer composition for use in practicing the method of claim 27, said composition comprising: one or more buffering agents; a source of magnesium ions; A chelating agent; Sugar and Antioxidants and a cytoprotective agent that binds to calcium ions; Membrane stabilizer and / or oxygen radical scavenger and / or Ca ２＋ an agent that acts as a binder of the fatty acids and / or a binder of free fatty acids; 1. A mitochondrial storage buffer composition comprising:

61. 61. The mitochondrial storage buffer composition of claim 60, wherein the composition does not contain an antibiotic.

62. The use of allogeneic mitochondria for the treatment of traumatic brain injury (TBI).

63. 1. Use of allogeneic mitochondria for the treatment of traumatic brain injury (TBI), wherein the allogeneic mitochondria are administered to a subject suffering from traumatic brain injury (TBI)-associated mitochondrial damage.

64. 64. The use of claim 63, wherein the subject has experienced a TBI.

65. 64. The use of claim 63, wherein the subject is suffering from TBI symptoms.

66. 64. The use of claim 63, wherein the subject has experienced mild traumatic brain injury (mTBI).

67. 64. The use of claim 63, wherein the subject is suffering from mTBI symptoms.

68. 64. The use of claim 63, wherein said use comprises administering a composition comprising said allogeneic mitochondria at the time of injury that may result in TBI.

69. 64. The use of claim 63, wherein said use comprises administering a composition comprising said allogeneic mitochondria a period of time after TBI or suspected TBI.

70. 64. The use of claim 63, wherein the subject is suffering from mitochondrial loss.

71. 64. The use of claim 63, wherein the subject is suffering from a mitochondrial damage dysfunction.

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