CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 62/645,519 filed on March 20, 2018, which is incorporated by reference herein in its entirety.

BACKGROUND

[0002] Mitochondria are the power plants of eukaryotic cells, providing over 90% of the energy required for life. With the exception of one recently identified organism, mitochondria are essential for the survival of all eukaryotic life forms.

[0003] Mitochondria are decedents of ancient bacteria that once existed as independent cells, but later developed a partnership arrangement inside a host primordial eukaryotic cell. In the modem eukaryotic cell, the mitochondria genome only expresses ~3% of the -1,200 genes required for proper assembly and function. The balance of the required genes are encoded by and expressed from the nuclear genome. The mitochondrial genome is thus highly compact, consisting of only a -16,000 base pair circular assembly of DNA encoding only 37 genes: specifically, genes for complex I, complex III, cytb, complex IV, complex V, Cyt b, and various mitochondrial -specific rRNAs and tRNAs (Gorman et al. 2016).

[0004] Mitochondria are central to the metabolic function of eukaryotic cells, enabling the flow of electrons from energy -rich carbon-to-carbon bonds found in food molecules (such as glucose) ultimately onto molecular oxygen. This“downhill flow” of potential energy from carbon-to-carbon bonds to molecular oxygen provides the vast majority of the energy required by eukaryotic cells to live. In addition, mitochondrial function is required for the following processes: oxidation of fatty acids, Iron- sulftir cluster assembly, calcium regulation, and control of the NAD/NADH flux.

[0005] While the mitochondrial genome is small, mutations in it accumulate as we age and have profound effects. Because such mutations result in the diminished production of functional mitochondrial proteins, mitochondria from older organisms produce less energy than mitochondria from younger organisms. Mitochondrial mutations contribute to diseases such as cardiomyopathy, myopathy, dementia, optic atrophy, infertility, fibrosis, Parkinson’s Disease, Alzheimer’s disease, amyotrophic lateral sclerosis (ALS), Huntington's disease (HD) and Duchenne muscular dystrophy.

SUMMARY OF THE INVENTION

[0006] This invention creates a new paradigm for the treatment of age related conditions. As a consequence of aging, mutations arise in the genome of mitochondria in the body, which lessens their function as power providers for the cells in which they reside. To overcome this deficit, this disclosure provides new ways of preparing rejuvenated mitochondria that are tailored for administration to a subject who has manifest signs of an age related condition. Autologous mitochondria are obtained from the subject, replicated (for example, in a producer cell line), and sorted for enhanced mitochondrial function, such as capacity to generate ATP. The mitochondria are then transplanted back into the subject, with a view to rejuvenating energy generation capacity and ameliorating the signs and symptoms of the age- related condition.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Fig. 1 provides a non-limiting illustration of the AMEE approach for mitochondrial transplantation. The various steps can be done in any operable sequence and repeated as necessary. In Step #1, a tissue sample is obtained from the patient needing a mitochondrial transplant. The tissue can be skin or isolated cells such as bone marrow cells, fibroblasts, or cheek epithelial cells. These isolated cells will be the source of the mitochondria used for production (after the rejuvenation steps). In Step # la, as an alternative to select for a higher proportion of functional mitochondria, the mitochondria from the cells in Step # 1 are isolated, sorted and transplanted into cells isolated from patients depleted of their mitochondria.

[0008] Next, in step #2, the isolated cells will be expanded. Expansion of such cells requires transformation into iPSCs (Step #3) or expression of other genetic factors (not shown). The serial passaging of reprogrammed iPSCs selects for rejuvenated mitochondria (Step #3), which will then be sorted for cells containing highly functional mitochondria in Step #4. In Step #5, individual mitochondria will be purified from cells and FACS sorted to select for the most functional mitochondria (if necessary). In Step #6, the highly functional mitochondria are injected into the patient. There are many operable permutations to the steps shown. For example, completion of the steps may occur in any order and any number of times.

[0009] Fig. 2 provides an illustration of one possible permutation of the general approach. An objective of AMEE is to produce a large amount of rejuvenated mitochondria for transplantation. The steps are as follows. 1. Isolate cells from patient needing the mitochondria transplant. Any cell type could be used (e.g., fibroblasts, epithelial). 2. Reprogram cells to rejuvenate mitochondria. 3. Sort cells for highest mitochondria function and then expand. 4. Purify mitochondria. 5. Inject mitochondria into patient.

[0010] Figs. 3A and 3B illustrate two alternative permutations of the basic AMEE approach. The procedure shown in Fig. 3 A is as follows: 1. Isolate cells and mitochondria from patient needing the mitochondria transplant. 2.. Sort isolated mitochondria for highest function. 3. Transplant mitochondria into cells created from step 4. 4. Reprogram cells to enhance proliferative capacity and remove endogenous mitochondria. 5. Sort cells for high functioning mitochondria and then expand. 6. Purify mitochondria. 7. Inject mitochondria into patient.

[0011] The procedure shown in Fig. 3B is as follows: 1. Isolate cells from patient needing the mitochondria transplant. 2. Reprogram cells to rejuvenate mitochondria. 3. Expand cells. 4. Purify mitochondria. 4. Sort mitochondria for highest mitochondrial function. 5. Inject mitochondria into patient. INCORPORATION BY REFERENCE

[0012] All publications, patents, and patent applications referred to in this specification are hereby incorporated herein by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTION

[0013] This disclosure provides various ways to purify mitochondria for administration to a subject in need of mitochondrial transplantation therapy, pharmaceutical compositions comprising the purified mitochondria, and methods of treatment using these pharmaceutical compositions.

Mitochondrial transplantation

[0014] Artificial transfer of purified mitochondria into mammalian cells was described by Clark and Shay in 1982. They demonstrated that purified mitochondria containing genes conferring particular traits (in that instance, the transferred gene conferred resistance to a particular drug, chloramphenicol) could be taken up by cells, thereby conferring upon the recipient cells resistance to the drug. The cells seemed to take up mitochondria spontaneously, consistent with the idea that the uptake of mitochondria is a natural process. Studies by Kesner et al. (2016) using Transmission Electron Microscopy (TEM) revealed that isolated mitochondria interact directly with cells, which rapidly (in under 10 minutes) engulf the mitochondria using cellular extensions. This suggests that mitochondrial transformation occurs via an active cellular process known as macropinocytosis. Macropinocytosis inhibitors, but not clathrin- mediated endocytosis inhibition treatments, block mitochondria transfer. Damage to the integrity of the mitochondrial outer membrane or to the mitochondrial outer membrane proteins decreases mitochondrial transformation, suggesting that cells can distinguish mitochondria from similar particles.

[0015] Later studies demonstrated that transferred mitochondria are functional inside the recipient cells, increasing ATP production, oxygen consumption and proliferation. Transplanted mitochondria can also replace depleted mitochondrial DNA in rho zero cells.

[0016] Shi et al. (2017) demonstrated that mitochondria injected intravenously into mice are taken up by numerous tissues throughout the body, including brain, heart, liver, kidney, and muscle, suggesting a potential path to rejuvenate numerous tissues in the body via intravenous administration, resulting in a whole body“mitochondrial transplant.” It is noteworthy that, in that experiment, mitochondria appeared to cross the blood-brain barrier.

[0017] Mitochondrial transplantation has been performed in human clinical studies for the treatment of acute ischemic injury to the heart (Shin et al. 2017). Five children experiencing acute ischemic injury were injected directly into damaged cardiac tissue with purified mitochondria taken from their own pectoral muscles.. Autologous Mitochondrial Extraction and Expansion (AMEE)

[0018] Methods of preparing a mitochondrial compositions according to this invention can comprise: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one cell from the cells based on a measurement of mitochondrial function; (c) expanding the at least one cell produce a plurality of expanded cells; and (d) purifying mitochondria from expanded cells. Methods of preparing a

mitochondrial composition can also comprise: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one mitochondria from the cells based on a measurement of mitochondrial function; (c) expanding the at least one mitochondria in a cell culture; and (d) purifying mitochondria from the cell culture.

[0019] An exemplary approach for AMEE is illustrated by FIG. 1. In some cases, the method comprises obtaining a sample from a subject. The subject can be a subject in need of a mitochondrial transplantation therapy. The subject can be suffering from or at risk of suffering from an age-related or senescence associated condition. The subject can be suffering from a condition caused by a mutation in the mtDNA. The subject can be a eukaryote. The subject can be a human. The subject can comprise cells showing reduced mitochondrial function. The subject can be under 40 years of age, under 50 years of age, under 60 years of age, or under 70 years of age. The subject can be over 70 years of age.

[0020] The sample can be a blood, urine, saliva, hair, or tissue sample. The sample can comprise cells. The cells can be blood cells, muscle cells, marrow cells, gametes, fibroblasts, keratinocytes, hepatocytes, epithelial cells, adipose-derived cells, melanocytes, pancreatic cells, amniotic cells, progenitor cells, stem cells, or a combination thereof. Examples of progenitor cells include, but are not limited to, neural progenitor cells, endothelial progenitor cells, and hematopoietic progenitor cells. Example of stem cells include, but are not limited to, mesenchymal stem cells, hematopoietic stem cells, endothelial stem cells, neural stem cells, and neural crest stem cells.

[0021] The cells can comprise a heteroplasmic population of mitochondria. The heteroplasmic population of mitochondria can comprises high functioning mitochondria and low functioning mitochondria. In one example, each cell in the sample can comprise a heteroplasmic population of mitochondria. In another example, some cells in the sample can comprise high functioning mitochondria while other cells in the sample can comprise low functioning mitochondria. A measurement of mitochondrial function can be used to determine whether a mitochondria is a high functioning mitochondria or a low functioning mitochondria.

Selecting high functioning mitochondria

[0022] High functioning mitochondria and low functioning mitochondria can be distinguished based on comparison to a threshold. In some cases, mitochondrial function above the threshold indicates the mitochondria is a high functioning mitochondria and mitochondrial function below the threshold indicates the mitochondria is a low functioning mitochondria. In some cases, mitochondrial function below the threshold indicates the mitochondria is a high functioning mitochondria and mitochondrial function above the threshold indicates the mitochondria is a low functioning mitochondria. The threshold can be an average mitochondrial function determined from a population of individuals. The population of individuals can be a healthy population of individuals. The threshold can be adjusted based on age of the population of individuals used to determine the threshold. In other cases, threshold can be determined based on the average mitochondrial function of the subject from which the sample is obtained. For example, the average mitochondrial function of a subject may initially be determined from the cells in the sample collected from the subject.

[0023] In some cases, the method comprises processing the sample prior to selecting at least one cell from the sample. Processing can comprise: homogenization, for example sonication, manual pulverization, or blending, to produce a suspension comprising the cells; enzymatic digestion, for example with collagenase or proteinase K; or acid digestion. In some cases, the processing comprises centrifugation. The centrifugation can be used to remove dead cells or debris. The centrifugation to remove dead cells or debris can be at a low speed, such as 200-300x g.

[0024] In some cases, the method comprises selecting at least one cell from the sample. The selecting can be done using positive selection, wherein the at least one cell is selected from the sample, or negative selection, wherein cells not part of the at least one cell are removed from the sample. The at least one cell can be isolated by using a cell size, surface antigen binding properties, adhesion, or a combination thereof. The at least one cell can be selected from the sample based on a measurement of mitochondrial function.

[0025] Selecting the at least one cell by using the cell size can comprise density gradient separation or filtration. Density gradient separation methods can comprise density gradient centrifugation. In one example, density gradient separation can be used to separate peripheral blood mononuclear cells from granulocytes and erythrocytes. In some cases, density gradient separation comprises the use of a gradient media. The gradient media can comprise: polysucrose 400, Ficoll®, Percoll®, or Optiprep®.

[0026] Selecting the at least one cell by using surface antigen binding properties can comprise use of a detectable probe. The detectable probe can be bound to cells in the subset of cells. The detectable probe can be an antibody, an aptamer, a magnetic particle, a fluorophore, a luminophore, or a combination thereof. The antibody can be conjugated to a fluorophore or a magnetic particle. The aptamer can be a conjugated to a fluorophore or a magnetic particle. In some cases, the detectable probe is removed from the at least one cell after the selecting.

[0027] Selecting the at least one cell by using adhesion can comprise culturing the sample in the presence of a surface, wherein the at least one cell attaches to the surface. The surface can be a polystyrene surface. The polystyrene surface can be oxidized, coated with extracellular matrix proteins, coated with poly-lysine, or a combination thereof. The extracellular matrix proteins can comprise heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, collagen, elastin, fibronectin, laminin, or a combination thereof. Examples of cells which can be isolated using adhesion include, but are not limited to macrophages, fibroblasts, and mesenchymal cells. [0028] Immuno -magnetic sorting methods can be used to select the at least one cell from the sample. Immunomagnetic sorting methods can comprise magnetic-activated cell sorting (MACS) or Dynabead® separation.

[0029] Selecting the at least one cell based on a measurement of mitochondrial function can comprise the use of automated sorting or clonal sorting. The automated sorting can comprise the use of FACS. FACS can be used to select the at least one cell from the sample. FACS can be used to select the at least one cell based on a measurement of mitochondrial function. FACS can comprise the use of a detectable marker. The detectable marker can be a fluorescent probe. In some cases, the detectable marker can be used to measure a measurement of mitochondrial function. Measurements of mitochondrial function can include, but are not limited to, measurements of mitochondria density, mitochondrial membrane potential (MMP), production of reactive oxygen species (ROS), plasma membrane potential, ATP production, heteroplasmy, or a combination thereof.

[0030] The detectable marker can be marker that measures the membrane potential of a mitochondria. The detectable marker which can measure the membrane potential of a mitochondria can be a rhodamine dye, a carbocyanine dye, or a rosamine dye. Examples of rhodamine dyes include, but are not limited to Rhodamine 123 (R123), Rhodamine-6G, tetramethylrhodamine methyl ester (TMRM), and

tetramethylrhodamine ethyl ester (TMRE). Examples of carbocyanine dyes include, but are not limited to, 5,5',6,6'-tetrachloro-l,l',3,3'-tetraethylbenzimidazolcarboc yanine (JC-l), 3,3'-dimethyl-a- naphthoxacarbocyanine iodide (JC-9) and 3,3'-dihexyloxacarbocyanine iodide (DiOC ₆ (3)). Examples of rosamine dyes include, but are not limited to MitoTracker® Orange CMTMRos, MitoTracker® Orange CM-H ₂ TMROS, MitoTracker® Red CMXRos, and MitoTracker® Red CM-H ₂ XRos.

[0031] The detectable marker can be a marker that is not dependent on the membrane potential of the mitochondria for labeling. In some cases, using a detectable marker not dependent on the membrane potential of the mitochondria can allow the density of mitochondria in a cell to be measured. Examples of probes which can are not dependent on the mitochondria membrane potential include, but are not limited to, MitoTracker® Green FM, MitoTracker® Red 580, MitoTracker® Deep Red 633, MitoFluor™ Green, MitoFluor™ Red 589, MITO-ID® Red, and nonyl acridine orange (NAO).

[0032] The detectable probe can be used to measure the amount of a reactive oxygen species (ROS). Examples of ROS include, but are not limited to hydrogen peroxide and superoxide. The ROS can be produced by the mitochondria. A detectable probe for measuring production of ROS can be a non- fluorescent probe that becomes fluorescent upon oxidation. Examples of probes which can be used to measure production of ROS include, but are not limited to dichlorodihydrofluorescein diacetate (DCFH- DA), Amplex red, dihydroethidium, dihydrorhodamine, coumarin boronate, RedoxSensor™ Red CC-l, and Mito-SOX™. The detectable probe can be used to measure the amount of a reactive nitrogen species (RNS). Examples of RNS include, but are not limited to peroxynitrite.

[0033] Examples of probes which can be used to measure plasma membrane potential include, but are not limited to, FluVolt™. [0034] The detectable marker can be fluorescent protein (FP). The fluorescent protein can be a photoactivatable fluorescent protein. In some cases the fluorescent protein binds specifically to a mitochondria, for example, by binding to a mitochondria specific targeting signal or peptide. Examples of fluorescent proteins include, but are not limited to, a photoactivatable green fluorescent protein (PA- GFP), a kindling fluorescent protein (KFP), and a photoactivatable mCherry (PAmCherry).

[0035] At least one, two, three, four, or five detectable markers can be used. In one example, nonyl acridine orange can be used in combination with Rhodamine 123 to measure mitochondrial density and mitochondrial membrane potential, respectively.

[0036] In some cases, an uncoupler, a mitochondrially metabolizable substrate, or a combination thereof is used in a calibration of a measurement of mitochondrial function. Examples of uncouplers include, but are not limited to p-trifluoromethoxy carbonyl cyanide phenyl hydrazine (FCCP), carbonyl cyanide m- chloro phenyl hydrazine (CCCP), and 2,4-dinitrophenol (DNP). Examples of substrates include, but are not limited to, glutamine, pyruvate, and methyl succinate. An assay to calibrate a measurement of mitochondrial function can comprise at least one mitochondrially metabolizable substrate and at least one uncoupling agent. One example of an assay to calibrate a measurement of mitochondrial function can be an assay of cells in the absence of glucose plus mitochondrially metabolizable substrates glutamine and pyruvate in combination with the uncoupling agent FCCP.

[0037] Mitochondrial DNA can be heteroplasmic, wherein more than one mitochondrial genome is present in a subject, within a cell, or even within a single mitochondrion. In some cases, the method comprises selecting one or more cells based on a measurement of heteroplasmy. Heteroplasmy can be determined by normalizing a measure of mitochondrial function according to the number of

mitochondria. For example, heteroplasmy can be determined by normalizing TMRM to a fluorescent probe such as MitoTracker® Deep Red or Rhodamine 123.

[0038] A single-cell microscopic assay can be used to select one or more cells from the plurality of cells based on a measurement of mitochondrial function.

[0039] The measurement of mitochondrial function can be a measurement of oxygen consumption. A Seahorse XF Cell Mio Stress Kit can be used to measure mitochondrial oxygen consumption.

[0040] The measurement of mitochondrial function can be a measurement of the mitochondrial membrane potential against an increased workload. Measuring the mitochondrial membrane potential against an increase workload can comprise the use of a detectable marker which can measure the membrane potential of a mitochondria.

[0041] The measurement of mitochondrial function can be a measurement of ATP production. A cytosolic ATP sensor GFP -variant can be used to measure ATP production. ATP production can be measured under aerobic or anaerobic conditions. An example of conditions under which ATP production can be measured include: 1 mM pyruvate + 0.05 mM palmitoyl-L-camitine + 10 mM a- ketoglutarate + 1 mM malate + lmM ADP + Luciferase. The enzyme Luciferase from the firefly Photinus pyralis can be used to generate a light signal in proportion to ATP concentration. ATP production can be normalized to citrate synthase activity, mitochondrial protein content, or mitochondrial DNA copy number. The threshold for normal, low, and improved mitochondria function can be different for each subject. An example of appropriate ATP production is -17 nmols/min/mg protein.

[0042] Assays to measure mitochondrial function can be calibrated to account for plasma membrane potential, cell size effects, or a combination thereof. In one example, the absolute value of mitochondrial membrane potential in individual cells is measured with and without an uncoupler in the absence of glucose. In some cases, a small depolarization in the presence of the uncoupler indicates greater functional capacity.

Modifications to the mitochondrial genome

[0043] Modifying the mtDNA can comprise introducing a mutation, a variant of an endogenous gene, or a transgene into the mtDNA sequence or can comprise introducing an extrachromosomal element, such as a plasmid, into the mitochondria. The modified mitochondrial genome can comprise the birth mtDNA sequence. The modified mitochondrial genome can comprise a modification of at least one gene in the modified mitochondrial genome. Genes in the mitochondria are exemplified in Table l.The modified mitochondria can have an increase in the functionality of the mitochondria compared to a majority of the endogenous mtDNA of the recipient. The modified mitochondria can have an increase in the functionality of the mitochondria compared to the birth mtDNA.

Table 1. Mitochondrial genes.

[0044] A modification can be a mutation, such as a substitution, insertion, deletion, or duplication relative to the mtDNA of the subject. The mutation can occur in a non-coding region of the mtDNA. The non-coding region can be the control region (D-loop). The mutation can occur in a coding region of a gene encoding a protein. A substitution occurring in a coding region can result in a synonymous amino acid substitution or a non-synonymous amino acid substitution. A deletion can be a deletion of a non coding region, for example, an intron. All non-coding regions can be removed. The deletion can be a deletion of a nuclease site in the mtDNA.

[0045] The modified mitochondrial genome can comprise a transgene. The transgene can be a mitochondrial gene or a nuclear gene. The transgene can be expressed in the cells of the subject after uptake of the modified mitochondria by the subject. The transgene can be a therapeutic gene.

[0046] The modified mitochondrial genome can comprise a mutation in at least one mitochondrial gene involved in energy production. Examples of mitochondrial genes involved in energy production include, but are not limited to, genes encoding an ATP synthase, coenzyme Q -cytochrome c reductase/ cytochrome b, and NADH dehydrogenase. The mutation in the at least one gene involved in energy production can result in a modified mitochondria more efficient at generating ATP compared to the endogenous mitochondria of the subject. The modified mitochondrial genome can comprise a transgene, wherein expression of the transgene results in more efficient ATP generation in the modified mitochondria compared to the endogenous mitochondria of the subject. The transgene can be a mitochondrial gene involved in energy production or a nuclear gene involved in mitochondrial energy production. Non-limiting examples of nuclear genes involved energy production include genes encoding a nuclear control of ATPase (NCA; NCA1, NCA2, NCA3), succinate dehydrogenase, and the transmembrane protein TMEM70.

[0047] The modified mitochondrial genome can comprise at least one selectable marker. The selectable marker can allow for in vivo selection or amplification of the modified mitochondria. The selectable marker can be a transgene. The transgene can be a gene conferring resistance to an antibiotic. The antibiotic can be chloramphenicol, ampicillin, tetracycline, geneticin, efrapeptin, or kanamycin. For example, the modified mitochondria can comprise a chloramphenicol acetyltransferase, wherein the chloramphenicol acetyltransferase is expressed in the mitochondria of the subject after administration of a mitochondrial preparation comprising the modified mitochondria, wherein the expression of chloramphenicol acetyltransferase provides protection against chloramphenicol.

[0048] The modified mitochondrial genome can comprise a mutation conferring increased stability or resistance to random mutations of the mtDNA. The modified mitochondrial genome can comprise a transgene conferring increased stability or resistance to random mutations of the mtDNA. The transgene conferring increased stability or resistance to random mutations of the mtDNA can be a gene encoding a free radical scavenger. Examples of free radical scavenger include, but are not limited to, antioxidant proteins such as oxidation resistance 1 (OXR1), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase (CAT), and thoredoxin reductase (TPx). The transgene conferring increased stability or resistance to random mutations can be a mtDNA polymerase (e.g. POLG).

[0049] The modified mitochondrial genome can comprise a mutation rendering the modified mitochondria resistant to elimination. The mutation can be a mutation in an enzyme encoded by the mtDNA. The mutation in the enzyme can be a mutation in the coding region of the enzyme. In some instances, the mutation does not alter the activity of the enzyme. The modified mitochondrial genome can comprise a transgene rendering the mitochondria resistant to elimination. Selection of the modified mitochondria can comprise the use of a small molecule drug or endonuclease to selectively eliminate endogenous mitochondria leaving the modified mitochondria.

[0050] The modified mitochondrial genome can comprise a sequence identical to the mitochondrial sequence of the subject at the time of the subject’s birth (the birth mtDNA sequence). For example, a birth mtDNA of a subject could be determined, subsequently a mitochondria from a matrilineal relative to the subject (i.e. donor) can be extracted and methods of modifying the donor mtDNA could be used to modify the sequence of the donor mtDNA into the sequence of the mtDNA of the subject. The sequence can be a haplotype. The modified mitochondrial genome can comprise a sequence identical to the sequence of the birth mtDNA. The modified mitochondrial genome can comprise a sequence at least 90%, at least 95%, or at least 99% similar to the mitochondrial genome sequence (the birth mtDNA sequence) of the subject.

[0051] Any suitable method of modifying the mitochondrial genome can be used. Modifying a mitochondrial genome can comprise the use of gene editing techniques to introduce a mutation or a transgene into the genome. Gene editing techniques include the use of a mitochondria-targeted transcription activator-like effector nuclease (mitoTALEN), a mitochondrial zinc finger nuclease (mitoZFN), or an endonuclease system capable of recognizing a clustered regularly interspaced short palindromic repeat (CRISPR). Homologous recombination can be used to introduce mutations into the genome of the mitochondria.

[0052] Upon gRNA association with the target mtDNA sequence and CRISPR/CRISPR associated endonuclease complex, the endonuclease can create a single or double strand DNA break upstream of the PAM sequence. The single or double strand DNA break can occur 3 or 4 base pairs upstream of the PAM sequence. CRISPR/CRISPR associated endonuclease systems can be used in pairs to remove a section of nucleotides from a given nucleic acid or they can be used to create targeted breaks in the mtDNA without the use of an additional CRISPR/CRISPR associated endonuclease pair to allow for insertion of an editing template, such as nucleic acid sequences encoding a mutant gene variant or a transgene. The CRISPR/CRISPR associated endonuclease system can be CRISPR/Cas9.

[0053] A vector can be used transduce a DNA sequence into the mitochondria. The vector can be a viral vector or a plasmid. The viral vector can be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector. The viral vector can comprise a capsid. The capsid can comprise a mitochondrial targeting sequence (MTS). Electroporation can be used to introduce a DNA sequence or a plasmid into a mitochondria.

[0054] In some instances, the DNA sequence does not integrate into the mitochondrial genome. The DNA sequence can be an editing template. The DNA sequence can integrate into the mitochondrial genome, for example, when used as an editing template for CRISPR/Cas9 gene editing. [0055] A mitochondrion comprising the modified mitochondrial genome can have enhanced mitochondrial function. The enhanced mitochondrial function can be enhanced compared to the mitochondrial function of the endogenous mitochondria of the individual. Non-limiting examples of enhanced mitochondrial function is increased ATP production, increased NADH oxidation, and decreased oxygen consumption. The mtDNA can be engineered to increase proliferative capacity.

Replication of mitochondria and the use of producer cell lines

[0056] This invention includes expanding or replicating mitochondria before or after selection for optimal function. The at least one mitochondria can be at least one mitochondria obtained from at least one cell from a sample, a synthetic mitochondria, or a donor mitochondria. The expanding of the at least one mitochondria can be in a cell culture or in at least one cell from the sample. Prior to expanding the at least one mitochondria, the cells of the cell culture or the at least one cell from the sample can be demitochondrified and subsequently remitochondrified with the at least one mitochondria or the mtDNA to be expanded.

[0057] The term“demitochondrifying” refers to removal of endogenous mitochondria or endogenous mtDNA from a cell. Demitochondrifying can comprise overexpression of regulators of mitophagy, such as SIRT1 or Parkin/PINKl. Demitochondrifying can comprise contacting mitochondria within the cells with a conjugate comprising a constituent taken up by mitochondria (such as triphenyl phosphonium) and a constituent that selectively causes mitophagy of mitochondria with reduced function (such as lower membrane potential), or that selectively promotes survival of mitochondrial with an increased function (such as higher membrane potential). Demitochondrifying can comprise expression of a mitochondrial targeted nuclease or nuclease activator. Examples of mitochondrial targeted nucleases or nuclease activators include, but are not limited to, UL12.5, EndoG, ExoG, ExoIII, and mUNGl . 100% of the endogenous mtDNA can be eliminated during the demitochondrifying. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the endogenous mtDNA can be eliminated during the demitochondrifying .

[0058] The term“remitochondrifying” refers to transfer of an exogenous mitochondria or transduction of an exogenous mtDNA into a demitochondrified cell. The exogenous mitochondria can be a donor mitochondria. The exogenous mtDNA can be a synthetic mtDNA. The synthetic mtDNA can be a birth mtDNA or an engineered mtDNA. The synthetic mtDNA can be a metabolically optimal mtDNA.

[0059] Examples of a donor from which a donor mitochondria can be obtained include, but are not limited to, a younger relative, an unrelated-third party donor, an archived personal biological sample (e.g., cord blood), or a combination thereof. The donor can be a matrilineal relative of the subject. The donor can be a different age from the subject (a heterochronic donor). The heterochronic donor can be younger than the subject. The heterochronic donor can be at least 5, 10, 15, 20, or 30 years younger than the donor. The mitochondria from the donor can be compatible with the mitochondria from the subject. The mitochondria in the mitochondrial preparation can be dominant over the endogenous mitochondria in the unhealthy cells of the subject. The mitochondria in the mitochondrial preparation can have an improved functional capability compared to endogenous mitochondria of the subject.

[0060] A vector can be used to transduce an mtDNA sequence into a mitochondria. The vector can be a viral vector or a plasmid. The viral vector can be a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector. The viral vector can comprise a capsid. The capsid can comprise a mitochondrial targeting sequence (MTS). Electroporation can be used to introduce a DNA sequence or a plasmid into mitochondria. The DNA sequence can be the birth mtDNA.

[0061] To transduce mtDNA in living cells, recombinant proteins can be used, such as a human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction (i.e. 11 arginines) domain and a mitochondrial localization signal (MLS). The recombinant proteins can bind mtDNA and rapidly transport the mtDNA across plasma membranes to mitochondria.

[0062] The method can further comprise addition of a transgene to the synthetic mtDNA sequence. The transgene can be a selectable marker, for example, antibiotic resistance. Such selectable marker can be used to maintain homoplasmy during production and after transplant. For example, if an mtDNA comprising a gene providing chloramphenicol resistance is transduced into a cell culture, applying chloramphenicol to the culture will eliminate cells not containing the mtDNA with the marker.

[0063] In order to enhance homoplasmic selection, the synthetic mtDNA can be specifically engineered to not be susceptible to nuclease degradation.

[0064] The mitochondria with reduced immunogenicity can have mtDNA representative of a birth mtDNA sequence of the subject. The birth mtDNA can be a synthetic mtDNA. The mitochondria with reduced immunogenicity can be a mitochondria isolated from a donor. The donor can be a maternally related relative. The donor can be a donor younger than the recipient. The mitochondria with reduced immunogenicity can have a metabolically optimal mtDNA. The metabolically optimal mtDNA can be a synthetic mtDNA. Synthetic mtDNA can be physically reconstructed using a plurality of

oligonucleotides.

[0065] The method can comprise reprogramming the at least one cell selected from the sample into an induced pluripotent stem cell (iPSC). The method can comprise reprogramming the at least one remitochondrified cell into at least one iPSC. In some cases, the at least one cell selected from the sample or the at least one remitochondrified cell is not reprogrammed into an iPSC.

[0066] At least 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, or 4% of the at least one cell selected from the sample or the at least one remitochondrified cell can be reprogrammed into iPSCs. In some cases, at least 0.1% of the at least one cell selected from the sample or the at least one remitochondrified cell can be reprogrammed into iPSCs. In some cases, at least 0.5% of the at least one cell selected from the sample or the at least one remitochondrified cell can be reprogrammed into iPSCs. In some cases, at least 1% of the at least one cell selected from the sample or the at least one remitochondrified cell can be reprogrammed into iPSCs. [0067] Reprogramming can comprise transfecting at least one vector comprising at least one transgene into the at least one cell selected from the sample or the at least one remitochondrified cell. The at least one transgene can encode at least one transcription factor. Examples of transcription factors include, but are not limited to, Oct4 (Octamer binding transcription factor-4), Sox2 (Sex determining region Y)-box 2, Klf4 (Kruppel Like Factor-4), c-Myc, Nanog, Lin28, L-Myc, Esrrb, C/EBRa, UTF1, and any combination thereof. One vector can encode more than one transcription factor. For example, a vector can comprise transgenes encoding Oct4, Sox2, Klf4, and c-Myc. In another example, a vector can comprise transgenes encoding Oct4, Sox2, Nanog, and Lin28. In another example, a vector can comprise transgenes encoding Lin28 and c-Myc. In some cases, one vector encodes one transcription factor. Reprogramming can comprise transfecting at least one RNA into the at least one cell selected from the sample or the at least one remitochondrified cell, wherein the at least one RNA can be used to silence expression of a target gene. The at least one RNA can be an small interfering RNA (siRNA) or a short hairpin RNA (shRNA). The target gene can be p53, Pax5, DNMT, or a combination thereof. In one example, the reprogramming can be carried out by administering to the one or more cells: (a) a first vector comprising transgenes encoding Oct4, Sox2, Klf4, and c-Myc; (b) a second vector comprising a transgene encoding UTF1; and (c) a p53 siRNA. The at least one transgene can be a SV40 large T antigen. The at least one transgene can be a human telomerase (hTRT). The at least one transgene can be a Wnt3a.

[0068] The at least one vector can be a viral vector, a transposon, a plasmid, an episome, or an RNA vector. The viral vector can be a lentivirus, a retrovirus, an adenovirus, or a Sendai virus. The transposon can be a PiggyBac transposase. The episome can be an oriP/EBNAl episome. The RNA vector can be an mRNA vector. The RNA vector can be a synthetic, self-replicating RNA vector. One example of a synthetic, self-replicating RNA vector is the Venezuelan equine encephalitis (VEE) RNA replicon encoding the transcription factors Oct4, Klf4, Sox2, and c-Myc. In some cases, the at least one vector is an integrating vector. In cases where the at least one vector is an integrating vector, a Cre recombinase can be used to excise the transgene from the transfected cells. In some cases, the at least one vector can be a non-integrating vector. The at least one non-integrating vector can be a self-replicating vector. The at one vector can be one, two, three, four, five, or more than five vectors.

[0069] In some cases, the method comprises differentiating the iPSCs. The iPSCs can be differentiated prior to the expanding. The iPSCs can be differentiated prior to purifying the mitochondria.

Differentiating the iPSCs can eliminate or reduce immunogenicity of the mitochondria. The iPSCs can be induced to differentiate into, for example, neuronal cells, hippocampal progenitors, dentate granule cell neurons, MGE progenitors, cortical intemeurons, dorsal cortical progenitors, excitatory cortical neurons, glial progenitors, astrocytes, neural crest stem cells, dopaminergic neurons, oligodendrocytes, dopaminergic neurons, hematopoietic cells, B-cells, T-cells, NK cells, granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes, platelets, cardiomyocytes, hepatocytes, skeletal muscle cells, adipocytes, pancreatic beta-cells, or cells from the ectoderm, mesoderm, or endoderm. The iPSCs can be grown as embryoid bodies. The iPSCs can be grown in the presence or absence of pro differentiation agents. Non-limiting examples of pro -differentiation agents include retinoic acid, growth factors, and components of the extracellular matrix (ECM). The iPSCs can be grown under hypoxic conditions and high pressure conditions to induce differentiation of the iPSCs.

[0070] The method can comprise expanding the at least one cell selected from the sample or the at least one remitochondrified cell to produce a plurality of expanded cells. The method can comprise expanding at least one iPSC to produce a plurality of expanded cells. The expanding can occur in a medium.

Proliferating and replicating can also be used to refer to expanding.

[0071] The medium can be a medium that supports stem cell growth. The medium can comprise hypoxic conditions. Hypoxic conditions can comprise about 1%, 2%, 3%, 4% or 5% oxygen. Hypoxic conditions can comprise the absence of oxygen. The medium can comprise minimal oxidative substrates. The medium can be a feeder free media. The medium can comprise fibroblast growth factors (FGF), epidermal growth factor (EGF), or a combination thereof. The expanding can occur in a bioreactor. The bioreactor can be a stirred suspension bioreactor. The expanding can occur in a multi-well plate.

[0072] The at least one cell selected from the sample or the at least one remitochondrified cell can be expanded in a cell culture. The cell culture can comprise the at least one cell from the sample. The cell culture can be an immortalized cell culture. For example, cells can be immortalized by transduction of hTERT; a viral gene such as EBV, HPV-16 E6/7, and SV40T; or a combination thereof. A plasmid or viral vector can be used for the transduction. Telomerase can be applied to the cell culture

[0073] The cell culture can be a production cell line. The production cell line can comprise cells which have been demitochondrified, and subsequently remitochondrified with a synthetic mtDNA or donor mitochondria. The production cell line can be a non-human production cell line. Non-human production cell lines include, but are not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells, or murine myeloma cells (NS0 and Sp2/0). The production cell line can be a human production cell line. Human production cell lines include, but are not limited to, HEK293, HT-1080, PER.C6, CAP, HKB-l 1, and HuH-7.

[0074] The cell culture can be a homoplasmic cell culture, in which the mitochondria of each cell in the cell culture are homoplasmic. A“homoplasmic” mitochondria, as the term is used in this disclosure, means that substantially all of the multiple copies of the mitochondrial genome enclosed in the single mitochondria being referred to are identical with each other. This means that in a given preparation of mitochondrial DNA, at least 95% (and preferably 97%, 99%, or 100%) of the DNA have the same sequence. A“homoplasmic” cell is a cell in which all of the mitochondria are homoplasmic and have the same mitochondrial genome. A“birth” mitochondrial genome sequence is a sequence of one of a plurality of mitochondrial genomes in a particular subject at the time of birth.

[0075] The cell culture can be a heteroplasmic cell culture. Heteroplasmy can occur in two manners: 1) at the cellular level, wherein cells contain mitochondria with different genomes; and 2) at the mitochondrial level, wherein each mitochondrion contains multiple genomes. [0076] Expanding the at least one iPSC can comprise serial passaging. Passaging can involve transferring an amount of a first cellular culture into a new culture to produce a second culture . Serial passaging can comprise at least two rounds of passaging. The serial passaging can produce iPSCs having reduced heteroplasmy. The iPSCs with reduced heteroplasmy can comprise iPSCs with a reduction in low functioning mitochondria compared to the iPSCs prior to serial passaging. Heteroplasmy can be reduced after serial passaging by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%.

[0077] The method can further comprising selecting cells from the plurality of expanded cells using a measurement of mitochondrial function. Selecting cells from the plurality of expanded cells can comprise the use of FACS.

[0078] In some cases, the expanding further comprises seeding the at least one cell selected from the sample, the at least one remitochondrified cell, or the at least one iPSC. The seeding can be a seeding of the at least one cell selected from the sample, the at least one remitochondrified cell, or the at least one iPSC further selected based on the measurement of mitochondrial function. The seeding can be done using FACS or the single-cell microscopic assay. The at least one cell selected from the sample, the at least one remitochondrified cell, or at least one iPSC can be seeded into a multi -well plate. Seeding can comprise placing an individual cell from the at least one cell selected from the sample, the at least one remitochondrified cell, or the at least one iPSC into each well of the multi-well plate. Seeding can comprise seeding the at least one cell selected from the sample, the at least one remitochondrified cell, or the at least one iPSC at a low density in each well of the multi -well plate. The multi -well plate can comprise 6, 24, 96, or 384 wells. Seeding can be done in a gel. The gel can be a polyacrylamide gel. The gel can be Matrigel®. The gel can comprise extracellular matrix components. Extracellular matrix components include, but are not limited to, heparin sulfate, chondroitin sulfate, keratin sulfate, hyaluronic acid, collagen, elastin, fibronectin, and laminin.

[0079] In some cases, the method comprises purifying the mitochondria from the plurality of expanded cells. Purifying the mitochondria can comprise homogenizing the plurality of expanded cells to produce a homogenate. Centrifuging the homogenate can produce a supernatant comprising the mitochondria and a precipitate comprising unbroken cells and cell debris. The supernatant can be isolated and further centrifugation of the supernatant can produce a precipitate comprising the purified mitochondria.

Isolation and purification of the mitochondria can comprise gradient centrifugation of the supernatant using a density media. The gradient centrifugation can be sucrose gradient centrifugation or Ficoll® density gradient centrifugation. Purifying the mitochondria can further comprise chromatography. In some cases, the homogenate is passed through a filter. The filter can be a double layer of wet muslin. In some cases, the purified mitochondria do not comprise a deleterious mutation.

[0080] In some cases, the method further comprises selecting mitochondria based on a measurement of mitochondrial function from the purified mitochondria. As previously described, measurements of mitochondrial function can include measurements of mitochondria density, mitochondrial membrane potential, production of reactive oxygen species (ROS), plasma membrane potential, mitochondrial volume fraction, ATP production, heteroplasmy, and a combination thereof. The purified high functioning mitochondria can be sorted using FACS based on a measurement of mitochondrial function. The sorting of the purified high functioning mitochondria can be used to isolate higher functioning purified mitochondria from lower functioning purified mitochondria.

[0081] In some cases, the method further comprises removing defective mitochondria. Removal of defective mitochondria can comprise conditional over-expression of Parkin or tor kinase inhibition. In some cases, the method further comprises inducing mitochondrial biogenesis. Mitochondrial biogenesis can be induced by over-expression of mitochondrial transcription factor A (TFAM), expression or addition of PGC-la, or addition of 5 -aminoimidazole -4 -carboxamide riboside (AICAR). Removing defective mitochondria or inducing mitochondrial biogenesis can occur during the reprogramming, the serial passaging, the expanding, or a combination thereof.

Permutations of steps of Autologous Mitochondrial Extraction and Expansion (AMEE)

[0082] The steps to produce a mitochondrial composition can be performed in a variety of orders, and optionally some steps can be repeated. In some cases, not all steps are performed. The examples provided in this disclosure are meant to be illustrative only and are not an exhaustive list of ways in which AMEE can be performed.

[0083] In one example, the method comprises: (a) obtaining a sample comprising cells from a subject;

(b) selecting at least one cell from the cells based on a measurement of mitochondrial function; (c) expanding the at least one cell produce a plurality of expanded cells; and (d) purifying mitochondria from expanded cells. In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one mitochondria from the cells based on a measurement of mitochondrial function; (c) expanding the at least one mitochondria in a cell culture; and (d) purifying mitochondria from the cell culture.

[0084] In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) purifying mitochondria from the cells of the sample; and (d) selecting mitochondria from the purified mitochondria based on a measurement of mitochondrial function.

[0085] In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one cell based on a measurement of mitochondrial function; and (c) purifying mitochondria from the at least one cell.

[0086] In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one cell from the cells based on a measurement of mitochondrial function;

(c) expanding the at least one cell produce a plurality of expanded cells; (d) purifying mitochondria from the expanded cells; and (e) selecting the purified mitochondria based on a measurement of mitochondrial function. In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one mitochondria from the cells based on a measurement of mitochondrial function; (c) expanding the at least one mitochondria in a cell culture; (d) purifying mitochondria the cell culture; and (e) selecting the purified mitochondria based on a measurement of mitochondrial function.

[0087] In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) selecting at least one cell from the cells based on a measurement of mitochondrial function; (c) expanding the at least one cell produce a plurality of expanded cells; (d) selecting at least one cell from the plurality of expanded cells based on a measurement of mitochondrial function; and (d) purifying mitochondria from the at least one cell.

[0088] In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) expanding the cells from the sample to produce a plurality of cells; (c) selecting from the plurality of cells at least one cell using a measurement of mitochondrial function; and (d) purifying mitochondria from the at least one cell. In another example, the method comprises: (a) obtaining a sample comprising cells from a subject; (b) expanding the cells from the sample to produce a plurality of cells; (c) purifying mitochondria from the plurality of cells; and (d) selecting at least one mitochondria from the plurality of mitochondria using a measurement of mitochondrial function.

Pharmaceutical compositions

[0089] Pharmaceutical compositions of this invention can contain mitochondria produced according to any of the methods of this invention.

[0090] In some cases, the mitochondria in the mitochondrial preparation are packaged within liposomes. The mitochondria in mitochondrial preparation can be taken up by cells of the subject. The mitochondria in the mitochondrial preparation can enhance the properties of the cells of the subject, thereby preventing, halting, or reversing pathologies arising from reduced endogenous mitochondrial function. Since mitochondria can cross the blood-brain barrier in vivo, a composition can be administered to a subject to improve cognitive function or other neurodegenerative conditions.

[0091] The compositions can further comprise a pharmaceutically compatible excipient. Examples of excipients or carriers include, but are not limited to solubilizers, antioxidants, buffering agents, pH adjusting agents, co-solvents, chelating agents, stabilizers, preservatives, lubricants, tonicity adjusting agents, or a combination thereof.

[0092] The composition can further comprise at least one additional agent. The additional agent can be an antibody, a peptide, a protein, a nucleic acid, an enzyme, a small molecule drug, a vector encoding a transgene, or a combination thereof. The additional agent can be an additional therapeutic agent.

[0093] The additional agent can be an agent promoting the uptake of the mitochondria in the mitochondrial preparation by the cells of the subject. The cells of the subject can be a specific cell type. For example, the agent can target cells of the heart, brain, intestine, liver, kidney, or muscle. In some embodiments, the agent promoting uptake of the mitochondria by cells of the subject is a cell -penetrating peptide (CPP). Non-limiting examples of CPPs include transactivator of transcription (Tat) peptides, penetratin, transportan (TP), an arginine rich peptide, MPG, Pep-l, and variants thereof. In some embodiments, the CPP is a derived from a pathogen, such as a bacteria or a virus. Virally derived CPP can be derived from a virus in the genus Flavivirus. Bacterially derived CPP can derived from a bacteria in the genus Yersinia or Listeria. The CPP can be expressed on the liposome.

[0094] The additional agent can be an agent decreasing the immune response of the subject to the mitochondria in the composition. For example, a liposome comprising polyethylene glycol (PEG) can show a reduced immune response in the subject. In another example, anti-ICAMl or anti-LFAl can be used to decrease the immune response in the subject to the mitochondria in the composition.

[0095] The additional agent can be an agent to stimulate mitochondrial biogenesis. Examples of agents to stimulate mitochondrial biogenesis include, but are not limited to, mitochondrial transcription factor A (TFAM), peroxisome proliferator-activated receptor gamma coactivator l-alpha (PGC-la), or a combination thereof. The agent to stimulate mitochondrial biogenesis can be a vector comprising a transgene encoding the agent. Alternatively, the agent to stimulate mitochondrial biogenesis can be a protein.

[0096] Preparation and formulation of pharmaceutical agents for use according to this invention can incorporate standard technology, as described, for example, in the current edition oΐ Remington: The Science and Practice of Pharmacy. The formulation will typically be optimized for administration to the target tissue, for example, by local administration, in a manner that enhances access of the active agent to the target senolytic cells and providing the optimal duration of effect, while minimizing side effects or exposure to tissues that are not involved in the condition being treated.

[0097] This invention provides commercial products that are kits that enclose unit doses of one or more of the agents or compositions described in this disclosure. Such kits typically comprise a pharmaceutical preparation in one or more containers. The preparations may be provided as one or more unit doses (either combined or separate). The kit may contain a device such as a syringe for administration of the agent or composition in or around the target tissue of a subject in need thereof. The product may also contain or be accompanied by an informational package insert describing the use and attendant benefits of the medicaments in treating the senescent cell associated condition, and optionally an appliance or device for delivery of the composition.

Methods of Treatment

[0098] Compositions can be administered to a subject with a condition. The composition can be administered to a subject to prevent the condition. The composition can be administered to reduce the effects of the condition.

[0099] In some cases, the method of treating a subject comprises: a. obtaining a sample comprising cells from the subject; b. selecting at least one cell from the cells based on a measurement of mitochondrial function; c. expanding the at least one cell produce a plurality of expanded cells; d. purifying mitochondria from the expanded cells; and e. administering the purified mitochondria to the subject. [0100] In some cases, the method of treating a subject comprises: a. obtaining a sample comprising cells from a subject; b. selecting at least one mitochondria from the cells based on a measurement of mitochondrial function; c. expanding the at least one mitochondria in a cell culture; d. purifying mitochondria from the cell culture; and e. administering the purified mitochondria to the subject.

[0101] The condition can be an age-related disorder, a senescence associated disorder, or a condition caused by a mutation in the mtDNA. Conditions caused by mutations in the mtDNA include, but are not limited to, Keams-Sayre syndrome, chronic progressive external ophthalmoplegia (CPEO); Pearson syndrome; mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS);

myoclonic epilepsy with ragged red fibers (MERRF); neuropathy, ataxia, and retinitis pigmentosa (NARP); maternally inherited Leigh syndrome (MILS); maternally inherited diabetes and deafness (MIDD); Leber’s hereditary optic neuropathy (LHON); myopathy; sensorineural hearing loss; exercise intolerance; rhabdomyolysis; and Leigh/Leigh-like syndrome. Mutations can be in the one or more mtDNA genes, such as TRNL1, ND1, ND2, ND4, ND5, ND6, TRNE, TRNK, TRNS1, CYB, ATP 6, and RNR1.

[0102] The compositions can be delivered either via local injection into a tissue or intravenously to access numerous body tissues. In some embodiments, the composition is placed in a device to facilitate administration. The device can be a syringe.

[0103] The composition can be administered locally. Local routes of administration include, without limitation, local injection, intracranial, intracerebroventricular, intracerebral, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracistemal, intraperitoneal, intranasal, inhalation, or topical administration. The composition can be administered systemically. Systemic routes of administration include, without limitation, injection or infusion performed intravenously, intra-atrially, intramuscularly, or subcutaneously.

[0104] The composition can be administered once or more than once. In some embodiments, the composition is administered 1, 2, 3, 4, or at least 5 times a day. In some embodiments, the composition is administered at least once a week for 1, 2, 3, 4, or at least 5 weeks.

[0105] The composition can be packaged as unit doses effective for treatment of one or more conditions. The composition can be packaged with or accompanied by information about its use in clinical medicine.

[0106] The composition can be stored at about -20°C, about 4°C, or at room temperature. The medicament or composition can be stored for 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before administration to a subject.

Terminology

[0107] As used herein and in the appended claims, the singular forms“a”,“an”, and“the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as“solely,”“only” and the like in connection with the recitation of claim elements, or use of a“negative” limitation.

[0108] Certain ranges are presented herein with numerical values being preceded by the term“about.” The term“about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating un-recited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions described herein are. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions described herein, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions described herein.

[0109] The terms“individual,”“patient,” or“subject” are used interchangeably. None of the terms require or are limited to situation characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly, or a hospice worker). Further, these terms refer to human or animal subjects.

[0110] “Treating” or“treatment” refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) a targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder, as well as those prone to have the disorder, or those in whom the disorder is to be prevented. For example, a subject or mammal is successfully“treated” for cancer, if, after receiving a therapeutic amount of a subject oligonucleotide conjugate according to this invention, the subject shows observable and/or measurable reduction in or absence of one or more of the following: reduction in the number of cancer cells or absence of the cancer cells; reduction in the tumor size; inhibition (i.e., slowing to some extent and preferably stopping) of cancer cell infiltration into peripheral organs, including the spread of cancer into soft tissue and bone; inhibition (i.e., slowing to some extent and preferably stopping) of tumor metastasis; inhibition, to some extent, of tumor growth; and/or relief to some extent of one or more of the symptoms associated with the specific cancer; reduced morbidity and/or mortality, and improvement in quality of life issues.

References

[0111] The following publications are referred to in the detailed description of this invention.

• Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Primer 2, 16080 (2016). • Clark, M. A. & Shay, J. W. Mitochondrial transformation of mammalian cells. Nature 295, 605-607 (1982).

• Kesner, E. E., Saada-Reich, A. & Lorberboum-Galski, H. Characteristics of Mitochondrial Transformation into Human Cells. Sci. Rep. 6, srep26057 (2016).

• Shi, X., Zhao, M., Fu, C. & Fu, A. Intravenous administration of mitochondria for treating experimental Parkinson’s disease. Mitochondrion 34, 91-100 (2017).

• Bacman, S. R., Williams, S. L., Pinto, M. & Moraes, C. T. The Use of Mitochondria- Targeted Endonucleases to Manipulate mtDNA. Methods Enzymol. 547, 373-397 (2014).

• Induction of pluripotent stem cells from adult human fibroblasts by defined factors. - PubMed - NCBI. 18035408. (Accessed: 8th July 2017)

• Dimri, G., Band, H. & Band, V. Mammary epithelial cell transformation: insights from cell culture and mouse models. Breast Cancer Res. BCR 7, 171-179 (2005).

• Suen, D.-F., Narendra, D. P., Tanaka, A., Manfredi, G. & Youle, R. J. Parkin overexpression selects against a deleterious mtDNA mutation in heteroplasmic cybrid cells. Proc. Natl. Acad. Sci. 107, 11835-11840 (2010).

• Scarpulla, R. C. Metabolic control of mitochondrial biogenesis through the PGC-l family regulatory network. Biochim. Biophys. Acta 1813, 1269-1278 (2011).

• Chang, J.-C. et al. Peptide -mediated delivery of donor mitochondria improves mitochondrial function and cell viability in human cybrid cells with the MELAS A3243G mutation. Sci. Rep. 7, 10710 (2017).

• Taylor, R. W. & Turnbull, D. M. Mitochondrial DNA mutations in human disease. Nat. Rev.

Genet. 6, 389-402 (2005).

[0112] The following examples provide non-limiting illustrations of certain aspects of the invention.

EXAMPLES

Example 1: Method for producing a mitochondrial composition comprising FACS before reprogramming.

[0113] A mitochondrial composition is produced using the following method:

• Step 1 : A tissue sample is taken from a patient.

• Step 2: Isolating a subset of cells from the tissue sample.

• Step 3: Using FACS to isolate cells from the subset of cells which contain high functioning mitochondria.

• Step 4: Reprogramming the cells with high functioning mitochondria isolated from step 3 into induced pluripotent stem cells (iPSCs) and serial passaging the iPSCs.

• Step 5: Mitochondria are extracted from these serial passaged iPSCs produce a mitochondria composition. Example 2: Method for producing a mitochondrial composition comprising FACS before reprogramming and after serial passaging.

[0114] A mitochondrial composition is produced using the following method:

• Step 1 : A tissue sample is taken from a patient.

• Step 2: Isolating a subset of cells from the tissue sample.

• Step 3: Using FACS to isolate cells from the subset of cells which contain high functioning mitochondria.

• Step 4: Reprogramming the cells with high functioning mitochondria isolated from step 3 into induced pluripotent stem cells (iPSCs) and serial passaging the iPSCs.

• Step 5: Using FACS to isolate iPSCs from the serial passaged iPSCs which contain high

functioning mitochondria.

• Step 6: Mitochondria are extracted from these serial passaged iPSCs which contain high

functioning mitochondria to produce a mitochondria composition.

Example 3: Method for producing a mitochondrial composition comprising FACS after serial passaging and after mitochondrial extraction.

[0115] A mitochondrial composition is produced using the following method:

• Step 1 : A tissue sample is taken from a patient.

• Step 2: Isolating a subset of cells from the tissue sample.

• Step 3: Reprogramming the cells with high functioning mitochondria isolated from step 3 into induced pluripotent stem cells (iPSCs) and serial passaging the iPSCs.

• Step 4: Using FACS to isolate iPSCs from the serial passaged iPSCs which contain high

functioning mitochondria.

• Step 5: Mitochondria are extracted from these serial passaged iPSCs which contain high

functioning mitochondria.

• Step 6: Using FACS to isolate higher functioning mitochondrial from the mitochondria extracted from the serial passaged iPSCs containing high functioning mitochondria in step 5 to produce a mitochondria composition.

Example 4: Method for producing a mitochondrial composition comprising a patient’s birth mtDNA sequence.

[0116] A mitochondrial composition is produced using the following method:

• Step 1 : A tissue sample is taken from a patient.

• Step 2: Determining the birth mtDNA sequence of the patient.

• Step 3: Using PCR-OE to assemble a synthetic mtDNA sequence representative of the birth mtDNA of the patent • Step 4: Removing the endogenous mtDNA from a HEK293 cell culture line and transducing the synthetic mtDNA assembled in step 3 into the demitochondrified HEK293 cells.

• Step 5: Expanding the cell culture.

• Step 6: Mitochondria are extracted from the expanded cell culture to produce a mitochondrial composition.

Example 5: Treatment of an individual at risk of developing Parkinson’s disease using AMEE to develop a personalized therapeutic

[0117] A blood sample from a 40 year old woman at risk of developing Parkinson’s disease is taken. From this blood sample, fibroblasts are isolated. Two oriP/EBNA episomes are transfected into fibroblasts isolated from the woman in hypoxic conditions. One episome contains the transgenes Oct4, Sox2, Nanog, and Klf4 while the other episome contains the transgenes c-Myc and Lin28. About 15 days after transfection, about .01% of fibroblasts are reprogrammed into iPSCs.

[0118] The iPSCs are serial passaged for 40 cycles and fluorescence automated cell sorting (FACS) is used to isolate the iPSCs with high functioning mitochondria. iPSCs with high functioning mitochondria are determined to be those that: 1) contain mitochondria high membrane potential, as determined using Rhodamine 123, 2) contain a high plasma membrane potential, as determined using FluVolt™ , and 3) contain a large mitochondrial volume fraction, as determined using nonyl acridine orange (NAO).

[0119] The iPSCs with high functioning mitochondria are homogenized and the high functioning mitochondria are purified. The purified mitochondria are subjected to another round of FACS sorting, to further isolate the highest functioning mitochondria based on the earlier used tests. The resulting isolated mitochondria are formulated into a pharmaceutical composition and intravenously administered to the woman.

[0120] While preferred embodiments of the invention have been shown and described, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.