WO2019060297A1 - Mitochondrial rejuvenation as a treatment for adverse age-related conditions and neurodegeneration - Google Patents

Abstract

Described herein are methods for mitochondrial transplantation wherein old, damaged, or mutated mitochondria are replaced with young, undamaged, or non-mutated mitochondria. The old, damaged, or mutated mitochondria can also be replaced with mitochondria comprising a desirable mutation or transgene or mitochondria comprising the birth mtDNA sequence. Substituting high functioning mitochondria into cells for low functioning mitochondria can improve function and help resolve conditions caused or propagated by mitochondrial defects that can be caused by aging.

Description

MITOCHONDRIAL REJUVENATION AS A TREATMENT FOR ADVERSE AGE- RELATED CONDITIONS AND NEURODEGENERATION

CROSS-REFERENCE

[0001] This application claims the priority benefit of U.S. provisional application 62/560,418, filed Sept. 19, 2017, which is hereby incorporated herein in its entirety for all purposes.

BACKGROUND OF THE INVENTION

[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 (Karnkowska et al. 2016).

[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 modern 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-sulfur cluster assembly, calcium regulation, and control of the NAD/NADH flux. Mutations in the mitochondrial genome can lead to dysfunction in any of these processes.

[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.

[0006] Additionally, mitochondrial mutations can also be inherited directly from one's mother (mitochondria are passed matrilineally) or can occur spontaneously at fertilization. Such inherited conditions include mitochondrial myopathy, Leber's hereditary optic neuropathy, Leigh syndrome, Neuropathy, ataxia, retinitis pigmentosa, myoneurogenic gastrointestinal

encephalopathy, myoclonic epilepsy with ragged red fiber, mitochondrial myopathy, and encephalomyopathy. But beyond these grievous illnesses, it has been suggested that gradual accumulation of mitochondrial mutations that occurs during the course of a human lifetime can contribute to numerous features of aging, including atherosclerosis, cardiovascular disease, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, Alzheimer's disease, sarcopenia, frailty, hair loss, hair graying, and presbycusis (hearing loss).

[0007] Mitochondrial dysfunction has also been detected in cancer cells, as part of the Warburg effect (i.e. a metabolic state in which cancer cells rely on glycolysis and thus show a low rate of mitochondrially-mediated pyruvate oxidation). Thus mtDNA mutations may have a role in cancer.

[0008] Artificial transfer of purified mitochondria into mammalian cells was first 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.

[0009] 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.

[0010] 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.

[0011] 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. [0012] The therapeutic potential of mitochondrial transplantation is promising, but numerous obstacles must be overcome before utilization of this therapy can be realized to its fullest potential. These barriers include how to reduce the immunogenicity of transplanted

mitochondria, determination of a metabolically optimal mitochondrial DNA sequence, physical reconstruction of a synthetic mitochondrial genome representative of the metabolically optimal mitochondrial DNA sequence, genomic engineering of mitochondrial genome for optimal function, creation of a cell line that contains a single mitochondrial genome (i.e., a cell line that is homoplasmic for a single mitochondrial genome), and industrial-scale production and purification of the homoplasmic mitochondria.

SUMMARY OF THE INVENTION

[0013] Disclosed herein, in certain embodiments, are methods for producing a mitochondrial preparation comprising: (a) obtaining a cell culture; (b) introducing a mitochondria having reduced immunogenicity into the cell culture; (c) expanding the cell culture; and (d) isolating and purifying the mitochondria having reduced immunogenicity to produce the mitochondrial preparation.

[0014] Disclosed herein, in certain embodiments, are methods of generating a homoplasmic cell culture, comprising: (a) obtaining a cell culture comprising a plurality of cells, where each of the plurality of cells comprises at least one endogenous mitochondria, and wherein each of the at least one endogenous mitochondria comprises an endogenous mitochondrial DNA (mtDNA); (b) eliminating the endogenous mtDNA in the plurality of cells in the cell culture; and (c) transducing a target mtDNA into the plurality of cells to produce a homoplasmic cell culture.

[0015] Disclosed herein, in certain embodiments, are methods for producing a mitochondrial preparation comprising: (a) obtaining a homoplasmic cell culture; (b) expanding the

homoplasmic cell culture; and (c) isolating and purifying mitochondria from the cell culture to produce a mitochondrial preparation.

[0016] Disclosed herein, in certain embodiments, are methods of determining a metabolically optimal mtDNA sequence of an individual comprising: (a) isolating a mitochondrial DNA (mtDNA) from a biological sample from the individual; (b) sequencing the isolated mtDNA to generate a plurality of reads; (c) assembling the plurality of reads based on a consensus mtDNA sequence to produce an mtDNA sequence comprising variant plurality of variants, wherein the plurality of variants comprises at least one mtDNA sequence variant present in the individual from birth; (d) determining the frequency of each of the plurality of variants; and (e)

determining the metabolically optimal mtDNA sequence of the individual based on the frequency of the plurality of variants wherein the metabolically optimal mtDNA sequence comprises the at least one mtDNA sequence variant present in the individual from birth.

[0017] Disclosed herein, in certain embodiments, are libraries comprising a plurality of oligonucleotides, wherein each oligonucleotide comprises: (a) a nucleic acid sequence homologous to a portion of a mitochondrial genome; and (b) at least one nucleic acid variant relative to the mitochondrial genome wherein the oligonucleotide represents a mitochondrial haplotype or a portion of a mitochondrial haplotype; wherein the plurality of oligonucleotides comprises at least one oligonucleotide for each known haplotype in the mitochondrial genome.

[0018] Disclosed herein, in certain embodiments, are methods of physically reconstructing a synthetic mitochondrial genome of an individual, comprising: (a) obtaining a metabolically optimal mitochondrial DNA (mtDNA) sequence of the individual comprising at least one mtDNA sequence variant present in the individual from birth; (b) selecting a subset of oligonucleotides, from a library comprising a plurality of oligonucleotides, wherein each oligonucleotide in the subset of oligonucleotides represents a portion of the metabolically optimal mtDNA sequence obtained in step (a) and the plurality of oligonucleotides represents the entirety of the metabolically optimal mtDNA sequence; and (c) physically assembling the plurality of oligonucleotides into the synthetic mitochondrial genome.

INCORPORATION BY REFERENCE

[0019] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative

embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0021] FIG. 1 illustrates an exemplary procedure to generate, propagate, and administer to an individual mitochondria comprising the birth mitochondrial DNA sequence of the individual. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present disclosure contemplates various wavs for solving problems in the design, production, purification, storage, stabilizations, development, and clinical use of non- immunogenic mitochondria.

Reducing Immunogenicity

[0023] Over time, mitochondrial DNA (mtDNA) can accumulate mutations, resulting in reduced mitochondria function. While exogenous mitochondria with increased function can be transplanted into cells showing reduced mitochondria function, this can result in a immunogenic reaction from the recipient's immune system due to differences between the recipient's natural mtDNA gene sequences (i.e., the mtDNA sequences the recipient was born with) and the mtDNA gene sequence of the mitochondria that are being transplanted into the recipient. It is therefore desirable to reduce or eliminate the immunogenicity of the transplanted mitochondria in order to maximize the therapeutic potential of mitochondrial transplantation. In some embodiments, the mitochondria in the compositions and methods described herein have reduced immunogenicity.

[0024] In some embodiments, the mitochondria are selected or modified for reduced

immunogenicity in an individual. The mitochondria having reduced immunogenicity in the individual can be mitochondria with a specific haplotype. The specific haplotype can be the haplotype of the individual at birth (birth mtDNA). In some embodiments, gene editing techniques are used to produce mitochondria with reduced immunogenicity in an individual. In some embodiments, automated genome assembly is used to produce mitochondria with reduced immunogenicity in an individual.

[0025] In some embodiments, the mitochondria has reduced immunogenicity in a plurality of individuals. In some embodiments, reduced immunogenicity in a plurality of individuals indicates that at least 50%, at least 60%>, at least 70%, at least 80%>, at least 90%, at least 95%, or at least 99% of the plurality of individuals would not show an immune response when administered the mitochondria. The mitochondria having reduced immunogenicity in a plurality of individuals can be mitochondria with a specific haplotype. In some embodiments, a mitochondria having reduced immunogenicity in a plurality of individuals is referred to as a "universal donor mitochondria." In some embodiments, gene editing techniques are used to produce mitochondria with reduced immunogenicity in a plurality of individuals. In some embodiments, automated genome assembly is used to produce mitochondria with reduced immunogenicity in a plurality of individuals. Determination of a metabolically optimal mitochondrial sequence

[0026] Disclosed herein, in certain embodiments, are methods of determining a metabolically optimal mtDNA sequence of an individual. In some embodiments, the method of determining the metabolically optimal mtDNA sequence of an individual comprises: (a) isolating a mitochondrial DNA (mtDNA) from a biological sample from the individual; (b) sequencing the isolated mtDNA to generate a plurality of reads; (c) assembling the plurality of reads based on a consensus mtDNA sequence to produce an mtDNA sequence comprising a plurality of variants, wherein the plurality of variants comprises at least one mtDNA sequence variant present in the individual from birth; (d) determining the frequency of each of the plurality of variants; and (e) determining the metabolically optimal mtDNA sequence of the individual based on the frequency of the plurality of variants wherein the metabolically optimal mtDNA sequence comprise the at least one mtDNA sequence variant present in the individual from birth.

[0027] In some embodiments, the metabolically optimal mitochondrial DNA (mtDNA) sequence is the mtDNA sequence present in the individual at birth (i.e. the birth mtDNA sequence). In some embodiments, the metabolically optimal mtDNA sequence is the mtDNA sequence of all protein coding regions present in the individual from birth. In some

embodiments, the metabolically optimal mtDNA sequence comprises a portion of the mtDNA sequence present in the individual at birth. In some embodiments, the at least one mtDNA sequence variant present in the individual from birth is a metabolically optimal mtDNA sequence variant. In some embodiments, the metabolically optimal mtDNA sequence variant is a variant in a gene encoding a protein. For example, the at least one metabolically optimal mtDNA sequence variant can be a non-synonymous mutation a gene encoding a protein. A

mitochondrion comprising the metabolically optimal mtDNA sequence 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.

[0028] Mitochondrial DNA can be heteroplastic, wherein more than one mitochondrial genome is present in a subject, within a cell, or even within a single mitochondrion. Deep sequencing of a biological sample from the subject (i.e., performing multiple redundant reads of the same sequence space) can produce mitochondrial sequence data representative of a plurality of mitochondrial genomes. The mitochondrial sequence data can comprise a plurality of single nucleotide polymorphisms (SNPs). The mitochondrial sequence data can be used to reconstruct a plurality of haplotypes within the individual. [0029] The birth mtDNA sequence of the subject can be determined by sequencing the mtDNA of cells from a biological sample from the subject. The biological sample can comprise a germ cell sample or a somatic cell sample. The somatic cell sample can be a blood, saliva, or urine sample.

[0030] The birth mtDNA sequence of the subject can be determined by sequencing the mtDNA of cells from a biological sample from a related family member of the subject. Comparing the mtDNA sequence of the subject to the mtDNA sequence of the related family member can identify mutations in the mtDNA of the subject relative to their original mtDNA sequence. Comparing the mtDNA sequence of the subject to the mtDNA sequence of the related family member can identify mutations in the mtDNA of the subject that occurred spontaneously at fertilization. In some embodiments, the related family member is a matrilineally related family member.

[0031] In some embodiments, the method comprises isolating mitochondrial nucleic acid. The mitochondrial nucleic acid can be mitochondrial DNA (mtDNA) or mitochondrial RNA (mtRNA). In some embodiments, the mtDNA sequence is complementary mtDNA produced by reverse transcribing the mtRNA. Determining the birth mitochondrial DNA sequence can comprise sequencing the exome or a whole genome of the mitochondria. Determining the birth mitochondrial DNA sequence can comprise determining a plurality of SNPs or a plurality of haplotypes present in the mitochondrial genome.

[0032] In some embodiments, mtDNA is isolated from a total DNA sample (a sample comprising mtDNA and nuclear DNA). Any suitable method can be used to isolate the mtDNA or mtRNA, including the use of commercial kits. In some embodiments, isolating mtDNA from nuclear DNA comprises: a) obtaining a solution comprising mtDNA and nuclear DNA, and b) using density-gradient ultracentrifugation to separate the mtDNA from the nuclear DNA. The density-gradient ultracentrifugation can be a cesium chloride density-gradient

ultracentrifugation. In some embodiments, isolating mtDNA from nuclear DNA comprises: a) obtaining a solution comprising mtDNA and nuclear DNA, b) digesting nuclear DNA with DNAse to produce a nuclear DNA free solution, lysing mitochondria in the solution to release mtDNA, and c) removal of protein from the solution to produce a substantially pure mtDNA extract.

[0033] In some embodiments, sequencing further comprises amplifying the mtDNA after isolation and prior to sequencing. In some embodiments, whole genome amplification (WGA) is used to amplify the mtDNA or complementary mtDNA prior to sequencing. In some

embodiments, mtDNA is amplified in combination with the nuclear DNA in a total DNA sample. In some embodiments, the mtDNA is selectively amplified from a total DNA sample. For example, a REPLI-g Mitochondrial DNA Kit (Qiagen) can be used to selectively amplify mtDNA from a total DNA sample without the need for mtDNA isolation.

[0034] In some embodiments, sequencing does not comprise amplifying the mtDNA after isolation and prior to sequencing. For example, if a particular isolation method produces a substantially pure mtDNA extract in an amount sufficient for sequencing, no amplification step is necessary. Not amplifying the mtDNA prior to sequencing can reduce the amount of sequencing error.

[0035] In some embodiments, determining the birth mtDNA sequence comprises sequencing the mitochondrial genome using a sequencing technology. Examples of sequencing technologies include Sanger sequencing and next generation sequencing (NGS). Examples of next generation sequencing include, but are not limited to, massively-parallel signature sequencing,

pyrosequencing (e.g., using a Roche 454 sequencing device), Ulumina (Solexa) sequencing, sequencing by synthesis (Dlumina), Ion torrent sequencing, sequencing by ligation (e.g., SOLiD sequencing), single molecule real-time (SMRT) sequencing (e.g., Pacific Bioscience), polony sequencing, DNA nanoball sequencing, heliscope single molecule sequencing (Helicos

Biosciences), and nanopore sequencing (e.g., Oxford Nanopore).

[0036] Computational assembly of the plurality of reads can comprise assembly of the plurality of reads relative to a mitochondrial reference sequence. Any suitable mitochondrial reference sequence can be used. Non-limiting examples of mitochondrial reference sequences include the Cambridge Reference Sequence (CRS) and the Reconstructed Sapiens Reference Sequence (RSRS). In some embodiments, computational assembly of the plurality of reads comprises generating a consensus sequence from the plurality of reads without comparison to a

mitochondrial reference sequence. Noise can be introduced through errors in copying

(amplification errors) or determining a nucleic acid identity during sequencing (sequencing errors). Prior to computational assembly of the plurality of reads, reads comprising amplification or sequencing errors can be eliminated. For example, sequences can have a per-base sequencing error rate of 0.5-1%. As a non-limiting example, sequence variants whose frequency is less than the sequencing error rate are determined to be errors and are eliminated. A threshold frequency can be used to eliminate reads. In some embodiments, any of the plurality of reads comprising a sequence variant whose frequency is less a threshold frequency can be eliminated. The threshold frequency can be about 1%, about 5%, about 10%, about 15%, or about 20%. A frequency from known reference sample can be used to set a threshold frequency. The known reference sample can be the Human Mitochondrial Genome Database (mtDB). The threshold frequency can be different for different variants. [0037] Sequencing the mitochondrial genome can produce a plurality of sequence reads. Each of the plurality of sequence reads can be at least 100 base pairs (bp), at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, or at least 1000 bp in length. The plurality of sequence reads can comprise paired end reads. The plurality of sequence reads can be generated using a plurality of sequencing technologies. For example, half of the plurality of reads can comprise paired end reads of approximately 150bp in length generated by Illumina sequencing and the other half of the plurality of reads can comprise Sanger sequence reads approximately 700 bp in length.

[0038] In some embodiments, coverage, or depth, in sequencing is the number of reads that include a given nucleotide in the computationally assembled sequence. Coverage of a given nucleotide in the mtDNA sequence can be at least 5, at least 10, at least 20, at least 50, at least 100, at least 1000, or at least 10,000.

[0039] In some embodiments, a variant is a sequence variant. Sequence variants present in the plurality of reads can comprise SNPs or haplotypes. In some embodiments, the sequence variant with majority frequency is determined to be the sequence variant present in the birth mtDNA sequence. The haplotype frequencies of each of the plurality of haplotypes can be used to determine the sequence of the birth mtDNA. The birth mtDNA can comprise the most frequent haplotypes. For example, in a particular mtDNA region, if haplotype A (for example,

ATTGCCC) is represented at 75%, haplotype B (for example, TTTCCCG) at 20%, and haplotype C (for example, TTTGCCG) at 5%, the birth haplotype is determined to be haplotype A. Single nucleotide polymorphism (SNP) frequencies can also be used to determine the sequence of the birth mtDNA. The birth mtDNA can comprise the most SNPs. For example, in a particular mtDNA nucleic acid position, if C (cysteine) is represented at 75%, A (adenine) at 20%), G (guanine) at 2.5%, and T (thymine) at 2.5%, the nucleic acid at that position in the birth mtDNA sequence is determine to be C. In some embodiments, the sequence variant with minority frequency is determined to be the sequence variant present in the birth mtDNA sequence. For example, in a subject suspected of having an mtDNA sequence mutation having occurred shortly after conception, the birth mtDNA sequence can comprise the minor frequency sequence variants identified during sequencing.

Physical reconstruction of a synthetic mitochondrial DNA

[0040] Disclosed herein, in certain embodiments, are libraries comprising a plurality of oligonucleotides. In some embodiments, each oligonucleotide of the plurality of

oligonucleotides comprises: (a) a nucleic acid sequence homologous to a portion of a

mitochondrial genome; and (b) at least one nucleic acid variant relative to the mitochondrial genome wherein the oligonucleotide represents a mitochondrial haplotype or a portion of a mitochondrial haplotype; wherein the plurality of oligonucleotides comprises at least one oligonucleotide for each known haplotype in the mitochondrial genome.

[0041] Further disclosed herein, in certain embodiments, are methods of physically

reconstructing a synthetic mitochondrial genome functionally identical to a mitochondrial genome present at the time the individual was born. In some embodiments, the method of physically reconstructing a synthetic mitochondrial genome comprises: (a) obtaining a metabolically optimal mtDNA sequence of an individual comprising at least one mtDNA sequence variant present in the individual from birth; (b) selecting a subset of oligonucleotides, from a library comprising a plurality of oligonucleotides, wherein each oligonucleotide in the subset of oligonucleotides represents a portion of the metabolically optimal mtDNA DNA sequence obtained in step (a) and the plurality of oligonucleotides represents the entirety of the metabolically optimal mitochondrial DNA sequence; and (c) physically assembling the plurality of oligonucleotides into the synthetic mitochondrial genome.

[0042] A library can comprise at least 1000, at least 2000, at least 3000, at least 4000, or at least 5000 oligonucleotides. The plurality of oligonucleotides in the library can comprise at least one oligonucleotide for each variant in the mitochondrial genome with a population frequency above about 0.5%, about 1%, about 5%, about 10%, about 20%, or about 30%. The variant can be a haplotype or a SNP. The plurality of oligonucleotides can comprise at least one oligonucleotide that does not comprise a variant. In some embodiments, each of the plurality of oligonucleotides is a double stranded oligonucleotide. In some embodiments, the plurality of oligonucleotides represents all known mitochondrial haplotypes found in humans. The plurality of

oligonucleotides can represent the entire human mitochondrial genome. The plurality of oligonucleotides can represent a portion of the human mitochondrial genome, for example the protein-coding regions.

[0043] Each oligonucleotide of the plurality of nucleotides can be at least 100 bp, at least 200 bp, at least 300 bp, at least 400 bp, at least 500 bp, at least 750 bp, or at least 1000 bp in length. Each oligonucleotide of the plurality of oligonucleotides can represent a portion of the mitochondrial genome, wherein the plurality of portions of the mitochondrial genome represented by the plurality of oligonucleotides is the entire mitochondrial genome. In some embodiments, oligonucleotides representing adjacent portions of the mitochondrial genome overlap by at least 10 nucleotides, at least 20 nucleotides, least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides.

[0044] In some embodiments, each of the oligonucleotides comprises a nucleic acid overhang. The nucleic acid overhang can be a 5' nucleic acid overhang, a 3' nucleic acid overhang, or a combination thereof. In some embodiments, the nucleic acid overhang comprises an overhang of at least 5bp, at least lObp, at least 15bp, at least 20bp, at least 25bp, or at least 30 bp. In some embodiments, the nucleic acid overhang is generated by digesting the oligonucleotide with an exonuclease. The exonuclease can be a 5' exonuclease. The 5' exonuclease can be T5 exonuclease.

[0045] In some embodiments, groups of oligonucleotides from the subset of plurality of nucleotides representing the birth mtDNA sequence are assembled to produce an intermediate assembly. Groups of oligonucleotides can comprise 2, 3, 4, 5, 6, 7, 8, 9, or 10 oligonucleotides. Each of the oligonucleotides in the group of oligonucleotides can comprise an overlapping nucleotide sequence with at least one other oligonucleotide in the group.

[0046] Assembling can comprise assembling a plurality of groups of oligonucleotides in parallel to produce a plurality of intermediate assemblies. Assembling can comprise annealing of complementary overhangs between adjacent oligonucleotides. In some embodiments, the intermediate assemblies are amplified. Amplification of the intermediate assemblies can be done using polymerase chain reaction (PCR). Assembling can further comprise assembling a plurality of intermediate assemblies, to create a larger intermediate assembly. The process of joining larger intermediate assemblies can be repeated as needed until the entire mtDNA sequence has been assembled. In some embodiments, assembly of the intermediate assemblies occurs after amplification of the intermediate assemblies. In some embodiments, assembly of the

intermediate assemblies does not require amplification of the intermediate assemblies. In some embodiments, the assembling produces a reconstructed mtDNA. The reconstructed mtDNA can be the birth mtDNA.

[0047] In some embodiments, assembly of the subset of oligonucleotides comprising the birth mtDNA sequence is automatic. Automatic assembly can comprise the use of a robot to select the oligonucleotides for assembly from the plurality of oligonucleotides.

[0048] Assembling the oligonucleotides representing each of the plurality of birth haplotypes into a reconstructed birth mitochondrial sequence can comprise any suitable assembly technology. Examples of suitable assembly technologies include, but are not limited to, Gibson assembly, sequence and ligase independent cloning (SLIC), circular polymerase extension cloning (CPEC), seamless ligation cloning extract (SLiCE), and polymerase cycling assembly (PCA). In some embodiments, a DNA ligase is used to create a circularized reconstructed birth mitochondrial sequence.

Mitochondria genomic engineering

[0049] Disclosed herein, in certain embodiments, are methods of modifying the genome of a mitochondria to produce a modified mitochondria comprising a modified mitochondrial genome. In some embodiments, the modified mitochondria have reduced immunogenicity. [0050] Modifying the genome 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. In some embodiments, the modified mitochondrial genome comprises the birth mtDNA sequence. In some embodiments, the modified mitochondrial genome comprises a modification of at least one gene in the modified mitochondrial genome. Genes in the mitochondria are exemplified in Table 1. In some embodiments, the modified mitochondria have an increase in the functionality of the

mitochondria compared to the endogenous mtDNA. In some embodiments, the modified mitochondria have an increase in the functionality of the mitochondria compared to the birth mtDNA.

Table 1. Mitochondrial genes.

[0051] 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. In some embodiments, all non-coding regions are removed. In some embodiments, the deletion is a deletion of a nuclease site in the mtDNA.

[0052] In some embodiments, the modified mitochondrial genome comprises a transgene. The transgene can be a mitochondrial gene or a nuclear gene. In some embodiments, the transgene is expressed in the cells of the subject after uptake of the modified mitochondria by the subject. The transgene can be a therapeutic gene.

[0053] In some embodiments, the modified mitochondrial genome comprises 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. In some embodiments, the modified mitochondrial genome comprises 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.

[0054] In some embodiments, the modified mitochondrial genome comprises 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. In some embodiments, the antibiotic is 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.

[0055] In some embodiments, the modified mitochondrial genome comprises a mutation conferring increased stability or resistance to random mutations of the mtDNA. In some embodiments, the modified mitochondrial genome comprises 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).

[0056] In some embodiments, the modified mitochondrial genome comprises 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 embodiments, the mutation does not alter the activity of the enzyme. In some embodiments, the modified mitochondrial genome comprises a transgene rendering the mitochondria resistant to elimination. In some embodiments, selection of the modified mitochondria comprises the use of a small molecule drug or endonuclease to selectively eliminate endogenous mitochondria leaving the modified mitochondria.

[0057] In some embodiments, the modified mitochondrial genome comprise a sequence identical to the mitochondrial sequence of the subject at the time of the subject's birth (the birth mtDNA sequence). The sequence can be a haplotype. In some embodiments, the modified mitochondrial genome comprise a sequence identical to the sequence of the birth mtDNA. In some embodiments, the modified mitochondrial genome 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.

[0058] 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). In some embodiments, homologous

recombination can be used to introduce mutations into the genome of the mitochondria.

[0059] Modifying the mitochondrial genome can comprise targeted editing using

CRISPR/CRISPR associated endonuclease complex. The CRISPR associated endonuclease can be an RNA guided endonuclease. Examples of RNA guided endonucleases include, but are not limited to Casl, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, CaslO, Csel, Cse2, Csyl, Csy2, Csy3, Csm2, Cmr5, CsxlO, Csxl l, Csfl, Csn2, C2cl, C2c2, C2c3, and Cpfl . The RNA guided endonuclease can be Cas9. The RNA guided endonuclease can be Cpfl . A guide RNA (gRNA) can be designed to target a specific location in the mtDNA where editing is desired. The gRNA associate directly with an mtDNA sequence of approximately 20 nucleotides in length, including sequences 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, and further associate with a CRISPR/CRISPR associated endonuclease complex, to recognize a specific sequence of DNA that can be 3 nucleotides in length, known as a protospacer adjacent motif (PAM).

[0060] 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 upsteam 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. In some embodiments, the

CRISPR/CRISPR associated endonuclease system is CRISPR/Cas9.

[0061] A vector can be used transduce a DNA sequence into the mitochondria. In some embodiments, the vector is 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. In some embodiments, the capsid comprises a mitochondrial targeting sequence (MTS). Electroporation can be used to introduce a DNA sequence or a plasmid into a mitochondria.

[0062] In some embodiments, the DNA sequence does not integrate into the mitochondrial genome. The DNA sequence can be an editing template. In some embodiments, the DNA sequence integrates into the mitochondrial genome, for example, when used as an editing template for CRISPR/Cas9 gene editing.

[0063] 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.

Creation of homoplasmic mitochondria for large scale production

[0064] In some embodiments, a homoplastic cell culture is a cell culture in which only one mitochondrial genomic sequence is present. Described herein, in certain embodiments, are methods of generating a homoplasmic cell culture, comprising: (a) obtaining a cell culture comprising a plurality of cells, where each of the plurality of cells comprises at least one endogenous mitochondria, and wherein each of the at least one endogenous mitochondria comprises an endogenous mitochondrial DNA (mtDNA); eliminating the endogenous mtDNA in the plurality of cells in the cell culture; and (c) transducing a synthetic mtDNA into the plurality of cells to produce a homoplasmic cell culture.

[0065] 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.

[0066] The endogenous mtDNA can be removed to create homoplasmy at both the cellular and mitochondrial level. Following transduction with the synthetic mtDNA, each of the plurality of cells in the homoplasmic cell culture can comprise the synthetic mtDNA sequence.

[0067] 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.

[0068] In some embodiments, 100% of the endogenous mtDNA is eliminated. In some embodiments, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the endogenous mtDNA is eliminated. Endogenous mtDNA can be eliminated by applying a mitochondrial targeted DNA endonuclease to the cell culture. The mtDNA nuclease can be derived from Herpes (HSV) protein UL12. Nuclease expression can occur prior to the transformation of the reconstructed mtDNA by use of an inducible promoter. Degradation tags can be used to further control the active nuclease life-time.

[0069] In some embodiments, in order to enhance homoplasmic selection, the regenerated mtDNA are specifically engineered to not be susceptible to nuclease degradation.

[0070] A vector can be used to transduce a DNA sequence into the mitochondria. In some embodiments, the vector is 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. In some embodiments, the capsid comprises 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.

[0071] To transform reconstructed mtDNA in living cells, some embodiments use recombinant proteins, such as a human mitochondrial transcription factor A (TF AM) engineered with an N- terminal protein transduction (i.e. 11 arginines) domain and a mitochondrial localization signal (MLS). Such proteins can bind mtDNA and rapidly transport the mtDNA across plasma membranes to mitochondria.

[0072] The method can further comprise addition of a transgene to the reconstructed birth mitochondrial 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. Industrial-scale Production of Purified Mitochondria

[0073] Disclosed herein, in certain embodiments, are methods for producing a mitochondrial preparation. In some embodiments, the method for producing a mitochondrial preparation comprises: (a) obtaining a cell culture; (b) introducing a mitochondria having reduced immunogenicity into the cell culture; (c) expanding the cell culture; and isolating and purifying the mitochondria having reduced immunogenicity to produce the mitochondrial preparation. In some embodiments, methods for producing a mitochondrial preparation comprise: (a) obtaining a homoplasmic cell culture; (b) expanding the homoplasmic cell culture; and (c) isolating and purifying mitochondria from the cell culture to produce a purified and stable mitochondrial preparation.

[0074] The mitochondria introduced into the cell culture can be mitochondria comprising the birth mtDNA sequence of the subject, modified mitochondria, or donor mitochondria. 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. In some embodiments, the heterochronic donor is 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. In some embodiments, the mitochondria in the mitochondrial preparation are dominant over the endogenous mitochondria in the unhealthy cells of the subject. In some embodiments, the mitochondria in the mitochondrial preparation have an improved functional capability compared to endogenous mitochondria of the subject.

[0075] The cell culture can comprise a plurality of cells. In some embodiments, the

mitochondria in a mitochondrial preparation are propagated within the plurality of cells. In some embodiments, the cell culture is a cell culture from a host cell line comprising nuclear encoded mitochondria proteins that are immunologically compatible with the subject. In some embodiments, telomerase is applied to the cell culture.

[0076] 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.

[0077] The cell culture can be a pluripotent stem cell (iPSC) culture. Reprogramming of cells into iPS cells can comprise transfecting cells with, for example, a vector encoding at least one stem cell transformation factor. In some embodiments, the vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector. Stem cell transformation factors include, but are not limited to, Oct4, Sox2, KLF-4, GLIS1, c-MYC, Nanog, and Lin28. In one example, fibroblasts are reprogrammed into iPSC by transfecting the fibroblasts with an adenoviral encoding Oct4, Sox2, KLF-4, and cMYC. In another example, fibroblasts are reprogrammed into iPSC by transfecting the fibroblasts with a retroviral vector encoding Oct4, Sox2, Nanog, and Lin28. In some embodiments, reprogramming of cells into iPSC further comprises using p53 to overcome reprogramming barriers such as cellular senescence. Reprogramming of cells into iPS cells can comprise somatic cell nuclear transfer (SCNT) into oocytes.

[0078] The cell culture can be an embryonic stem (ES) cell culture. The ES cell culture can be derived from a compatible donor.

[0079] In some embodiments, the cells in an iPSC culture or ES cell culture are differentiated prior to isolating and purifying the mitochondria. Differentiating the cells prior to isolation can eliminate or reduce immunogenicity of the mitochondria. The cells can be induced to differentiate into, for example, neuronal cells, hippocampal progenitors, dentate granule cell neurons, MGE progenitors, cortical interneurons, 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 cells can be grown as embryoid bodies. The cells 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 cells can be grown under hypoxic and high pressure conditions to induce differentiation of the cells.

[0080] The cell culture can be derived from cells obtained from the subject or a donor. The cells can be any suitable somatic cells, such as fibroblasts or keratinocytes. The cells can be obtained from a biopsy (e.g. a skin biopsy), a blood draw, or from plucked hair.

[0081] In some embodiments, a donor mitochondrial sequence is introduced into the cell culture. In some embodiments, mitochondria comprising birth mtDNA sequence is introduced into the cell. In some embodiments, modified mitochondria is introduced into the cell culture. In some embodiments, a the modified mitochondria comprising a synthetic mtDNA. The modified mitochondria can comprise a gene conferring a competitive advantage over the endogenous mitochondria. The modified mitochondria can comprise a gene encoding a selectable marker, such as a gene providing drug resistance. Prior to expansion, cells comprising the modified mitochondria can be selected for by applying a selective pressure. For example, if modified mitochondria comprising a gene providing chloramphenicol resistance is introduced into a cell culture, applying chloramphenicol to the culture will eliminate endogenous mitochondria not containing the marker.

[0082] The mitochondria introduced into the cell culture can be mitochondria having an increased function. In some embodiments, producing a mitochondrial preparation comprises enriching the cell culture for cells comprising mitochondria having an increased function. The increased function can be higher membrane potential. Enriching the cell culture for cells comprising mitochondria having an increased function can comprise engineering cells able to more efficiently eliminate defective mitochondria via selective mitophaphy. Enriching a cell culture via enhanced selective mitophagy can comprise cells engineered an overexpress regulators of mitophagy, such as SIRT1 or Parkin/PINKl . Enriching the cell culture for cells comprising mitochondria having an increased function 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).

[0083] Expansion of the cell culture can increase the amount of mitochondria. Expansion of the cell culture can occur before or after enriching the cell culture for cells comprising the mitochondria introduced into the cell culture. Isolation and purification of the mitochondria can occur after expansion of the cell culture. The cell culture may be allowed to expand for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, or 14 days. In some embodiments, the cells are grown in the cell culture until the cells are approximately 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater than 95% confluent. In some embodiments, the cells are grown in the cell culture until the cells are 100% confluent.

[0084] In some embodiments, isolation and purification of the mitochondria comprises homogenizing the cells in the cell culture to produce a homogenate. Centrifuging the homogenate can produce a supernatant comprising the mitochondria and a precipitate comprising unbroken cells and cell debris. Isolation and purification of the mitochondria can comprise isolating the supernatant and further centrifuging the supernatant to produce a precipitate comprising the purified mitochondria. In some embodiments, isolation and purification of the mitochondria comprises gradient centrifugation of the supernatant using a density media. The gradient centrifugation can be sucrose gradient centrifugation or ficoll gradient centrifugation. In some embodiments, isolation and purification of the mitochondria comprises chromatography. [0085] An exemplary method for determination of the birth mtDNA sequence of an individual, reconstructing this sequence, creation and propagation of a homoplasmic cell culture containing this reconstructed sequence, producing and purifying mitochondria containing the reconstructed sequence, and administering the purified mitochondria to a patient is illustrated in FIG. 1.

[0086] In some embodiments, the mitochondrial function of the mitochondrial preparation is determined. Mitochondrial function can be characterized by assays measuring: ATP production, oxygen consumption, NADH oxidation, membrane potential, or a combination thereof. An assay to determine mitochondrial function can be a standard colorimetric or a fluorescent assays. An example of conditions under which ATP production can be measured include: 1 mM pyruvate + 0.05 mM palmitoyl-L-carnitine + 10 mM a- ketoglutarate + 1 mM malate + ImM 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

[0087] 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.

Preparation of Medicaments and use in Therapy

[0088] Disclosed herein, in certain embodiments, are methods of treating a condition in a subject, comprising: administering to the subject a composition comprising a mitochondrial preparation. In some embodiments, the condition is an age-related condition.

[0089] In some embodiments, the mitochondria in the mitochondrial preparation are packaged within liposomes for delivery to the subject.

[0090] In some embodiments, the composition further comprises at least one excipient or carrier. Any pharmaceutically acceptable excipient or carrier can be used. 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.

[0091] In some embodiments, the mitochondria in mitochondrial preparation are 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 described herein can be administered to a subject to improve cognitive function or other neurodegenerative conditions.

[0092] The composition can further comprise an additional agent. The additional agent can be expressed in the liposome or administered in conjugation with the composition. In some embodiments, the additional agent is an antibody, a peptide, a nucleic acid, an enzyme, a small molecule drug, or a combination thereof. The additional agent can be an additional therapeutic agent.

[0093] The additional agent can be an agent decreasing the immune response of the subject to the mitochondria in the mitochondrial preparation. 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 mitochondrial preparation.

[0094] 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-1, 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.

[0095] The compositions described herein can be administered to a subject with a condition. In some embodiments, the composition is administered to a subject to prevent the condition. In some embodiments, the composition is administered to reduce the effects of the condition. In some embodiments, the condition is cancer, cardiomyopathy, myopathy, optic atrophy, infertility, fibrotic organs, Duchenne muscular dystrophy, or neurodegeneration.

Neurodegeneration can be dementia, Parkinson's disease (PD), Alzheimer's disease,

Huntington's disease (HD), or amyotrophic lateral sclerosis (ALS). The condition can be an age- related condition. The condition can be a condition caused by a mutation in the mtDNA.

Conditions caused by mutations in the mtDNA include, but are not limited to, Kearns-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. [0096] The compositions described herein 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.

[0097] The composition can be administered locally. Local routes of administration include, without limitation, local injection, intracranial, intracerebroventricular, intracerebral,

intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, intracisternal, 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.

[0098] 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.

[0099] A medicament or a 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.

[0100] The medicament or 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. Certain terminology

[0101] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. The below terms are discussed to illustrate meanings of the terms as used in this specification, in addition to the understanding of these terms by those of skill in the art. 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.

[0102] 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.

[0103] 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.

[0104] "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 the methods of the present disclosure, 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.

[0105] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions described herein belong. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions described herein, representative illustrative methods and materials are now described. EXAMPLES

[0106] Example 1: Determination of a birth mitochondrial sequence

[0107] A blood sample is drawn from an 80 year old patient suffering from atherosclerosis. From this sample, the mitochondrial genome of the patient is sequenced to 50X coverage. Reads showing nucleic acid variants known to be present in the human mitochondrial genome at a frequency of 1% or less are discarded. The remaining reads are assembled using the Cambridge Reference Sequence (CRS) as a reference mitochondrial sequence and the frequency of the remaining nucleic acid variants are determined. The patient's birth mtDNA sequence at each variant position is considered to be the variant with the highest frequency.

[0108] Example 2: Reconstruction of a birth mitochondrial sequence

[0109] A library of synthetic oligonucleotides is obtained comprising 2,000 oligonucleotides. Each oligonucleotide represents a fragment of the mitochondrial genome and a known haplotype within that fragment. A subset of the 2,000 oligonucleotides are chosen which collectively represent the birth mitochondrial DNA sequence as determined in Example 1. Groups of 5 adjacent oligonucleotides from this subset are first digested with a 5' exonuclease to create 30 bp overhangs, and then are annealed together to create an intermediate assembly. This process is repeated with the resulting intermediate assemblies until the full length birth mitochondrial DNA generated. A transgene encoding chloramphenicol acetyltransferase is added to the birth mitochondrial DNA for use as a selectable marker (chloramphenicol resistance).

[0110] Example 3: Generation of a homoplasmic cell culture

[0111] The birth mtDNA from Example 2 is transduced into the mitochondria of cells in an iPSC culture with the addition of a recombinant protein, such as transcription factor A (TFAM) engineered with an N-terminal protein transduction (i.e., 11 arginines) domain and a

mitochondrial localization signal (MLS). The endogenous mtDNA is removed by inducible expression of mitochondrial targeted nucleases, while the birth mtDNA is selected by the addition of chloramphenicol. The result is a homoplasmic cell culture where all cells have identical mitochondrial genomes representing the birth mtDNA sequence of the patient.

[0112] Example 4: Administration of a medicament to the patient

[0113] Once the homoplastic cell culture from Example 3 reaches 100% confluence, the mitochondria comprising the reconstructed birth mtDNA are isolated and purified to produce a mitochondrial preparation. The resulting mitochondrial preparation is formulated for intravenous injection and administered to the patient.

[0114] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that 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 described herein 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.

References

1. Karnkowska, A. et al. A Eukaryote without a Mitochondrial Organelle. Curr. Biol.

CB 26, 1274-1284 (2016).

2. Bratic, A. & Larsson, N.-G. The role of mitochondria in aging. J. Clin. Invest.

123, 951-957 (2013).

3. Gorman, G. S. et al. Mitochondrial diseases. Nat. Rev. Dis. Primer 2, 16080

(2016).

4. Lezi, E. & Swerdlow, R. H. Mitochondria in neurodegeneration. Adv. Exp. Med.

Biol. 942, 269-286 (2012).

5. Kelly-Worden, M. & Thomas, E. Mitochondrial Dysfunction in Duchenne

Muscular Dystrophy. Open J. Endocr. Metab. Dis. 4, 211 (2014).

6. Masuzawa, A. et al. Transplantation of autologously derived mitochondria

protects the heart from ischemia-reperfusion injury. Am. J. Physiol. Heart Circ. Physiol. 304, H966-982 (2013).

7. Kesner, E. E., Saada-Reich, A. & Lorberboum-Galski, H. Characteristics of

Mitochondrial Transformation into Human Cells. Sci. Rep. 6, srep26057 (2016).

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

9. Takahashi, K. et al. Induction of pluripotent stem cells from adult human

fibroblasts by defined factors. Cell 131, 861-872 (2007).

10. Y. Buganim, D.A. Faddah and R. Jaenisch. Mechanisms and models of somatic cell reprogramming. Nature Reviews Genetics 14, 427-439 (2013).

11. Lanza, I. R. & Nair, K. S. Functional Assessment of Isolated Mitochondria In Vitro. Methods Enzymol. 457, 349-372 (2009).

12. Jo, A. et al. Efficient Mitochondrial Genome Editing by CRISPR/Cas9. Biomed Res Int. 2015;2015:305716.

13. Clark M.A. and Shay J.W. Mitochondrial transformation of mammalian cells.

Nature 295, 605-607 (1982).

14. Pacak, C. A. et al. Actin-dependent mitochondrial internalization in

cardiomyocytes: evidence for rescue of mitochondrial function. Biol Open 4, 622-626 (2015).

15. Kitani, T., Kami, D., Matoba, S. & Gojo, S. Internalization of isolated functional mitochondria: involvement of macropinocytosis. J Cell Mol Med 18, 1694-1703 (2014). Caicedo, A. et al. MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function. Sci Rep 5, 9073 (2015).

Katrangi, E. et al. Xenogenic transfer of isolated murine mitochondria into human rhoO cells can improve respiratory function. Rejuvenation Res 10, 561-570 (2007).

Masuzawa, A. et al. Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 304, H966-982 (2013).

Dinesh C. Joshi and Joanna C. Bakowska. Determination of Mitochondrial Membrane Potential and Reactive Oxygen Species in Live Rat Cortical Neurons. J Vis Exp. 2011; (51): 2704.

Poole OV, Hanna MG, Pitceathly RD. Mitochondrial disorders: disease mechanisms and therapeutic approaches. Discov Med. 2015 Nov;20(l 11):325-31 Shin B, Cowan DB, Emani SM, Del Nido PJ, McCully JD. Mitochondrial Transplantation in Myocardial Ischemia and Reperfusion Injury. Adv Exp Med Biol. 2017; 982:595-619.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A pharmaceutical composition comprising a homoplasmic preparation of human

mitochondria in a pharmaceutically compatible excipient.

2. The pharmaceutical composition of claim 1, wherein the homoplasmic preparation of mitochondria is prepared specifically for administration to a particular human subject in need thereof.

3. A method of preparing a pharmaceutical composition according to claim 2, comprising:

(a) sequencing the subject's mitochondrial genome with at least 20-fold coverage;

(b) determining a consensus sequence of the mitochondrial genome of the subject; and

(c) providing a homoplasmic preparation of human mitochondria in which the sequence of the mitochondrial genome is identical to a predetermined subject-specific genome template;

wherein the genome template consists of the consensus sequence determined in step (b), or a version of the consensus sequence in which one or more nucleotides in one or more mitochondrial genes has been added, changed, or deleted in comparison with the consensus sequence.

4. The method of claim 3, wherein step (a) comprises sequencing the subject's

mitochondrial genomic DNA with at least 100-fold, 500-fold, 2,000-fold, or 10,000-fold coverage.

5. The method of claim 3 or claim 4, wherein step (a) comprises sequencing the genomes of mitochondria obtained from several different tissues in the subject.

6. The method of any of claims 3 to 5, wherein the consensus sequence is a birth

mitochondrial genome sequence.

7. The method of any of claims 3 to 6, wherein the genome template of the homoplasmic mitochondria lacks a mutation in a mitochondrial gene identified in the consensus sequence.

8. The method of any of claims 3 to 7, wherein the genome template of the homoplasmic mitochondria contains at least one change in the sequence of a mitochondrial gene that confers improved functional capability to the mitochondria compared with mitochondria endogenous to the subject.

9. The method of claim 8, wherein the change confers to the mitochondria at least one property selected from increased stability, resistance to mutation, resistance to elimination, higher membrane potential, and higher energy production capability.

10. The method of any of claims 3 to 9, wherein step (c) includes assembling the genome of the homoplasmic mitochondria from a library of mitochondrial gene fragments.

11. The method of any of claims 3 to 10, wherein step (c) includes preparing multiple copies of the genome template, and inserting the copies into mitochondria from which endogenous DNA has been removed.

12. The method of any of claims 3 to 11, wherein step (c) includes harvesting the

homoplasmic mitochondria from a culture of homoplasmic cells.

13. A method of treating an age-related condition or neurodegeneration in a subject,

comprising obtaining a preparation of homoplasmic mitochondria according to any of claims 3 to 12, and administering the preparation to the subject.

14 A method of treating an age-related condition or neurodegeneration in a subject,

comprising administering to the subject a preparation of mitochondria having improved functional capability compared with mitochondria endogenous to the subject.

15. The method of claim 14, wherein the preparation of mitochondria has been prepared by:

obtaining cells from the subject;

proliferating the cells in vitro;

harvesting mitochondria from the proliferated cells; and

enriching the harvested mitochondria for mitochondria having higher membrane potential.

16. The method of any preceding clam, wherein the age-related condition is selected from cardiomyopathy, myopathy, dementia, optic atrophy, infertility, organ fibrosis,

Parkinson's disease and Alzheimer's disease.