WO2016115632A1 - Method for treating mitochondrial disease - Google Patents

Method for treating mitochondrial disease Download PDF

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WO2016115632A1
WO2016115632A1 PCT/CA2016/050046 CA2016050046W WO2016115632A1 WO 2016115632 A1 WO2016115632 A1 WO 2016115632A1 CA 2016050046 W CA2016050046 W CA 2016050046W WO 2016115632 A1 WO2016115632 A1 WO 2016115632A1
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mitochondrial
subunit
nadh
mitochondrially encoded
trna
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French (fr)
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Mark TARNOPOLSKY
Adeel SAFDAR
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Exerkine Corporation
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention generally relates to treatment of mitochondrial disease, and more particularly relates to a method of treating mitochondrial disease using exosomes.
  • Mitochondria are intracellular organelles that have a variety of functions. They are best known for their ability to produce ATP from reducing equivalents derived from fat, protein and carbohydrates.
  • the reducing equivalents, FAD3 ⁇ 4 and NADH+H + are delivered to the respiratory chain and used to pump a proton from the matrix of the mitochondria to the inter- membrane space.
  • the electrons are transported from complex I and II to coenzyme Q10 and then on to complex III, cytochrome c and complex IV where oxygen is reduced to molecular water. The flow of electrons leads to the pumping of the proton complex I, III and IV.
  • the build-up of protons in the inter-membrane space leads to a proton motive force where the electrons flow through complex V and re-phosphorylate ADP to ATP.
  • the mitochondria are also involved in other cellular processes including: calcium buffering, apoptosis, oxidative stress, telomere maintenance, and activation of inflammatory pathways such as the inflammasome.
  • the mitochondria are thought to have their origin as bacteria that took on a symbiotic relationship with a proto-eukaryotic cell 1.5 billion years ago.
  • the human mitochondrial DNA retains 37 of these 1500 genes in a small circular piece of DNA called mitochondrial DNA (mtDNA).
  • mtDNA mitochondrial DNA
  • This circular DNA resembles bacterial DNA (likely from its origin) and undergoes polycistronic replication.
  • Most of the mitochondrial DNA contains exons and the repair mechanisms are not as sophisticated as those in the nuclear DNA. This is associated with an increased propensity for mutagenesis in mtDNA verses nuclear DNA.
  • the nuclear mutations can be inherited in an autosomal recessive, autosomal dominant or X-linked recessive manner, Dysfunction of the mitochondria leads to anaerobic ATP generation with an increased reliance on anaerobic pathways. This leads to inefficient energy generation and the production of lactic acid (through glycolysis).
  • mtDNA mutations both sporadic and familial
  • mitochondrial dysfunction is becoming increasingly apparent in broad range of metabolic and degenerative diseases, cancer, and aging.
  • Mitochondrial Encephalomyopathy Lactic Acidosis and Stroke-like episodes (MELAS) resulting from the mutation, 3243A>G; Leber Hereditary Optic Neuropathy (LHON) resulting from the mutation, 11778G>A; as well as large scale deletions that result in Kearn-Sayre- Syndrome (KSS). Subsequently, many hundreds of mtDNA mutations have been described ( " http://www.mitomap.org/MITOMAP).
  • NDUF mutations in Leigh syndrome SC02 mutations in infantile cardiomyopathy
  • POLG mutations mutations in gene that codes for DNA polymerase gamma
  • SANDO sensor ataxic neuropathy, dysarthria, and ophthalmoparesis
  • SPG7 mutations in hereditary spastic paraparesis MFN2 mutations in peripheral neuropathy, and others f http : //www.mitomap. org/MITOMAP
  • Mitochondrial diseases are heterogeneous and often multi-systemic due to the fact that mitochondria are present in all tissues in the human body with the exception of mature red blood cells.
  • mitochondrial disorders preferentially affect tissues with high energy demand, including the brain, muscle, and heart, although any organ (including liver, pancreas, bone marrow, etc.) can be affected. Consequently, mitochondrial defects are implicated in forms of blindness, deafness, movement disorders, dementias, cardiomyopathy, myopathy, renal dysfunction, and aging. At the molecular level, in addition to energy crisis, mitochondrial dysfunction can also lead to telomere shortening, oxidative stress, apoptosis and inflammasome activation.
  • exosomes may be effectively used as a vehicle to deliver a mitochondrial product to a mammal to treat pathological conditions such as a mitochondrial disease resulting from a deficiency of a functional mitochondrial product.
  • exosomes are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
  • a method of increasing the amount of a mitochondrial product in mitochondria in a mammal comprising administering to the mammal exosomes that are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
  • a method of increasing the activity of a target mitochondrial product in a mammal comprising administering to the mammal a composition comprising exosomes which are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
  • a method of treating a pathological condition in a mammal resulting from the deficiency of a functional mitochondrial product comprising administering to the mammal a composition comprising exosomes genetically engineered to incorporate the mitochondrial product and/or nucleic acid encoding the mitochondrial product.
  • a method of treating a mitochondrial disease in a mammal comprising administering to the mammal a composition comprising exosomes genetically engineered to incorporate a mitochondrial product useful to treat the mitochondrial disease and/or nucleic acid encoding the mitochondrial product.
  • nucleic acid comprising a nucleotide sequence that encodes the functional mitochondrial product or precursor thereof;
  • A4 The exosome according to paragraph Al to A3, wherein the biological sample is from a mammal, or the cell is from a mammal or a mammalian ceil line. [0022] A5. The exosome according to any one of paragraphs Al to A4, wherein the isolating removes vesicles and cellular debris less than 20 nm in diameter.
  • A6 An exosome that comprises a modification selected from the group consisting of:
  • nucleic acid comprising a nucleotide sequence that encodes the functional mitochondrial product or precursor thereof;
  • exosome according to any of paragraphs Al - A6, that comprises a nucleic acid comprising a nucleotide sequence encoding a functional mitochondrial product or precursor thereof, wherein the nucleic acid is present in a lumen of the exosome.
  • nucleic acid comprises a species of RNA or a species of modified RNA (modRNA, e.g. 5 methyl cytosine, or N6 methyladenine) encoding for a mitochondrial product set forth in Table 1 and/or Table 2.
  • modified RNA e.g. 5 methyl cytosine, or N6 methyladenine
  • the mitochondrial product is selected from the group consisting of Lon peptidase 1, mitochondrial (LONP1), NADH:ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADH:ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH:ubiquinone oxidoreductase complex assembly factor 3 ( DUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH:ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl-CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1),
  • LONP1 Lon peptidase 1, mitochondrial
  • NDUFAF1 NADH
  • composition according to paragraph CI wherein the composition is substantially free of vesicles having a diameter less than 20 nm.
  • C3. The composition according to paragraph CI or C2, wherein the composition is substantially free of vesicles having a diameter greater than 120nm.
  • composition according to claim C4 which exhibits a zeta potential having a magnitude of up to 200 mV, or up to 175 mV, or up to 150 mV, or up to 140 mV, or up to 130 mV, or up to 120 mV, or up to 110 mV, or up to 100 mV.
  • Dl A method of increasing the amount of a mitochondrial product in mitochondria in a mammal, comprising administering to the mammal an exosome according to any one of paragraphs Al - B6, or a composition according to any one of paragraphs CI - C5.
  • a method of treating a mitochondrial disease in a mammal comprising administering to the mammal an exosome according to any one of paragraphs Al - B6, or a composition according to any one of paragraphs CI - C5.
  • D6 The method or use according to paragraph D5, wherein the human has a mitochondrial disease selected from the group consisting of Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQ10D), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG (e.g.
  • CPEO Chronic Progressive External Ophthalmoplegia syndrome
  • AHS Alpers-Huttenlocher syndrome
  • MCHS Childhood Myocerebrohepatopathy Spectrum
  • MEMSA Myoclonic Epilepsy Myopathy Sensory Ataxia
  • ANS Ataxia Neuropathy Spectrum (ANS) (including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO)), adPEO due to mutations in ANT or due to C10orf2 (twinkle) mutations, Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1/MyoNeurogenic Gastrointestinal Encephalopathy (MNGIE), Mohr-Tranebjaerg syndrome, 3-methylglutaconic aciduria, Combined
  • Glutaric acidemia IIA Glutaric acidemia ⁇ or Glutaric acidemia IIC
  • Pyruvate dehydrogenase deficiency e.g. Pyruvate dehydrogenase El -alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3-binding protein deficiency, Pymvate dehydrogenase E2 deficiency or Pyruvate dehydrogenase El -beta deficiency
  • 3-hydroxyacyl-CoA dehydrogenase SCHAD
  • FIG. 1 graphically illustrates that (A) mitochondria-encoded NADH dehydrogenase 4 (ND4) mRNA or ND4 protein-loaded exosomes rescue mitochondrial complex I deficiency in primary fibroblasts isolated from Leber Hereditary Optic Neuropathy (LHON) patients; (B) Mean ⁇ SD of experiments in (A) independently repeated three experiments. *P ⁇ 0.05. Data were analyzed using an unpaired /-test;
  • Figure 2 graphically illustrates that ND4 mRNA or ND4 protein-loaded exosomes improves oxygen consumption rate of primary fibroblasts isolated from LHON (m.11778 G>A) patients. Mean ⁇ SD of independently repeated three experiments. *P ⁇ 0.05. Data were analyzed using one-way ANOVA, followed by Tukey post hoc test;
  • POLG DNA polymerase gamma
  • Figure 4 graphically illustrates that (A) tRNALeu (UUR) -loaded exo somes rescue mitochondrial complex IV deficiency in primary fibroblasts isolated from MELAS (m.3243A>G) patients; (B) Mean ⁇ SD of experiments in (A) independently repeated three experiments. *P ⁇ 0.05. Data were analyzed using an unpaired Mest; and
  • FIG. 5 graphically illustrates that Lon Peptidase 1, mitochondrial (LONPl) mR A-loaded exosomes rescue mitochondrial complex IV deficiency in primary fibroblasts isolated from patients with impaired and/or reduced LONPl protein activity and/or levels (LONPl patients). *P ⁇ 0.05. Data were analyzed using an unpaired t-test.
  • a method of treating a mitochondrial disease in a mammal in which the mitochondrial disease results from a nucleic acid mutation that results in a dysfunctional mitochondrial product.
  • the method comprises administering to the mammal a therapeutically effective amount of exosomes engineered to comprise a functional target mitochondrial product or nucleic acid encoding a functional target mitochondrial product.
  • a target mitochondrial product is used herein to refer to a protein or nucleic acid product which retains innate biological activity, including but not limited to, catalytic, metabolic, regulatory, binding, transport and the like.
  • a target mitochondrial product need not exhibit an endogenous level of biological activity, but will exhibit sufficient activity to render it useful to treat a mitochondrial disease, e.g. at least about 25% of the biological activity of the corresponding endogenous mitochondrial product, and preferably at least about 50% or greater of the biological activity of the corresponding endogenous mitochondrial product.
  • the present method may be used to treat any form of mitochondrial disease or disorder resulting from mitochondrial dysfunction.
  • Mitochondrial dysfunction results from a nucleic acid mutation, e.g. familial or sporadic nucleic acid mutation, including, for example, a gene or other nucleic acid mutation (such as nucleic acid rearrangements, deletions, point mutations, nonsense mutations and the like) that results in a dysfunctional mitochondrial product.
  • mitochondrial product is used herein to refer to mitochondrial proteins or precursors or functional sub-units of mitochondrial proteins, and mitochondrial RNA species, including but not limited to, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), micro RNA (miRNA), small nuclear RNA (snRNA), small cytoplasmic RNA (scRNA), signal recognition particle (SRP) RNA, small nucleolar (sno) RNA, guide RNA (gRNA), ribonuclease P (RNase P), ribonuclease MRP (RNase MRP), yRNA, telomerase RNA component (TERC), spliced leader RNA (SLRNA), long non-coding RNA (IncRNA), piwi-interacting RNA (piRNA) and total mitochondrial RNA (e.g.
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • mRNA messenger RNA
  • miRNA messenger RNA
  • miRNA micro
  • mitochondrial product also encompasses protein and RNA species synthesized outside of the mitochondria, e.g. in the nucleus (i.e. nuclear-encoded mitochondrial product), which is subsequently imported into the mitochondria.
  • mitochondrial protein is used herein to refer to proteins, or sub-units thereof, synthesized in the mitochondria from mitochondrial DNA (e.g. mitochondria-encoded), as well as proteins synthesized outside of the mitochondria which are imported into and functional in the mitochondria.
  • mitochondrial RNA encoding a mitochondrial protein may be synthesized outside of the mitochondria and subsequently imported as RNA, or its protein product, into the mitochondria.
  • exosome refers to cell-derived vesicles having a diameter of between about 20 and 120 nm, for example, a diameter of about 50-100 nm, including exosomes of a diameter of about 60 nm, 70 nm, 80 nm, 90 nm, and/or 100 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g.
  • immature dendritic cells wild-type or immortalized
  • induced and non-induced pluripotent stem cells fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like.
  • cultured cell samples will be in the cell-appropriate culture media (using exosome- free serum).
  • Exosomes include specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g.
  • Exosomes may also be obtained from a non- mammalian biological sample, including cultured non-mammalian cells.
  • exosomes from non-mammalian sources include surface markers which are isoforms of mammalian surface markers, such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles.
  • mammalian surface markers such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles.
  • the term "mammal” is meant to encompass, without limitation, humans, domestic animals such as dogs, cats, horses, cattle, swine, sheep, goats and the like, as well as non-domesticated animals such as, but not limited to, mice, rats and rabbits.
  • non-mammal is meant to encompass, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruit/vegetables (e.g. corn, pomegranate) and yeast.
  • Exosomes may be obtained from the appropriate biological sample using a combination of isolation techniques, for example, centrifugation, filtration and ultracentrifugation methodologies.
  • the isolation protocol includes the steps of: i) exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation; ii) subjecting the supernatant from step i) to centrifugation to remove microvesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the microfiltered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a physiological solution and conducting a second ultracentrifugation in a density gradient and removing the exosome pellet fraction there
  • the process of isolating exosomes from a biological sample includes a first step of removing undesired large cellular debris from the sample, i.e. cells, cell components, apoptotic bodies and the like greater than about 7-10 microns in size.
  • This step is generally conducted by centrifugation, for example, at 1000-4000x g for 10 to 60 minutes at 4 °C, preferably at 1500-2500x g, e.g. 2000x g, for a selected period of time such as 10-30 minutes, 12-28 minutes, 14-24 minutes, 15-20 minutes or 16, 17, 18 or 19 minutes.
  • a suitable commercially available laboratory centrifuge e.g.
  • Thermo- ScientificTM or Cole-ParmerTM is employed to conduct this isolation step.
  • the resulting supernatant is subjected to a second optional centrifugation step to further remove cellular debris and apoptotic bodies, such as debris that is at least about 7-10 microns in size, by repeating this first step of the process, i.e. centrifugation at 1000-4000x g for 10 to 60 minutes at 4 °C ⁇ preferably at 1500-2500x g, e.g. 2000x g, for the selected period of time.
  • the supernatant resulting from the first centrifugation step(s) is separated from the debris- containing pellet (by decanting or pipetting it off) and may then be subjected to an optional additional (second) centrifugation step, including spinning at 12,000-15,000x g for 30-90 minutes at 4 °C to remove intermediate-sized debris, e.g. debris that is greater than 6 microns size.
  • this centrifugation step is conducted at 14,000x g for 1 hour at 4 °C.
  • the resulting supernatant is again separated from the debris- containing pellet.
  • the resulting supernatant is collected and subjected to a third centrifugation step, including spinning at between 40,000-60,000x g for 30-90 minutes at 4 °C to further remove impurities such as medium to small-sized microvesicles greater than 0.3 microns in size e.g. in the range of about 0.3-6 microns.
  • the centrifugation step is conducted at 50,000x g for 1 hour.
  • the resulting supernatant is separated from the pellet for further processing.
  • the supernatant is then filtered to remove debris, such as bacteria and larger microvesicles, having a size of about 0.22 microns or greater, e.g, using microfiltration.
  • the filtration may be conducted by one or more passes through filters of the same size, for example, a 0.22 micron filter.
  • filters of the same or of decreasing sizes e.g. one or more passes through a 40-50 micron filter, one or more passes through a 20-30 micron filter, one or more passes through a 10-20 micron filter, one or more passes through a 0.22-10 micron filter, etc.
  • Suitable filters for use in this step include the use of 0.45 and 0.22 micron filters.
  • the microfiltered supernatant may then be combined with a suitable physiological solution, preferably sterile, for example, an aqueous solution, a saline solution or a carbohydrate-containing solution in a 1 : 1 ratio, e.g. 10 mL of supernatant to 10 mL of physiological solution, to prevent clumping of exosomes during the subsequent ultracentrifugation and to maintain the integrity of the exosomes.
  • a suitable physiological solution preferably sterile, for example, an aqueous solution, a saline solution or a carbohydrate-containing solution in a 1 : 1 ratio, e.g. 10 mL of supernatant to 10 mL of physiological solution, to prevent clumping of exosomes during the subsequent ultracentrifugation and to maintain the integrity of the exosomes.
  • the exosomal solution is then subjected to ultracentrifugation to pellet exosomes and any remaining contaminating microvesicles
  • This ultracentrifugation step is conducted at 110,000- 170,000x g for 1-3 hours at 4 °C, for example, 170,000x g for 3 hours.
  • This ultracentrifugation step may optionally be repeated, e.g. 2 or more times, in order to enhance results.
  • Any commercially available ultracentrifuge e.g. Thermo-ScientificTM or BeckmanTM, may be employed to conduct this step.
  • the exosome-containing pellet is removed from the supernatant using established techniques and re-suspended in a suitable physiological solution.
  • the re-suspended exosome-containing pellet is subjected to density gradient separation to separate contaminating microvesicles from exosomes based on their density.
  • density gradients may be used, including, for example, a sucrose gradient, a colloidal silica density gradient, an iodixanol gradient, or any other density gradient sufficient to separate exosomes from contaminating microvesicles (e.g. a density gradient that functions similar to the 1.100-1.200 g/ml sucrose fraction of a sucrose gradient).
  • density gradients include the use of a 0.25-2.5 M continuous sucrose density gradient separation, e.g.
  • sucrose cushion centrifugation comprising 20-50% sucrose and a colloidal silica density gradient, e.g. PercollTM gradient separation (colloidal silica particles of 15-30 nm diameter, e.g. 30%/70% w/w in water (free of RNase and DNase), which have been coated with polyvinylpyrrolidone (PVP)).
  • PercollTM gradient separation colloidal silica particles of 15-30 nm diameter, e.g. 30%/70% w/w in water (free of RNase and DNase), which have been coated with polyvinylpyrrolidone (PVP)
  • PVP polyvinylpyrrolidone
  • the re-suspended exosome pellet resulting from the density gradient separation may be ready for use,
  • the density gradient used is a sucrose gradient
  • the exosome pellet is removed from the appropriate sucrose gradient fraction, and is ready for use, or may preferably be subjected to an ultracentrifugation wash step at a speed of 110,000- 170,000x g for 1-3 hours at 4 °C.
  • the density gradient used is, for example, a colloidal silica density gradient
  • the resuspended exosome pellet may be subjected to additional wash steps, e.g.
  • the exosome pellet from any of the centrifugation or ultracentrifugation steps may be washed between centrifugation steps using an appropriate physiological solution, e.g. saline.
  • the final pellet is removed from the supernatant and may be re-suspended in a physiologically acceptable solution for use.
  • the exosome pellet may be stored for later use, for example, in cold storage at 4°C, in frozen form or in lyophilized form, prepared using well-established protocols.
  • the exosome pellet may be stored in any physiological acceptable carrier, optionally including cryogenic stability and/or vitrification agents (e.g. DMSO, glycerol, trehalose, polyhydroxylated alcohols (e.g. methoxylated glycerol, propylene glycol), M22 and the like).
  • cryogenic stability and/or vitrification agents e.g. DMSO, glycerol, trehalose, polyhydroxylated alcohols (e.g. methoxylated glycerol, propylene glycol), M22 and the like).
  • the described exosome isolation protocol advantageously provides a means to obtain mammalian exosomes which are at least about 90% pure, and preferably at least about 95% or greater pure, i.e. referred to herein as "essentially free" from cellular debris, apoptotic bodies and microvesicles having a diameter less than 20 or greater than 120 nm, for example, free from particles having a diameter of less than 40 or greater than 120 nm (as measured, for example, by dynamic light scattering), and which are biologically intact, e.g. not clumped or in aggregate form, and not sheared, leaky or otherwise damaged.
  • Exosomes isolated according to the methods described herein exhibit a high degree of stability, evidenced by the zeta potential of a mixture/solution of such exosomes, for example, a zeta potential of at least a magnitude of 30 mV, e.g. ⁇ -30 or > +30, and preferably, a magnitude of at least 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, or greater.
  • zeta potential refers to the electrokinetic potential of a colloidal dispersion, and the magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles (exosomes) in a dispersion.
  • a zeta potential of magnitude 30 mV or greater indicates moderate stability, i.e. the solution or dispersion will resist aggregation, while a zeta potential of magnitude 40-60 mV indicates good stability, and a magnitude of greater than 60 mV indicates excellent stability.
  • exosomes in an amount of about 100-2000 g total protein can be obtained from 1-4 mL of mammalian serum or plasma, or from 15-20 mL of cell culture spent media (from at least about 2 x 10 6 cells).
  • solutions comprising exosomes at a concentration of at least about 5 pg pL, and preferably at least about 10-25 pg pL may readily be prepared due to the high exosome yields obtained by the present method.
  • the term "about” as used herein with respect to any given value refers to a deviation from that value of up to 10%, either up to 10% greater, or up to 10% less.
  • Exosomes isolated in accordance with the methods herein described which beneficially retain integrity, and exhibit a high degree of purity (being "essentially free” from entities having a diameter less than 20 ran and greater than 120 nm), stability and biological activity both in vitro and in vivo, have not previously been achieved.
  • the present exosomes are uniquely useful, for example* diagnostically and/or therapeutically, e.g. for the in vivo delivery of protein and/or nucleic acid. They have also been determined to be non- allergenic/non-immunogenic, and thus, safe for autologous, allogenic, and xenogenic use.
  • isolated exosomes are genetically engineered to incorporate exogenous nucleic acid or exogenous protein suitable to treat the disease, for example, nucleic acid (e.g. DNA, or mRNA) encoding a functional mitochondrial product or total mitochondrial RNA, or a functional mitochondrial product (e.g. protein, mRNA, tRNA, rRNA, mi RNA, SRP RNA, snRNA, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, or piRNA) itself.
  • nucleic acid e.g. DNA, or mRNA
  • a functional mitochondrial product e.g. protein, mRNA, tRNA, rRNA, mi RNA, SRP RNA, snRNA, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, or piRNA
  • exogenous is used herein to refer to a mitochondrial product originating from a source external to the exosomes.
  • the desired nucleic acid may be produced using known synthetic techniques, incorporated into a suitable expression vector using well established methods to form a mitochondrial product-encoding expression vector which is introduced into isolated exosomes using known techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like.
  • isolated exosomes may be engineered to incorporate total mammalian mitochondrial RNA, which is free from pathogenic mitochondrial nucleic acid mutations.
  • Total mammalian mitochondrial RNA may be isolated from biological tissue using known techniques and incorporated into isolated exosomes, for example, by electroporation or transfection.
  • the selected protein may be produced using recombinant techniques, or may be otherwise obtained, and then may be introduced directly into isolated exosomes by electroporation or other transfection methods. More particularly, electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid or protein into exosomes.
  • Transfection using cationic lipid-based transfection reagents such as, but not limited to, Lipofectamine® MessengerMAXTM Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUSTM Reagent, may also be used.
  • the amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced.
  • Lipofectamine® MessengerMAXTM Transfection Reagent an amount in the range of about 0.15 L to 10 ⁇ , may be used to load 100 ng to 2500 ng nucleic acid or protein into exosomes.
  • Other methods may also be used to load mitochondrial products into exosomes including, for example, the use of cell-penetrating peptides.
  • Exosomes isolated in accordance with the methods herein described which beneficially retain integrity, and exhibit a high degree of purity and stability, readily permit loading of exogenous protein and/or nucleic acid in an amount of at least about 1 ng nucleic acid (e.g. mR A) per 10 ug of exosomal protein or 30 ug protein per 10 ug of exosomal protein.
  • nucleic acid e.g. mR A
  • a mitochondrial product-encoding expression vector as above described may be introduced directly into exo some-producing cells, e.g. autologous, allogenic, or xenogenic cells, such as immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like, by electroporation or other transfection method as described above. Following a sufficient period of time, e.g. 3-7 days to achieve stable expression of the mitochondrial product, exosomes incorporating the expressed protein may be isolated from the exosome-producing cells as described herein.
  • exosomes incorporating the expressed protein may be isolated from the exosome-producing cells as described herein.
  • the desired mitochondrial product, nucleic acid encoding the mitochondrial product or both may be introduced into isolated exosomes, as previously described, using electroporation or other transfection methods. Introduction to the exosome of both the desired mitochondrial product and nucleic acid encoding the same mitochondrial product may increase delivery efficiency of the mitochondrial product. In addition, introduction of a combination of mitochondrial products, and/or nucleic acid encoding one or more mitochondrial products, may be desirable to treat a mitochondrial disease resulting from different DNA mutations, or for the treatment of secondary mitochondrial dysfunction such as type 2 diabetes, obesity, or muscular dystrophy, for example,
  • exosomes prior to incorporation into exosomes of a selected mitochondrial product, or nucleic acid encoding a mitonchondrial product, exosomes may be modified to express or incorporate a target-specific fusion product which provides targeted delivery of the exosomes to the mitochondria.
  • a target-specific fusion product comprises a sequence that targets mitochondria, i.e. a mitochondrial targeting sequence, linked to an exosomal membrane marker.
  • the exosomal membrane marker of the fusion product will localize the fusion product within the membrane of the exosome to enable the targeting sequence to direct the exosome to the intended target.
  • exosome membrane markers include, but are not limited to: tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151; targeting or adhesion markers such as integrins, ICAM-1 and CDD31; membrane fusion markers such as armexins, TSG101, ALIX; and other exosome transmembrane proteins such as LAMP (lysosome-associated membrane protein), e.g. LAMP 1 or 2, and LIMP (lysosomal integral membrane protein). All or a fragment of an exosomal membrane marker may be utilized in the fusion product, provided that the fragment includes a sufficient portion of the membrane marker to enable it to localize within the exosome membrane, i.e. the fragment comprises at least one intact transmembrane domain to permit localization of the membrane marker into the exosomal membrane.
  • tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151
  • targeting or adhesion markers
  • the target-specific fusion product also includes a mitochondrial targeting sequence, i.e. a protein or peptide sequence which facilitates the targeted delivery of the exosome to mitochondria.
  • a mitochondrial targeting sequence i.e. a protein or peptide sequence which facilitates the targeted delivery of the exosome to mitochondria.
  • suitable mitochondrial targeting proteins include, but are not limited to, aconitase or superoxide dismutase 2, or a targeting fragment thereof, e.g. a portion of the C-terminal sequence thereof.
  • mitochondrial targeting peptide sequences include, but are not limited to, MLSARSAIKRPIVRGLATV (SEQ ID NO: 1), MLRFTNCSCKTFV SSYKLNIRRMNTV (SEQ ID NO: 2),
  • MLRFTNCSCKTFVKSSYKLNIRRMNSSFRT (SEQ ID NO: 5), MLSRAVCGTSRQLAPV (SEQ ID NO: 6), or a targeting fragment of any one of these, e.g. a portion of the C-terminal sequence thereof.
  • Exosomes incorporating a mitochondrial-targeting fusion product may be produced, as described above, using recombinant technology.
  • an expression vector encoding the fusion product is introduced by electi poration or other transfection methods into exosome-producing cells isolated from an appropriate biological sample.
  • the desired mitochondrial product may be introduced into isolated exosomes incorporating a mitochondrial-targeting fusion product (modified mitochondrial-targeting exosomes) as previously described, using electroporation or other transfection methods. Addition to the exosomes incorporating the mitochondrial-targetting sequence and the desired mitochondrial product may increase delivery efficiency of the mitochondrial product.
  • Exosomes genetically engineered to incorporate a mitochondrial product, and/or nucleic acid encoding the mitochondrial product may be used to deliver the mitochondrial product and/or nucleic acid to a mammal in vivo in the treatment of a mitochondrial disease, e.g. a pathological condition or disease in which a mitochondrial product is dysfunctional or absent, to upregulate the activity of the target mitochondrial product and thereby treat the disease.
  • a mitochondrial disease e.g. a pathological condition or disease in which a mitochondrial product is dysfunctional or absent
  • Examples of mitochondrial diseases that are caused by mutations in nuclear- encoded mitochondrial genes and that may be treated using the present engineered exosomes are set out in Table 1 below.
  • Table 1 identifies the gene encoding the mitochondrial product involved in each disease, and gene mutations that result in dysfunctional/absent mitochondrial product, the Online Mendelian Inheritance in Man (OMIM) reference number as well as providing mRNA transcript sequence information and protein sequence information (e.g. NCBI (National Centre for Biotechnology Information) GenBank accession numbers for the mitochondrial products useful to treat each disease. Table 1.
  • OMIM Online Mendelian Inheritance in Man
  • Fatal infantile COX 15 e.700C>T 603646 NM 004376.5 NP 004367.2 cardioencephalomyopath (Cytochrome c oxidase or NM_078470.4 NP_510870.1 yand and Leigh assembly homolog 15) .4470G
  • Encephalomyopathy FAST D2 .12460T 612322 NM 001136193.1 NP 001129665.1 due to mitochondrial (FAST kinase domains NM 001136194.1 NP 001129666.1 complex IV deficiency 2) NMJI14929.3 NPJ155744.2
  • Carnitine CPT1A (Carnitine P.D454G 600528 NM 001031847.2 NP 001027017.1 palmitoyltransferase 1 palmitoyltransferase 1A NM classroom001876.3 NP_001867.2 (CPT I) deficiency (liver))
  • Cerebral creatine SLC6A8 (Solute carrier P.R514X 300036 NM 001142805.1 NP 001136277.1 deficiency syndrome- 1 family 6 NM 001142806.1 NP 001136278.1
  • mitochondrial diseases resulting from mutations in mitochondrial genes expressed in the mitochondria are set out in Table 2 below.
  • Table 2 identifies the mitochondrial gene product involved in each disease, and mutations thereof, as well as providing gene sequence information for the mitochondrial products useful to treat each disease by reference to the nucleotide position range of the gene for said mitochondrial product in the complete genome of the Homo sapiens mitochondrion (NCBI Genbank Reference Sequence: NC 012920.1) and the Online Mendelian Inheritance in Man (OMIM) reference number.
  • Mitochondrial myopathy MT-TM (Mitochondrially m.4409T C 590065 4402..4469 encoded tRNA Methionine)
  • Mitocliondrial encephalopathy MT-TW (Mitocliondrial ly m.5549G>A 590095 5512..5579 encoded tRNA tryptophan)
  • Myotonic dystrophy-like myopathy MT-TA (Mitochondrially m.5650G>A 590000 5587..5655 encoded tRNA alanine)
  • Isolated ophthalmoplegia MT-TN (Mitochondrially m.5703G>A 590010 56S7..5729 encoded fRNA asparagtne)
  • MELAS syndrome MT-TC (Mitochondrially m.5814A>G 590020 5761..5826 encoded tRNA cysteine)
  • MERRF/MELAS overlap syndrome MT-TS1 (Mitochondrially m.7512T C 590080 7446..7514 and encoded tRNA serine 1 (UCN))
  • Isolated mitochondrial myopathy MT-TD (Mitochondrially m.7526A>G 590015 7518..7585 encoded tRNA asparlic acid)
  • MERRF syndrome and MT-TK (Mitochondrially m.8344A>G 590060 S295..8364 Leigh syndrome and encoded tRNA lysine)
  • Hypertrophic cardiomyopathy MT-TG (Mitochondrially m.9997T>C 590035 9991..10058 encoded tRNA glycine)
  • Mitochondrial encepha!omyopathy MT-TR (Mitochondrially m. i 0438A>G 590005 10405..10469 encoded tRNA avginine)
  • Hypertrophic cardiomyopathy MT-TH ⁇ Mitochondrially m.l2192G>A 590040 12138..12206 encoded tRNA histidine
  • Mitochondrial encepha!omyopathy MT-TL2 (Mitochondrial ly m, 12315G>A 590055 12266..12336 encoded tRNA Leucine 2 (CUN))
  • Mitochondrial myopathy with diabetes MT-TE (Mitochondrially m.l4709T>C 590025 14674..14742 mellitus encoded tRNA glutamic acid)
  • exosomes are used to deliver to a mammal one or more mitochondrial products selected from the group consisting of nuclear-encoded mitochondrial products such as Lon peptidase 1, mitochondrial (LONPl), NADH ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADH ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH:ubiquinone oxidoreductase complex assembly factor 3 (NDUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH: ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl- CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase
  • NUBPL Nucleot
  • the present method is useful to treat a mitochondrial disease selected from the group consisting of Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQ10D), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG (e.g.
  • CPEO Chronic Progressive External Ophthalmoplegia syndrome
  • AHS Alpers-Huttenlocher syndrome
  • MCHS Childhood Myocerebrohepatopathy Spectrum
  • MEMSA Myoclonic Epilepsy Myopathy Sensory Ataxia
  • ANS Ataxia Neuropathy Spectrum (ANS) (including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO)), adPEO due to mutations in ANT or due to C10orf2 (twinkle) mutations, Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1/MyoNeurogenic Gastrointestinal Sideroblastic Encephalopathy (MNGIE), Mohr-Tranebjaerg syndrome, 3-methylglutaconic aciduri
  • Optic atrophy type 1 Optic atrophy type 1
  • Ethylmalonic encephalopathy Carnitine-acylcamitine translocase deficiency
  • Primary systemic carnitine deficiency Creatine deficiency syndromes (e.g.
  • Glutaric acidemia IIA, Glutaric acidemia IIB or Glutaric acidemia IIC Pyruvate dehydrogenase deficiency (e.g. Pyruvate dehydrogenase El-alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3 -binding protein deficiency, Pyruvate dehydrogenase E2 deficiency or Pyruvate dehydrogenase El -beta deficiency), 3-hydroxyacyl- CoA dehydrogenase (SCHAD) deficiency/(HADH) deficiency, and Perrault syndrome.
  • Pyruvate dehydrogenase deficiency e.g. Pyruvate dehydrogenase El-alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydr
  • the present method may also be effective to treat pathologies that result from secondary mitochondrial dysfunction including, but not limited to, metabolic syndrome, obesity, diabetes, blindness, deafness, cardiovascular diseases, movement disorders, neurometabolic and neuromuscular pathologies, epilepsy, autoimmune diseases, dementia, schizophrenia, bipolar disorder, neurodegenerative disease such as Alzheimer's disease and Parkinson's disease, sarcopenia, cancer, stroke, cachexia, cataracts, infertility, skin aging, and other aging-associated co -morbidities.
  • metabolic syndrome including, but not limited to, metabolic syndrome, obesity, diabetes, blindness, deafness, cardiovascular diseases, movement disorders, neurometabolic and neuromuscular pathologies, epilepsy, autoimmune diseases, dementia, schizophrenia, bipolar disorder, neurodegenerative disease such as Alzheimer's disease and Parkinson's disease, sarcopenia, cancer, stroke, cachexia, cataracts, infertility, skin aging, and other aging-associated co -morbidities.
  • the mitochondrial products for incorporation into exosomes according to the invention may be. a functional native mammalian mitochondrial product, including for example, a mitochondrial product from human and non- human mammals, or a functionally equivalent mitochondrial product.
  • the term "functionally equivalent” is used herein to refer to a protein which exhibits the same or similar function to the native protein (e.g. retains at least about 30% of the activity of the native protein), and includes all isoforms, variants, recombinant produced forms, and naturally-occurring or artificially modified forms, i.e. including modifications that do not adversely affect activity and which may increase cell uptake, stability, activity and/or therapeutic efficacy.
  • nucleic acid e.g. mRNA, rRNA, tRNA, DNA, or cDNA
  • encoding a mitochondrial product any nucleic acid sequence which encodes a functional mitochondrial product, including all transcript variants, valiants that encode mitochondrial product isoforms, variants due to degeneracy of the genetic code, artificially modified variants, and the like.
  • Protein modifications may include, but are not limited to, one or more amino acid substitutions (for example, with a similarly charged amino acid, e.g.
  • substitution of one amino acid with another each having non-polar side chains such as valine, leucine, alanine, isoleucine, glycine, methionine, phenylalanine, tryptophan, proline
  • substitution of one amino acid with another each having basic side chains such as histidine, lysine, arginine
  • substitution of one amino acid with another each having acidic side chains such as aspartic acid and glutamic acid
  • substitution of one amino acid with another each having polar side chains such as cysteine, serine, threonine, tyrosine, asparagine, glutamine), additions or deletions;
  • MLSARSAI RPIVRGLATV (SEQ ID NO: 1), MLRFTNCSCKTF V S SYKL I RRMNTV (SEQ ID NO: 2), MLRS S WRS RATLRPLLRRAYSS SFRT (SEQ ID NO: 3),
  • Suitable modifications will generally maintain at least about 70% sequence similarity with the active site and other conserved domains of a native mitochondrial protein, and preferably at least about 80%, 90%, 95% or greater sequence similarity.
  • Nucleic acid modifications may include one or more base substitutions or alterations, addition of 5' or 3' protecting groups, and the like, preferably maintaining significant sequence similarity, e.g. at least about 70%, and preferably, 80%, 90%, 95% or greater.
  • Engineered exosomes incorporating a mitochondrial product, and/or nucleic acid encoding the mitochondrial product, in accordance with the invention may be formulated for therapeutic use by combination with a pharmaceutically or physiologically acceptable earner.
  • pharmaceutically acceptable or “physiologically acceptable” means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable for physiological use.
  • the selected carrier will vary with intended utility of the exosome formulation.
  • exosomes are formulated for administration by infusion or injection, e.g.
  • a medical-grade, physiologically acceptable carrier such as an aqueous solution in sterile and pyrogen-free form, optionally, buffered or made isotonic.
  • the carrier may be distilled water (DNase- and RNase-free), a sterile carbohydrate-containing solution (e.g. sucrose or dextrose) or a sterile saline solution comprising sodium chloride and optionally buffered.
  • Suitable sterile saline solutions may include varying concentrations of sodium chloride, for example, normal saline (0.9%), half-normal saline (0,45%), quarter-normal saline (0.22%), and solutions comprising greater amounts of sodium chloride (e.g. 3%-7%, or greater).
  • Saline solutions may optionally include additional components, e.g. carbohydrates such as dextrose and the like. Examples of saline solutions including additional components, include Ringer's solution, e.g.
  • PBS phosphate buffered saline
  • TRIS hydroxymethyl) aminomethane hydroxymethyl) aminomethane
  • TBS TRIS-buffered saline
  • HBSS Hank's balanced salt solution
  • EBSS Earle's balanced solution
  • SSC standard saline citrate
  • HBS HEPES- buffered saline
  • GBSS Gey's balanced salt solution
  • the present exosomes are formulated for administration by routes including, but not limited to, oral, intranasal, enteral, topical, sublingual, intra-arterial, intramedullary, intrauterine, intrathecal, inhalation, ocular, transdermal, vaginal or rectal routes, and will include appropriate carriers in each case.
  • exosomes may be formulated in normal saline, complexed with food, in a capsule or in a liquid formulation with an emulsifying agent (honey, egg yolk, soy lecithin, and the like).
  • Oral compositions may additionally include adjuvants including sugars, such as lactose, trehalose, glucose and sucrose; starches such as com starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and com oil; polyols such as propylene glycol, glycerine, sorbital, mannitoi and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions.
  • sugars such as lactose, trehalose, glucose and sucrose
  • starches such as com starch and potato starch
  • Exosome compositions for topical application may be prepared including appropriate carriers. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents, anti-oxidants and other preservatives may be added to the composition to prevent microbial growth and/or degradation over prolonged storage periods.
  • the present engineered exosomes are useful in a method to treat a pathological condition involving a defective mitochondrial product, or a condition involving lack of expression of a mitochondrial product, e.g. a mitochondrial disease.
  • the terms "treat”, “treating” or “treatment” are used herein to refer to methods that favourably alter a mitochondrial disease or disorder, including those that moderate, reverse, reduce the severity of, or protect against, the progression of a mitochondrial disease or disorder.
  • a therapeutically effective amount of exosomes engineered to incorporate a functional mitochondrial product, and/or nucleic acid encoding the functional mitochondrial product, useful to treat the disease are administered to a mammal.
  • terapéuticaally effective amount is an amount of exosome required to treat the disease, while not exceeding an amount that may cause significant adverse effects
  • Exosome dosages that are therapeutically effective will vary on many factors including the nature of the condition to be treated as well as the particular individual being treated.
  • Appropriate exosome dosages for use include dosages sufficient to result in an increase in the amount or activity of the target mitochondrial product in the patient by at least about 10%, and preferably an increase in activity of the target mitochondrial product of greater than 10%, for example, at least 20%, 30%, 40%, 50% or greater.
  • the dosage may be a dosage in an amount in the range of about 1 ug to about 500 mg of total exosomal protein for the delivery of a mitochondrial protein, or an amount in the range of about 20 ng to about 200 mg of total exosomal protein for the delivery of RNA species such as mRNA, tRNA, rRNA, miRNA, SRP RNA, snR A, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, piRNA or total mitochondrial RNA.
  • RNA species such as mRNA, tRNA, rRNA, miRNA, SRP RNA, snR A, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, piRNA or total mitochondrial RNA.
  • a dosage of exosomes sufficient to deliver about 0.1 mg/kg to about 100 mg/kg of a protein mitochondrial product, or a dosage of exosomes sufficient to deliver about 1 ng/kg to about 100 ug kg of a nucleic acid mitochondrial product (e.g. an RNA species), is administered to the mammal in the treatment of a target mitochondrial disease.
  • a nucleic acid mitochondrial product e.g. an RNA species
  • exosomes comprising a mitochondrial product, and/or nucleic acid encoding the mitochondrial product may be used in conjunction with (at different times or simultaneously, either in combination or separately) one or more additional therapies to facilitate treatment, including but not limited to; anti-oxidants (i.e., coenzyme Q10, alpha lipoic acid, vitamin E, synthetic coenzyme Q10 analogues, resveratrol, N-acetylcysteine, etc.), creatine monohydrate, exercise (endurance, resistance or sprint-interval), mitochondrial targeting sequence motifs, or AMPK activators.
  • anti-oxidants i.e., coenzyme Q10, alpha lipoic acid, vitamin E, synthetic coenzyme Q10 analogues, resveratrol, N-acetylcysteine, etc.
  • creatine monohydrate i.e., coenzyme Q10, alpha lipoic acid, vitamin E, synthetic coenzyme Q10 analogues,
  • exosomes in a therapy to treat mitochondrial disease advantageously results in delivery of nucleic acid (mRNA, lRNA and tRNA) or protein safely to a cell to treat either mitochondrial or nuclear defects.
  • nucleic acid mRNA, lRNA and tRNA
  • the use of exosomes overcomes the challenges of delivery to the mitochondria, including the double mitochondrial membrane and the elaborate system of import that includes the TIMM and TOMM complex as well as various chaperone proteins.
  • exosomes were engineered to treat two of the most common and representative examples of mitochondrial DNA-based disease: LHON m.l l778G>A and MELAS m.3243A>G.
  • Exosome isolation methodology Immature dendritic cells from human and mice are grown to 65-70% confluency in alpha minimum essential medium supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 niM sodium pyruvate and 5 ng/ml murine GM-CSF, and 20% fetal bovine serum.
  • ribonucleosides deoxyribonucleosides
  • 4 mM L-glutamine 4 mM L-glutamine
  • 1 niM sodium pyruvate 1 niM sodium pyruvate
  • 5 ng/ml murine GM-CSF murine GM-CSF
  • 20% fetal bovine serum 20% fetal bovine serum.
  • conditioned media collection cells were washed twice with sterile PBS (pH 7.4, Life Technologies) and aforementioned media (with exosome depleted fetal bovine serum) was added.
  • the media (10 mL) was spun at 2,000x g for 15 min at 4°C to remove any cellular debris. This is followed by 2000x g spin for 60 min at 4°C to further remove any contammating non-adherent cells. The supernatant was then spun at 14,000x g for 60 min at 4°C. The resulting supernatant was then filtered through a 40 ⁇ filter, followed by filtration through a 0.22 ⁇ syringe filter (twice) and finally filtered with a 0.10 ⁇ syringe filter.
  • the resultant filtered supernatant was spun at 50,000x g for 60 min at 4°C, The supernatant was then carefully transferred into ultracentrifuge tubes and diluted with an equal amount of sterile PBS (pH 7.4, Life Technologies). This mixture was then subjected to ultracentrifugation at 100,000x-170,000x g for 2 hours at 4°C using a fixed-angle rotor. The resulting pellet was re-suspended in PBS and re-centrifuged at 100,000x- 170,000x g for 2 hours at 4°C.
  • the pellet was resuspended carefully with 600 ⁇ ] ⁇ of sterile PBS (pH 7.4, Life Technologies) and then gently added on top of 300 L of 30% Percoll cushion in an ultracentrifuge tube. This mixture was spun at 100,000x-170,000x g for 90 minutes at 4°C. With a syringe/pipette, the exosomal fraction (pellet-containing) was isolated carefully followed by a final spin for 90 minutes at 100,000x-170,000x g at 4°C to obtain purified exosomes. The resulting exosomes were resuspended in sterile PBS or sterile 0.9% saline for downstream use. Exosomal fraction purity was confirmed by sizing using a NanoSight LM10 instrument, and by immuno-gold labelling/Western blotting using the exosome membrane markers, CD9, CD63, TSG101 and ALIX.
  • ND4 NCBI Reference Sequence: NC Ol 1137.1
  • cDNA from skeletal muscle was sub-cloned into the mammalian vector, pGEX GST-fusion vector (GE Healthcare Life Sciences) to form an ND4-pGEX vector.
  • the vector was maintained using the competent E. coll DHSalpha cell-line (Life Technologies).
  • the pGEX mammalian vector was then transfected into Chinese Hamster Ovary Cells (CHO; ATCC Cat. CCL-661) for mass production of recombinant human ND4 protein.
  • CHO cells transfected with ND4- pGEX vector were lysed using known techniques and CHO cell iysate was cleared using ultra- performance resins for GST-tagged fusion protein purification (GE Healthcare Life Sciences). Over 75% of the recombinant protein was eluted after 3 washes. Elution #1 and Elution #2 were combined to obtain a high yield of protein. GST tag was removed from recombinant ND4 using PreScission Protease (GE Healthcare Life Sciences).
  • ND4 cDNA from skeletal muscle was sub-cloned into pCMV6 entry vector (Origene) and amplified. Using conventional PCR, start codon (ATG) and Kozak sequence (GCCACC) were introduced. This cDNA was then cloned into the pMRNA p plasmid (System Biosciences) using EcoRI and BamHI restriction enzymes sites and the plasmids were used to transform competent E. coli DH5alpha cell-line (Life Technologies). Colonies containing the ND4 vector were amplified.
  • the vector was isolated from these colonies (Qiagen) and T7 RNA polymerase-based in vitro transcription reaction was carried out to yield ND4 mRNA.
  • An anti-reverse cap analog (ARCA), modified nucleotides (5-Methylcytidine-5'- Triphosphate and Pseudouridine-5'-Triphosphate) and poly-A tail were incorporated within the mRNAs to enhance their stability and to reduce the immune response of host cells.
  • DNase I digest and phosphatase treatment was carried out to remove any DNA contamination and to remove the 5' triphosphates at the end of the RNA to further reduce innate immune responses in mammalian cells, respectively.
  • the clean-up spin columns were used to recover ND4 mRNA for downstream encapsulation in engineered exosomes.
  • Percoll-pmified mitochondrial fraction (from healthy human subject muscle biopsy) was sub- cloned into pCMV6 entry vector (Origene) and amplified. T7 RNA polymerase-based in vitro transcription reaction was carried out followed by charging of tRNA eu ⁇ m) with Leu amino acid using aminoacyl tRNA synthetase, LARS2, which was over- expressed and isolated from CHO cells.
  • LARS2 aminoacyl tRNA synthetase
  • Electroporation mixture was prepared by carefully mixing exosomes and ND4 mRNA, protein or tRNALeu ⁇ in 1 :1 ratio (for example, 150 ⁇ x exosome suspension (10-15 £/ ⁇ 3_, of total protein concentration as determined by BCA) to 150 ⁇ i of mRNA, tRNA, or protein suspension (at a concentration of 100-1000 in electroporation buffer. Electroporation was earned out in 0,4 mm electroporation cuvettes at 400 mV and 125 capacitance (pulse time 14 ms for mRNA and 24 ms for protein) using Gene Pulse XCell electroporation system (BioRad).
  • exosomes were resuspended in 20 mL of 0.9% saline solution followed by ultracentrifugation for 2 hours at 170,000x g at 4°C.
  • ND4 mRNA or protein
  • tRNA-Leu-loaded exosomes were re-suspended in 5% (wt/vol) glucose in 0.9% saline solution.
  • the resulting homogenate was centrifuged for 15 min at 700x g at 4°C, and the resulting supernatants were centrifuged for 20 min at 12,000x g at 4°C.
  • the mitochondrial pellets from the 12,000x g spin were washed and then resuspended in a small volume of ice-cold isolation buffer B (10 mM sucrose, 0.1 mM EGTA/Tris, and 10 M Tris/HCl (pH 7.4), supplemented with Complete ETDA-free protease inhibitor mixture (Roche Applied Science)). All centrifugation steps were carried out at 4 °C.
  • the mitochondrial pellets were then immediately flash frozen with liquid nitrogen and thawed 3 times to break open the mitochondria.
  • Mitochondrial total RNA (including mRNA, rRNA, tRNA, and miRNA species) was isolated using Qiagen total RNeasy kit and packaged into exosomes as described above.
  • CS mitochondrial complex I/citrate synthase
  • OCR oxygen consumption rate
  • exosomes engineered to treat mitochondrial disease resulting from mutations in mitochondrial genes expressed in the nucleus exosomes engineered to treat LONP1 deficiency, PolG deficiency and Friedreich ataxia (representative of such mitochondrial disease) were prepared and tested as described in the foregoing and further below.
  • Quadriceps femoris were harvested from all mice for Western blotting using mito-cocktail antibody (Mitosciences) to probe for subunits of mitochondrial electron transport chain (mitochondrial OXPHOS subunits) as previously described (Safdar et al., PNAS 108(10), 4135- 40, 2011).
  • mito-cocktail antibody Mitosciences
  • mitochondrial electron transport chain mitochondrial electron transport chain
  • NCBI Reference Sequence: NM 004793 from skeletal muscle was sub-cloned into pCMV6 entry vector (Origene) and amplified as described for ND4 mRNA above.
  • the cDNA was then cloned separately into the pMRNA* p plasmid (System Biosciences) using EcoRl and BamHI restriction enzymes sites and the plasmids were used to transform competent E. coli DH5alpha cell-line (Life Technologies). Colonies containing the LONPl vectors were amplified, isolated (Qiagen) and a T7 RNA polymerase- based in vitro transcription reaction was carried out to yield mRNA. Further processing was as described for ND4 mRNA.

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Abstract

A method of treating a mitochondrial disease in a mammal is provided, wherein the mitochondrial disease results from a mutation in nucleic acid encoding a mitochondrial product, The method comprises administering to the mammal a therapeutically effective amount of non-naturally occurring exosomes engineered to comprise total mammalian mitochondrial RNA, nucleic acid encoding the mitochondrial product, or the functional mitochondrial product.

Description

METHOD FOR TREATING MITOCHONDRIAL DISEASE
Field of the Invention
[0001] The present invention generally relates to treatment of mitochondrial disease, and more particularly relates to a method of treating mitochondrial disease using exosomes.
Background of the Invention
[0002] Mitochondria are intracellular organelles that have a variety of functions. They are best known for their ability to produce ATP from reducing equivalents derived from fat, protein and carbohydrates. The reducing equivalents, FAD¾ and NADH+H+, are delivered to the respiratory chain and used to pump a proton from the matrix of the mitochondria to the inter- membrane space. Through a series of oxidation/reduction reactions, the electrons are transported from complex I and II to coenzyme Q10 and then on to complex III, cytochrome c and complex IV where oxygen is reduced to molecular water. The flow of electrons leads to the pumping of the proton complex I, III and IV. The build-up of protons in the inter-membrane space leads to a proton motive force where the electrons flow through complex V and re-phosphorylate ADP to ATP. In addition to the function as an aerobic source of ATP, the mitochondria are also involved in other cellular processes including: calcium buffering, apoptosis, oxidative stress, telomere maintenance, and activation of inflammatory pathways such as the inflammasome.
[0003] The mitochondria are thought to have their origin as bacteria that took on a symbiotic relationship with a proto-eukaryotic cell 1.5 billion years ago. Throughout evolution the approximate 1500 genes that are required for mitochondrial biogenesis and maintenance have been transferred to the nuclear DNA, whilst the human mitochondrial DNA retains 37 of these 1500 genes in a small circular piece of DNA called mitochondrial DNA (mtDNA). This circular DNA resembles bacterial DNA (likely from its origin) and undergoes polycistronic replication. Most of the mitochondrial DNA contains exons and the repair mechanisms are not as sophisticated as those in the nuclear DNA. This is associated with an increased propensity for mutagenesis in mtDNA verses nuclear DNA.
[0004] Mitochondrial diseases can be caused by genetic defects in many of the mitochondrial (mtDNA) or nuclear DNA (nDNA) genes that encode a mitochondrial localized protein or NA species (mtDNA only). Mutations in mtDNA can affect any of the 2 ribosomal RNAs (rRNA), 22 of the transfer RNAs (tRNA) or 13 protein-coding subunits (N = 7 complex I, N = 1 complex III, N = 3 complex IV and N= 2 complex V). Mitochondrial DNA is maternally inherited. nDNA mutations can affect genes involved in mitochondrial DNA replication or maintenance or structural components. The nuclear mutations can be inherited in an autosomal recessive, autosomal dominant or X-linked recessive manner, Dysfunction of the mitochondria leads to anaerobic ATP generation with an increased reliance on anaerobic pathways. This leads to inefficient energy generation and the production of lactic acid (through glycolysis). The role of mtDNA mutations (both sporadic and familial) and mitochondrial dysfunction is becoming increasingly apparent in broad range of metabolic and degenerative diseases, cancer, and aging.
[0005] The first mutations were found in mtDNA in the late 1980s, including
Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS) resulting from the mutation, 3243A>G; Leber Hereditary Optic Neuropathy (LHON) resulting from the mutation, 11778G>A; as well as large scale deletions that result in Kearn-Sayre- Syndrome (KSS). Subsequently, many hundreds of mtDNA mutations have been described ("http://www.mitomap.org/MITOMAP). Many nuclear mutations have now been discovered including; NDUF mutations in Leigh syndrome, SC02 mutations in infantile cardiomyopathy, POLG mutations (mutations in gene that codes for DNA polymerase gamma) in many disorders such as SANDO (sensory ataxic neuropathy, dysarthria, and ophthalmoparesis), ataxia and Alper Syndrome, SPG7 mutations in hereditary spastic paraparesis, MFN2 mutations in peripheral neuropathy, and others f http : //www.mitomap. org/MITOMAP) . Mitochondrial diseases are heterogeneous and often multi-systemic due to the fact that mitochondria are present in all tissues in the human body with the exception of mature red blood cells. Because the mitochondrion provides much of the energy for the cell, mitochondrial disorders preferentially affect tissues with high energy demand, including the brain, muscle, and heart, although any organ (including liver, pancreas, bone marrow, etc.) can be affected. Consequently, mitochondrial defects are implicated in forms of blindness, deafness, movement disorders, dementias, cardiomyopathy, myopathy, renal dysfunction, and aging. At the molecular level, in addition to energy crisis, mitochondrial dysfunction can also lead to telomere shortening, oxidative stress, apoptosis and inflammasome activation. [0006] Although the identification of the first mtDNA mutations occurred over 20 years ago, treatment of mitochondrial diseases has been largely supportive (i.e., anti-seizure medications, nutrition, spasticity control, etc.). Minimal success has been achieved using nutraceuticals, such as coenzyme Q10, alpha lipoic acid, creatine monohydrate and riboflavin, that target the cellular consequences of mitochondrial dysfunction. Synthetic anti-oxidants, such asidebenone and other CoQlO-like substances, as well as compounds that enhance mitochondrial biogenesis, and lower lactic acidosis, have also been used to treat individuals with mitochondrial disease. However, all of the above options are supportive measures at best and far from curative.
[0007] Thus, there is a need to develop improved methods of treating mitochondrial dysfunction.
Summary of the Invention
[0008] It has now been determined that exosomes may be effectively used as a vehicle to deliver a mitochondrial product to a mammal to treat pathological conditions such as a mitochondrial disease resulting from a deficiency of a functional mitochondrial product.
[0009] Thus, in one aspect of the invention, exosomes are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
[0010] In another aspect, a method of increasing the amount of a mitochondrial product in mitochondria in a mammal is provided, comprising administering to the mammal exosomes that are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
[0011] In another aspect, a method of increasing the activity of a target mitochondrial product in a mammal is provided, comprising administering to the mammal a composition comprising exosomes which are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product. [0012] In another aspect, a method of treating a pathological condition in a mammal resulting from the deficiency of a functional mitochondrial product is provided comprising administering to the mammal a composition comprising exosomes genetically engineered to incorporate the mitochondrial product and/or nucleic acid encoding the mitochondrial product.
[0013] In a further aspect, a method of treating a mitochondrial disease in a mammal is provided comprising administering to the mammal a composition comprising exosomes genetically engineered to incorporate a mitochondrial product useful to treat the mitochondrial disease and/or nucleic acid encoding the mitochondrial product.
[0014] Additional aspects of the invention include aspects and variations set forth in the following lettered paragraphs:
[0015] A 1. An exosome produced by a process that comprises: (a) isolating exosomes from a biological sample from an organism or from a conditioned medium from a cultured cell; and (b) introducing a modification into the exosome selected from the group consisting of:
[0016] (i) at least one functional mitochondrial product or precursor thereof;
[0017] (ii) at least one nucleic acid comprising a nucleotide sequence that encodes the functional mitochondrial product or precursor thereof;
[0018] (iii) the combination of (i) and (ii).
[0019] A2. The exosome according to paragraph Al , wherein the isolating includes at least one density gradient centrifugation step ideally using Percoll or other colloidal silica product.
[0020] A3. The exosome according to any one of paragraphs Al to A2, wherein the isolating removes vesicles that are greater than 120 nm in diameter.
[0021] A4. The exosome according to paragraph Al to A3, wherein the biological sample is from a mammal, or the cell is from a mammal or a mammalian ceil line. [0022] A5. The exosome according to any one of paragraphs Al to A4, wherein the isolating removes vesicles and cellular debris less than 20 nm in diameter.
[0023] A6. An exosome that comprises a modification selected from the group consisting of:
[0024] (i) at least one functional mitochondrial product or precursor thereof;
[0025] (ii) at least one nucleic acid comprising a nucleotide sequence that encodes the functional mitochondrial product or precursor thereof;
[0026] (iii) the combination of (i) and (ii).
[0027] Bl . The exosome according to any of paragraphs Al - A6, having a diameter of 20- 120 nm.
[0028] B2. The exosome according to any of paragraphs Al - A6, that comprises a functional mitochondrial product or precursor thereof, wherein the mitochondrial product is present in a lumen of the exosome.
[0029] B3. The exosome according to any of paragraphs Al - A6, that comprises a nucleic acid comprising a nucleotide sequence encoding a functional mitochondrial product or precursor thereof, wherein the nucleic acid is present in a lumen of the exosome.
[0030] B3.1. The exosome according to paragraph B3, wherein the nucleic acid comprises a species of RNA or a species of modified RNA (modRNA, e.g. 5 methyl cytosine, or N6 methyladenine) encoding for a mitochondrial product set forth in Table 1 and/or Table 2.
[0031] B4. The exosome according to paragraph B2 or B3 or B3.1, wherein the mitochondrial product comprises one or more of the mitochondrial products set forth in Table 1 and/or Table 2.
[0032] B5. The exosome according to any one of paragraphs B2 - B3.1, wherein the mitochondrial product is selected from the group consisting of Lon peptidase 1, mitochondrial (LONP1), NADH:ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADH:ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH:ubiquinone oxidoreductase complex assembly factor 3 ( DUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH:ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl-CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1), Succinate dehydrogenase complex assembly factor 2 (SDHAF2), BCS1 homolog, ubiquinol-cytochrome c reductase complex chaperone (BCS1L), Surfeit 1 (SURFl), SCOl cytochrome c oxidase assembly protein (SCOl), COXIO heme A:farnesyltransferase cytochrome c oxidase assembly factor (COXIO), Cytochrome c oxidase assembly homolog 15 (COX15), Leucine rich pentatricopeptide repeat containing (LRPPRC), FAST kinase domains 2 (FAST D2), Translationai activator of mitochondrially encoded cytochrome c oxidase I (TACOl), ATP synthase mitochondrial Fl complex assembly factor 2 (ATPAF2), Transmembrane protein 70 (TMEM70), Polymerase (DNA directed), gamma or DNA polymerase gamma (POLG), Polymerase (DNA directed), gamma 2, accessory subunit (POLG2), MpV17 mitochondrial inner membrane protein (MPV17), Chromosome 10 open reading frame 2 (C10orf2), Thymidine phosphorylase (TYMP), Deoxyguanosine kinase (DGUOK), Ribonucleotide reductase M2 B (RRM2B), Succinate-CoA ligase, ADP-forming, beta subunit (SUCLA2), Succinate-CoA ligase, alpha subunit (SUCLG1), Thymidine kinase 2, mitochondrial (TK2), Translocase of inner mitochondrial membrane 8 homolog A (TIMM8A), DnaJ heat shock protein family (Hsp40) member C19 (DNAJC19), G elongation factor, mitochondrial 1 (GFMl), Aminoacyl tRNA synthetase 2 (e.g. Leucyl-tRNA synthetase 2 (LARS2), TyrosyL-tRNA synthetase 2 (YARS2), Seryl-tRNA synthetase 2, mitochondrial (SARS2), Aspartyl-tRNA synthetase 2, mitochondrial (DARS2), and Arginyl-tRNA synthetase 2, mitochondrial (RARS2)), Mitochondrial ribosomal protein S16 (MRPS16), Mitochondrial ribosomal protein S22 (MRPS22), Ts translation elongation factor, mitochondrial (TSFM), Tu translation elongation factor, mitochondrial (TUFM), Frataxin (FXN), ATP-binding cassette subfamily B member 7 (ABCB7), Solute carrier family 25 member 38 (SLC25A38), Iron-sulfur cluster assembly enzyme (ISCU), BolA family member 3 (BOLA3), NFU1 iron-sulfur cluster scaffold (NFU3), Coenzyme Q2 4- hydroxybenzoate polyprenyltransferase (COQ2), Coenzyme Q4 (COQ4), Coenzyme Q9 (COQ9), Aprataxin (APTX), Prenyl (decaprenyl) diphosphate synthase, subunit 1 (PDSS1), Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2), AarF domain containing kinase 3 (ADCK3), Paraplegin (SPG7), Heat shock protein family D (Hsp60) member 1 (HSPD1), Optic atrophy 1 (autosomal dominant) (OPA1), Mitofusin 2 ( FN2), Dynamin 1-like (DNM1L), Tafazzin (TAZ), Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Ethylmalonic encephalopathy 1 (ETHE1), Pseudouridylate synthase 1 (PUS1), NADH:ubiquinone oxidoreductase core subunit SI (NDUFSl), NADH:ubiquinone oxidoreductase core subunit S2 (NDUFS2), NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), NADH:ubiquinone oxidoreductase subunit S6 (NDUFS6), NADH:ubiquinone oxidoreductase core subunit S7 (NDUFS7), NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8), NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3), NADH:ubiquinone oxidoreductase core subunit VI (NDUFVl), NADH:ubiquinone oxidoreductase core subunit V2 (NDUFV2), NADHmbiquinone oxidoreductase subunit Al (NDUFA1), NADH:ubiquinone oxidoreductase subunit A2 (NDUFA2), NADH ubiquinone oxidoreductase subunit A10 (NDUFA10), NADH:ubiquinone oxidoreductase subunit Al l (NDUFA11), Succinate dehydrogenase complex subunit A, flavoprotein (Fp) (SDHA), Succinate dehydrogenase complex subunit B, iron sulfur (Ip) (SDHB), Succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa (SDHC), Succinate dehydrogenase complex subunit D, integral membrane protein (SDHD), Ubiquinol-cytochrome c reductase binding protein (UQCRB), Ubiquinol-cytochrome c reductase complex III subunit VII (UQCRQ), Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) (COX6B1), ATP synthase, H+ transporting, mitochondrial Fl complex, epsilon subunit (ATP5E), Solute carrier family 22 (organic cation/camitine transporter), member 5 (SLC22A5), Carnitine palmitoyltransferase 2 (CPT2), Carnitine palmitoyltransferase 1A (liver) (CPT1A), Solute carrier family 6 (neurotransmitter transporter), member 8 (SLC6A8), Guanidinoacetate N-methyltransferase (GAMT), Glycine amidinotransferase (GATM), Pyruvate carboxylase (PC), hydroxyacyl-CoA dehydrogenase/3 -ketoacyl-Co A thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA)/(LCHAD), Acyl-CoA dehydrogenase, very long chain (ACADVL), Acyl- CoA dehydrogenase, C-2 to C-3 short chain (ACADS), Hydroxyacyl-CoA dehydrogenase (HADH)/(SCHAD), Electron transfer flavoprotein alpha subunit (ETFA), Electron transfer flavoprotein beta subunit (ETFB), Electron transfer flavoprotein dehydrogenase (ETFDH), Solute earner family 25 (SLC25A20), Pyruvate dehyrogenase phosphatase catalytic subunit 1 (PDP1), Pyruvate dehydrogenase complex component X (PDHXD), Dihydrolipoamide S- acetyltransferase (DLAT), Pyruvate dehydrogenase (lipoamide) beta (PDHB), Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member (4SLC25A4)/(ANT), Mitochondrially encoded NADH dehydrogenase 1 (MT-NDl), Mitochondrially encoded NADH dehydrogenase 2 (MT-ND2), Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3), Mitochondrially encoded NADH dehydrogenase 4 (MT-ND4 or ND4)5 Mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), Mitochondrially encoded NADH dehydrogenase 5 (MT- ND5), Mitochondrially encoded NADH dehydrogenase 6 (MT-ND6 or ND6), Mitochondrially encoded cytochrome b (MT-CYB), Mitochondrially encoded cytochrome c oxidase I (MT-COI), Mitochondrially encoded cytochrome c oxidase II (MT-COII), Mitochondrially encoded cytochrome c oxidase III (MT-COIII), ATPase6 (MT-ATP6), ATPase8 (MT-ATP8), 12S rRNA (MT-RNR1), 16S rRNA (MT-RNR2), Mitochondrially encoded tRNA threonine (MT-TT), Mitochondrially encoded tRNA proline (MT-TP), Mitochondrially encoded tRNA phenylalanine (MT-TF), Mitochondrially encoded tRNA valine (MT-TV), Mitochondrially encoded tRNA leucine 1 (UUA/G) or tRNALeu UUR) (MT-TL1), Mitochondrially encoded tRNA isoleucine (MT-TI), Mitochondrially encoded tRNA glutamine (MT-TQ), Mitochondrially encoded tRNA Methionine (MT-TM), Mitochondrially encoded tRNA tryptophan (MT-TW), Mitochondrially encoded tRNA alanine (MT-TA), Mitochondrially encoded tRNA asparagine (MT-TN), Mitochondrially encoded tRNA cysteine (MT-TC), Mitochondrially encoded tRNA tyrosine (MT-TY), Mitochondrially encoded tRNA serine 1 (UCN) (MT-TS1), Mitochondrially encoded tRNA aspartic acid (MT-TD), Mitochondrially encoded tRNA lysine (MT-TK), Mitochondrially encoded tRNA glycine (MT-TG), Mitochondrially encoded tRNA arginine (MT-TR), Mitochondrially encoded tRNA histidine (MT-TH), Mitochondrially encoded tRNA Serine 2 (AGY) (MT-TS2), Mitochondrially encoded tRNA Leucine 2 (CUN) (MT-TL2) and Mitochondrially encoded tRNA glutamic acid (MT-TE).
[0033] CI . A composition comprising exosomes according to any one of paragraphs
Al - A6, and a pharmaceutically acceptable carrier.
[0034] C2. The composition according to paragraph CI, wherein the composition is substantially free of vesicles having a diameter less than 20 nm. [0035] C3. The composition according to paragraph CI or C2, wherein the composition is substantially free of vesicles having a diameter greater than 120nm.
[0036] C4. The composition according to any one of claims CI - C3, which exhibits a zeta potential having a magnitude of at least 30 mV, or at least 40 mV, or at least 50 mV, or at least 60 mV, or at least 70 mV, or at least 80 mV, e.g. 30-80 mV, or -80 to -30 mV.
[0037] C5. The composition according to claim C4, which exhibits a zeta potential having a magnitude of up to 200 mV, or up to 175 mV, or up to 150 mV, or up to 140 mV, or up to 130 mV, or up to 120 mV, or up to 110 mV, or up to 100 mV.
[0038] Dl , A method of increasing the amount of a mitochondrial product in mitochondria in a mammal, comprising administering to the mammal an exosome according to any one of paragraphs Al - B6, or a composition according to any one of paragraphs CI - C5.
[0039] D2. Use of an exosome according to any one of paragraphs Al - B6, or a composition according to any one of paragraphs CI - C5, for increasing the amount of a mitochondrial product in mitochondria in a mammal.
[0040] D3. A method of treating a mitochondrial disease in a mammal comprising administering to the mammal an exosome according to any one of paragraphs Al - B6, or a composition according to any one of paragraphs CI - C5.
[0041] D4. Use of an exosome according to any one of paragraphs Al - B6, or a composition according to any one of paragraphs CI - C5, for treating a mitochondrial disease in a mammal.
[0042] D5. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human.
[0043] D6. The method or use according to paragraph D5, wherein the human has a mitochondrial disease selected from the group consisting of Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQ10D), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG (e.g. Chronic Progressive External Ophthalmoplegia syndrome (CPEO), Alpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS) (including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO)), adPEO due to mutations in ANT or due to C10orf2 (twinkle) mutations, Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1/MyoNeurogenic Gastrointestinal Encephalopathy (MNGIE), Mohr-Tranebjaerg syndrome, 3-methylglutaconic aciduria, Combined oxidative phosphorylation deficiency (COXPD), Myopathy, Lactic Acidosis, and Sideroblastic Anemia (ML AS A), Hyperuricemia, Pulmonary hypertension, Renal failure, and Alkalosis (HUPRA) syndrome, Leigh Syndrome, Leigh syndrome-French Canadian type, Friedreich ataxia, Gracile syndrome, Bjo nstad syndrome, Multiple Mitochondrial Dysfunctions Syndrome (MMDS), Early-onset Ataxia with Ocular motor apraxia and Hypoaibuminemia (EAOH), Charcot-Marie-Tooth Disease-2A2, Leber Hereditary Optic Neuropathy (LHON), Sudden Infant Death Syndrome, Myoclonic Epilepsy with Ragged Red Fibers (MERRF), MERRF/MELAS overlap syndrome, Neuropathy, Ataxia, Retinitis Pigmentosa (NARP), Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS), Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL), Mitochondrial disease resulting from SDH mutations (e.g. Pheochromocytoma and Paragangliomas), Optic atrophy type 1, Ethylmalonic encephalopathy, Carnitine-acylca nitine translocase deficiency, Primary systemic carnitine deficiency, Creatine deficiency syndromes (e.g. Cerebral creatine deficiency syndrome- 1, Cerebral creatine deficiency syndrome-2 or Cerebral creatine deficiency syndrome-3), Carnitine palmitoyltransferase 1 (CPT I) deficiency, Carnitine palmitoyltransferase 2 (CPT II) deficiency, Short-chain acyl-CoA dehydrogenase deficiency, Very long chain acyl-CoA dehydrogenase deficiency, Long-chain 3-hydroxyl-CoA dehydrogenase (LCHAD) deficiency, Pyruvate carboxylase deficiency, Multiple acyl-CoA dehydrogenase deficiency (e.g. Glutaric acidemia IIA, Glutaric acidemia ΠΒ or Glutaric acidemia IIC), Pyruvate dehydrogenase deficiency (e.g. Pyruvate dehydrogenase El -alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3-binding protein deficiency, Pymvate dehydrogenase E2 deficiency or Pyruvate dehydrogenase El -beta deficiency), 3-hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency/(HADH) deficiency, and Perrault syndrome.
[0044] D7. The method or use according to any one of paragraphs Dl - D4, wherein the mammal is human and has a disease set forth in Table 1 and/or Table 2, and the exosome contains the corresponding mitochondrial product in Table 1 and/or Table 2, or a nucleic acid encoding said mitochondrial product,
[0045] The invention further includes numerous embodiments, aspects, and variations that will be apparent from the drawings, detailed description, and claims that follow.
[0046] These and other aspects of the invention will be described by reference to the following figures.
Brief Description of the Figures
[0047] Figure 1 graphically illustrates that (A) mitochondria-encoded NADH dehydrogenase 4 (ND4) mRNA or ND4 protein-loaded exosomes rescue mitochondrial complex I deficiency in primary fibroblasts isolated from Leber Hereditary Optic Neuropathy (LHON) patients; (B) Mean ± SD of experiments in (A) independently repeated three experiments. *P < 0.05. Data were analyzed using an unpaired /-test;
[0048] Figure 2 graphically illustrates that ND4 mRNA or ND4 protein-loaded exosomes improves oxygen consumption rate of primary fibroblasts isolated from LHON (m.11778 G>A) patients. Mean ± SD of independently repeated three experiments. *P < 0.05. Data were analyzed using one-way ANOVA, followed by Tukey post hoc test;
[0049] Figure 3 graphically illustrates that exosomes packaged with mitochondrial total
RNA rescues mitochondrial dysfunction in a DNA polymerase gamma (POLG) mutator mouse model of systemic mitochondrial disease; [0050] Figure 4 graphically illustrates that (A) tRNALeu(UUR)-loaded exo somes rescue mitochondrial complex IV deficiency in primary fibroblasts isolated from MELAS (m.3243A>G) patients; (B) Mean ± SD of experiments in (A) independently repeated three experiments. *P < 0.05. Data were analyzed using an unpaired Mest; and
[0051] Figure 5 graphically illustrates that Lon Peptidase 1, mitochondrial (LONPl) mR A-loaded exosomes rescue mitochondrial complex IV deficiency in primary fibroblasts isolated from patients with impaired and/or reduced LONPl protein activity and/or levels (LONPl patients). *P < 0.05. Data were analyzed using an unpaired t-test.
Detailed Description of the Invention
[0052] A method of treating a mitochondrial disease in a mammal is provided in which the mitochondrial disease results from a nucleic acid mutation that results in a dysfunctional mitochondrial product. The method comprises administering to the mammal a therapeutically effective amount of exosomes engineered to comprise a functional target mitochondrial product or nucleic acid encoding a functional target mitochondrial product.
[0053] The term "functional" with respect to a target mitochondrial product is used herein to refer to a protein or nucleic acid product which retains innate biological activity, including but not limited to, catalytic, metabolic, regulatory, binding, transport and the like. As will be appreciated by one of skill in the art, to be functional, a target mitochondrial product need not exhibit an endogenous level of biological activity, but will exhibit sufficient activity to render it useful to treat a mitochondrial disease, e.g. at least about 25% of the biological activity of the corresponding endogenous mitochondrial product, and preferably at least about 50% or greater of the biological activity of the corresponding endogenous mitochondrial product.
[0054] The present method may be used to treat any form of mitochondrial disease or disorder resulting from mitochondrial dysfunction. Mitochondrial dysfunction results from a nucleic acid mutation, e.g. familial or sporadic nucleic acid mutation, including, for example, a gene or other nucleic acid mutation (such as nucleic acid rearrangements, deletions, point mutations, nonsense mutations and the like) that results in a dysfunctional mitochondrial product. The term "mitochondrial product" is used herein to refer to mitochondrial proteins or precursors or functional sub-units of mitochondrial proteins, and mitochondrial RNA species, including but not limited to, ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA (mRNA), micro RNA (miRNA), small nuclear RNA (snRNA), small cytoplasmic RNA (scRNA), signal recognition particle (SRP) RNA, small nucleolar (sno) RNA, guide RNA (gRNA), ribonuclease P (RNase P), ribonuclease MRP (RNase MRP), yRNA, telomerase RNA component (TERC), spliced leader RNA (SLRNA), long non-coding RNA (IncRNA), piwi-interacting RNA (piRNA) and total mitochondrial RNA (e.g. from a healthy mammal not having a target mitochondrial disease). Mitochondrial proteins and RNA species may or may not be synthesized in the mitochondria. Thus, the term "mitochondrial product" also encompasses protein and RNA species synthesized outside of the mitochondria, e.g. in the nucleus (i.e. nuclear-encoded mitochondrial product), which is subsequently imported into the mitochondria. For example, the term "mitochondrial protein" is used herein to refer to proteins, or sub-units thereof, synthesized in the mitochondria from mitochondrial DNA (e.g. mitochondria-encoded), as well as proteins synthesized outside of the mitochondria which are imported into and functional in the mitochondria. Likewise, mitochondrial RNA encoding a mitochondrial protein may be synthesized outside of the mitochondria and subsequently imported as RNA, or its protein product, into the mitochondria.
[0055] The term "exosome" refers to cell-derived vesicles having a diameter of between about 20 and 120 nm, for example, a diameter of about 50-100 nm, including exosomes of a diameter of about 60 nm, 70 nm, 80 nm, 90 nm, and/or 100 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g. immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like). As one of skill in the art will appreciate, cultured cell samples will be in the cell-appropriate culture media (using exosome- free serum). Exosomes include specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g. CD 9, CD37, CD44, CD53, CD63, CD81, CD82 and CD 151 ; targeting or adhesion markers such as integrins, ICAM-1, EpCAM and CD31; membrane fusion markers such as annexins, TSGlOl, ALIX; and other exosome transmembrane proteins such as RabSb, HLA-G, HSP70, LAMP2 (lysosome-associated membrane protein) and LIMP (lysosomal integral membrane protein). Exosomes may also be obtained from a non- mammalian biological sample, including cultured non-mammalian cells. As the molecular machinery involved in exosome biogenesis is believed to be evolutionarily conserved, exosomes from non-mammalian sources include surface markers which are isoforms of mammalian surface markers, such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles. As used herein, the term "mammal" is meant to encompass, without limitation, humans, domestic animals such as dogs, cats, horses, cattle, swine, sheep, goats and the like, as well as non-domesticated animals such as, but not limited to, mice, rats and rabbits. The term "non-mammal" is meant to encompass, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruit/vegetables (e.g. corn, pomegranate) and yeast.
[0056] Exosomes may be obtained from the appropriate biological sample using a combination of isolation techniques, for example, centrifugation, filtration and ultracentrifugation methodologies. In one embodiment, the isolation protocol includes the steps of: i) exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation; ii) subjecting the supernatant from step i) to centrifugation to remove microvesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the microfiltered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) re-suspending the exosome pellet from step iv) in a physiological solution and conducting a second ultracentrifugation in a density gradient and removing the exosome pellet fraction therefrom,
[0057] Thus, the process of isolating exosomes from a biological sample includes a first step of removing undesired large cellular debris from the sample, i.e. cells, cell components, apoptotic bodies and the like greater than about 7-10 microns in size. This step is generally conducted by centrifugation, for example, at 1000-4000x g for 10 to 60 minutes at 4 °C, preferably at 1500-2500x g, e.g. 2000x g, for a selected period of time such as 10-30 minutes, 12-28 minutes, 14-24 minutes, 15-20 minutes or 16, 17, 18 or 19 minutes. As one of skill in the art will appreciate, a suitable commercially available laboratory centrifuge, e.g. Thermo- Scientific™ or Cole-Parmer™, is employed to conduct this isolation step. To enhance exosome isolation, the resulting supernatant is subjected to a second optional centrifugation step to further remove cellular debris and apoptotic bodies, such as debris that is at least about 7-10 microns in size, by repeating this first step of the process, i.e. centrifugation at 1000-4000x g for 10 to 60 minutes at 4 °C} preferably at 1500-2500x g, e.g. 2000x g, for the selected period of time.
[0058] Following removal of cell debris, the supernatant resulting from the first centrifugation step(s) is separated from the debris- containing pellet (by decanting or pipetting it off) and may then be subjected to an optional additional (second) centrifugation step, including spinning at 12,000-15,000x g for 30-90 minutes at 4 °C to remove intermediate-sized debris, e.g. debris that is greater than 6 microns size. In one embodiment, this centrifugation step is conducted at 14,000x g for 1 hour at 4 °C. The resulting supernatant is again separated from the debris- containing pellet.
[0059] The resulting supernatant is collected and subjected to a third centrifugation step, including spinning at between 40,000-60,000x g for 30-90 minutes at 4 °C to further remove impurities such as medium to small-sized microvesicles greater than 0.3 microns in size e.g. in the range of about 0.3-6 microns. In one embodiment, the centrifugation step is conducted at 50,000x g for 1 hour. The resulting supernatant is separated from the pellet for further processing.
[0060] The supernatant is then filtered to remove debris, such as bacteria and larger microvesicles, having a size of about 0.22 microns or greater, e.g, using microfiltration. The filtration may be conducted by one or more passes through filters of the same size, for example, a 0.22 micron filter. Alternatively, filtration using 2 or more filters may be conducted, using filters of the same or of decreasing sizes, e.g. one or more passes through a 40-50 micron filter, one or more passes through a 20-30 micron filter, one or more passes through a 10-20 micron filter, one or more passes through a 0.22-10 micron filter, etc. Suitable filters for use in this step include the use of 0.45 and 0.22 micron filters.
[0061] The microfiltered supernatant (filtrate) may then be combined with a suitable physiological solution, preferably sterile, for example, an aqueous solution, a saline solution or a carbohydrate-containing solution in a 1 : 1 ratio, e.g. 10 mL of supernatant to 10 mL of physiological solution, to prevent clumping of exosomes during the subsequent ultracentrifugation and to maintain the integrity of the exosomes. The exosomal solution is then subjected to ultracentrifugation to pellet exosomes and any remaining contaminating microvesicles (between 100-220 nm). This ultracentrifugation step is conducted at 110,000- 170,000x g for 1-3 hours at 4 °C, for example, 170,000x g for 3 hours. This ultracentrifugation step may optionally be repeated, e.g. 2 or more times, in order to enhance results. Any commercially available ultracentrifuge, e.g. Thermo-Scientific™ or Beckman™, may be employed to conduct this step. The exosome-containing pellet is removed from the supernatant using established techniques and re-suspended in a suitable physiological solution.
[0062] Following ultracentrifugation, the re-suspended exosome-containing pellet is subjected to density gradient separation to separate contaminating microvesicles from exosomes based on their density. Various density gradients may be used, including, for example, a sucrose gradient, a colloidal silica density gradient, an iodixanol gradient, or any other density gradient sufficient to separate exosomes from contaminating microvesicles (e.g. a density gradient that functions similar to the 1.100-1.200 g/ml sucrose fraction of a sucrose gradient). Thus, examples of density gradients include the use of a 0.25-2.5 M continuous sucrose density gradient separation, e.g. sucrose cushion centrifugation, comprising 20-50% sucrose and a colloidal silica density gradient, e.g. Percoll™ gradient separation (colloidal silica particles of 15-30 nm diameter, e.g. 30%/70% w/w in water (free of RNase and DNase), which have been coated with polyvinylpyrrolidone (PVP)). The resuspended exosome solution is added to the selected gradient and subjected to ultracentrifugation at a speed between 110,000- 170,000x g for 1-3 hours. The resulting exosome pellet is removed and re-suspended in physiological solution.
[0063] Depending on the density gradient used, the re-suspended exosome pellet resulting from the density gradient separation may be ready for use, For example, if the density gradient used is a sucrose gradient, the exosome pellet is removed from the appropriate sucrose gradient fraction, and is ready for use, or may preferably be subjected to an ultracentrifugation wash step at a speed of 110,000- 170,000x g for 1-3 hours at 4 °C. If the density gradient used is, for example, a colloidal silica density gradient, then the resuspended exosome pellet may be subjected to additional wash steps, e.g. subjected to one to three ultracentrifugation steps at a speed of 110,000-170,000x g for 1-3 hours each at 4 °C, to yield an essentially pure exosome- containing pellet. As one of skill in the art will appreciate, the exosome pellet from any of the centrifugation or ultracentrifugation steps may be washed between centrifugation steps using an appropriate physiological solution, e.g. saline. The final pellet is removed from the supernatant and may be re-suspended in a physiologically acceptable solution for use. Alternatively, the exosome pellet may be stored for later use, for example, in cold storage at 4°C, in frozen form or in lyophilized form, prepared using well-established protocols. The exosome pellet may be stored in any physiological acceptable carrier, optionally including cryogenic stability and/or vitrification agents (e.g. DMSO, glycerol, trehalose, polyhydroxylated alcohols (e.g. methoxylated glycerol, propylene glycol), M22 and the like).
[0064] The described exosome isolation protocol advantageously provides a means to obtain mammalian exosomes which are at least about 90% pure, and preferably at least about 95% or greater pure, i.e. referred to herein as "essentially free" from cellular debris, apoptotic bodies and microvesicles having a diameter less than 20 or greater than 120 nm, for example, free from particles having a diameter of less than 40 or greater than 120 nm (as measured, for example, by dynamic light scattering), and which are biologically intact, e.g. not clumped or in aggregate form, and not sheared, leaky or otherwise damaged. Exosomes isolated according to the methods described herein exhibit a high degree of stability, evidenced by the zeta potential of a mixture/solution of such exosomes, for example, a zeta potential of at least a magnitude of 30 mV, e.g. < -30 or > +30, and preferably, a magnitude of at least 40 mV, 50 mV, 60 mV, 70 mV, 80 mV, or greater. The term "zeta potential" refers to the electrokinetic potential of a colloidal dispersion, and the magnitude of the zeta potential indicates the degree of electrostatic repulsion between adjacent, similarly charged particles (exosomes) in a dispersion. For exosomes, a zeta potential of magnitude 30 mV or greater indicates moderate stability, i.e. the solution or dispersion will resist aggregation, while a zeta potential of magnitude 40-60 mV indicates good stability, and a magnitude of greater than 60 mV indicates excellent stability.
[0065] Moreover, high quantities of exosomes are achievable by the present isolation method, e.g. exosomes in an amount of about 100-2000 g total protein can be obtained from 1-4 mL of mammalian serum or plasma, or from 15-20 mL of cell culture spent media (from at least about 2 x 106 cells). Thus, solutions comprising exosomes at a concentration of at least about 5 pg pL, and preferably at least about 10-25 pg pL, may readily be prepared due to the high exosome yields obtained by the present method. The term "about" as used herein with respect to any given value refers to a deviation from that value of up to 10%, either up to 10% greater, or up to 10% less.
[0066] Exosomes isolated in accordance with the methods herein described, which beneficially retain integrity, and exhibit a high degree of purity (being "essentially free" from entities having a diameter less than 20 ran and greater than 120 nm), stability and biological activity both in vitro and in vivo, have not previously been achieved. Thus, the present exosomes are uniquely useful, for example* diagnostically and/or therapeutically, e.g. for the in vivo delivery of protein and/or nucleic acid. They have also been determined to be non- allergenic/non-immunogenic, and thus, safe for autologous, allogenic, and xenogenic use.
[0067] For the treatment of mitochondrial disease, isolated exosomes are genetically engineered to incorporate exogenous nucleic acid or exogenous protein suitable to treat the disease, for example, nucleic acid (e.g. DNA, or mRNA) encoding a functional mitochondrial product or total mitochondrial RNA, or a functional mitochondrial product (e.g. protein, mRNA, tRNA, rRNA, mi RNA, SRP RNA, snRNA, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, or piRNA) itself. The term "exogenous" is used herein to refer to a mitochondrial product originating from a source external to the exosomes. The desired nucleic acid may be produced using known synthetic techniques, incorporated into a suitable expression vector using well established methods to form a mitochondrial product-encoding expression vector which is introduced into isolated exosomes using known techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like. Alternatively, isolated exosomes may be engineered to incorporate total mammalian mitochondrial RNA, which is free from pathogenic mitochondrial nucleic acid mutations. Total mammalian mitochondrial RNA may be isolated from biological tissue using known techniques and incorporated into isolated exosomes, for example, by electroporation or transfection. Similarly, the selected protein may be produced using recombinant techniques, or may be otherwise obtained, and then may be introduced directly into isolated exosomes by electroporation or other transfection methods. More particularly, electroporation applying voltages in the range of about 20-1000 V/cm may be used to introduce nucleic acid or protein into exosomes. Transfection using cationic lipid-based transfection reagents such as, but not limited to, Lipofectamine® MessengerMAX™ Transfection Reagent, Lipofectamine® RNAiMAX Transfection Reagent, Lipofectamine® 3000 Transfection Reagent, or Lipofectamine® LTX Reagent with PLUS™ Reagent, may also be used. The amount of transfection reagent used may vary with the reagent, the sample and the cargo to be introduced. For example, using Lipofectamine® MessengerMAX™ Transfection Reagent, an amount in the range of about 0.15 L to 10 μϊ, may be used to load 100 ng to 2500 ng nucleic acid or protein into exosomes. Other methods may also be used to load mitochondrial products into exosomes including, for example, the use of cell-penetrating peptides.
[0068] Exosomes isolated in accordance with the methods herein described, which beneficially retain integrity, and exhibit a high degree of purity and stability, readily permit loading of exogenous protein and/or nucleic acid in an amount of at least about 1 ng nucleic acid (e.g. mR A) per 10 ug of exosomal protein or 30 ug protein per 10 ug of exosomal protein.
[0069] In another embodiment, a mitochondrial product-encoding expression vector as above described, may be introduced directly into exo some-producing cells, e.g. autologous, allogenic, or xenogenic cells, such as immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like, by electroporation or other transfection method as described above. Following a sufficient period of time, e.g. 3-7 days to achieve stable expression of the mitochondrial product, exosomes incorporating the expressed protein may be isolated from the exosome-producing cells as described herein.
[0070] The desired mitochondrial product, nucleic acid encoding the mitochondrial product or both may be introduced into isolated exosomes, as previously described, using electroporation or other transfection methods. Introduction to the exosome of both the desired mitochondrial product and nucleic acid encoding the same mitochondrial product may increase delivery efficiency of the mitochondrial product. In addition, introduction of a combination of mitochondrial products, and/or nucleic acid encoding one or more mitochondrial products, may be desirable to treat a mitochondrial disease resulting from different DNA mutations, or for the treatment of secondary mitochondrial dysfunction such as type 2 diabetes, obesity, or muscular dystrophy, for example,
[0071] In another embodiment, prior to incorporation into exosomes of a selected mitochondrial product, or nucleic acid encoding a mitonchondrial product, exosomes may be modified to express or incorporate a target-specific fusion product which provides targeted delivery of the exosomes to the mitochondria. Such a target-specific fusion product comprises a sequence that targets mitochondria, i.e. a mitochondrial targeting sequence, linked to an exosomal membrane marker. The exosomal membrane marker of the fusion product will localize the fusion product within the membrane of the exosome to enable the targeting sequence to direct the exosome to the intended target. Examples of exosome membrane markers include, but are not limited to: tetraspanins such as CD9, CD37, CD53, CD63, CD81, CD82 and CD151; targeting or adhesion markers such as integrins, ICAM-1 and CDD31; membrane fusion markers such as armexins, TSG101, ALIX; and other exosome transmembrane proteins such as LAMP (lysosome-associated membrane protein), e.g. LAMP 1 or 2, and LIMP (lysosomal integral membrane protein). All or a fragment of an exosomal membrane marker may be utilized in the fusion product, provided that the fragment includes a sufficient portion of the membrane marker to enable it to localize within the exosome membrane, i.e. the fragment comprises at least one intact transmembrane domain to permit localization of the membrane marker into the exosomal membrane.
[0072] The target-specific fusion product also includes a mitochondrial targeting sequence, i.e. a protein or peptide sequence which facilitates the targeted delivery of the exosome to mitochondria. Examples of suitable mitochondrial targeting proteins include, but are not limited to, aconitase or superoxide dismutase 2, or a targeting fragment thereof, e.g. a portion of the C-terminal sequence thereof. Examples of mitochondrial targeting peptide sequences include, but are not limited to, MLSARSAIKRPIVRGLATV (SEQ ID NO: 1), MLRFTNCSCKTFV SSYKLNIRRMNTV (SEQ ID NO: 2),
MLRSSVVRSRATLRPLLRRAYSSSFRT (SEQ ID NO: 3),
MLSARSAIKRPIVRGLATV S SFRT (SEQ ID NO: 4),
MLRFTNCSCKTFVKSSYKLNIRRMNSSFRT (SEQ ID NO: 5), MLSRAVCGTSRQLAPV (SEQ ID NO: 6), or a targeting fragment of any one of these, e.g. a portion of the C-terminal sequence thereof.
[0073] Exosomes incorporating a mitochondrial-targeting fusion product may be produced, as described above, using recombinant technology. In this regard, an expression vector encoding the fusion product is introduced by electi poration or other transfection methods into exosome-producing cells isolated from an appropriate biological sample. As one of skill in the art will appreciate, it is also possible to produce the fusion product using recombinant techniques, and then introduce the fusion product directly into exosome-producing cells using similar techniques, e.g. electroporation, transfection using cationic lipid-based transfection reagents, and the like. Following a sufficient period of time, exosomes generated by the exosome-producing cells, and including the fusion product, may be isolated as described. The desired mitochondrial product may be introduced into isolated exosomes incorporating a mitochondrial-targeting fusion product (modified mitochondrial-targeting exosomes) as previously described, using electroporation or other transfection methods. Addition to the exosomes incorporating the mitochondrial-targetting sequence and the desired mitochondrial product may increase delivery efficiency of the mitochondrial product.
[0074] Exosomes genetically engineered to incorporate a mitochondrial product, and/or nucleic acid encoding the mitochondrial product, may be used to deliver the mitochondrial product and/or nucleic acid to a mammal in vivo in the treatment of a mitochondrial disease, e.g. a pathological condition or disease in which a mitochondrial product is dysfunctional or absent, to upregulate the activity of the target mitochondrial product and thereby treat the disease. Examples of mitochondrial diseases that are caused by mutations in nuclear- encoded mitochondrial genes and that may be treated using the present engineered exosomes are set out in Table 1 below. Table 1 identifies the gene encoding the mitochondrial product involved in each disease, and gene mutations that result in dysfunctional/absent mitochondrial product, the Online Mendelian Inheritance in Man (OMIM) reference number as well as providing mRNA transcript sequence information and protein sequence information (e.g. NCBI (National Centre for Biotechnology Information) GenBank accession numbers for the mitochondrial products useful to treat each disease. Table 1.
Figure imgf000024_0001
Figure imgf000025_0001
mitochondrial complex NM 001257342.1 NP 001244271.1 111 deficiency NM 001257343.1 NP 001244272.1
NM 001257344.1 NP 001244273.1 NM 004328.4 NP 004319.1
Gracile syndrome BCS1L c.232A>G 603647 NM 001079866.1 NP 001073335.1
NM 001257342.1 NP 001244271.1 NM 001257343.1 NP 001244272.1 NM 001257344.1 NP 001244273.1 NM 004328.4 NP 004319.1
Bjornstad syndrome BCS1L .5470T 603647 NM 001079866.1 NP 001073335.1 due to mitochondrial NM 001257342.1 NP 001244271.1 complex III deficiency NM 001257343.1 NP 001244272.1
NM 001257344.1 NP 001244273.1 NM 004328.4 NP 004319.1
Leigh Syndrome due to SURF1 c.337T C 185620 NM 001280787.1 NP 001267716.1 mitochondrial complex (Surfeit 1) or NM_003172.3 NPJ)03163.1 IV deficiency .7560T
Leigh Syndrome due to SURF! c.855de!CT 185620 NM 001280787.1 NP 001267716.1 mitochondrial complex NM_003172.3 NP_003163.1 IV deficiency
Leigh Syndrome due to SURFl c.868insT 185620 NM 001280787.1 NP 001267736.1 mitochondrial complex NM 003172.3 NP_003163.1 IV deficiency
Cardiac failure due to SCOl c.394G>A 603644 NM_004589.3 NPJ04580.1 cardiac hypertrophy (SCOl cytochrome c
caused by oxidase assembly
mitochondrial complex protein)
IV deficiency
Severe neurologic SCOl c.363delGA 603644 NM_004589.3 NP .004580.1 disorder and metabolic or
acidosis due to c.520C>T
mitochondrial complex
IV deficiency
c.l211A>T
Leigh syndrome due to COX 10 602125 NM 001303.3 NP_001294.2
(COXlO hcme or
mitochondrial complex
Aifamesyitransferase c.l211A>G
IV deficiency
cytochrome c oxidase
assembly factor)
Severe muscle COX 10 .6120A 602125 NM_001303.3 NPJJ01294.2 weakness, hypotonia,
ataxia, ptosis,
pyramidal syndrome,
and status epileptieus
due to mitochondrial
complex IV deficiency
Sensorineural hearing coxio .7910A 602125 NM_001303.3 NP_001294.2 loss, severe or
biventricular .8780T
hypertrophic
cardiomyopathy, and
macrocytic anemia due
to mitochondrial
complex IV deficiency
Fatal infantile COX 15 e.700C>T 603646 NM 004376.5 NP 004367.2 cardioencephalomyopath (Cytochrome c oxidase or NM_078470.4 NP_510870.1 yand and Leigh assembly homolog 15) .4470G
syndrome due to
mitochondrial complex
IV deficiency Leigh syndrome due to CO 15 .4520G 603646 NM 004376.5 NP 004367.2 mitochondrial complex or NM 078470.4 NP_ 10870.1 IV deficiency c.l030T>C
Leigh syndrome, French LRPPRC .111 0T 607544 NM__133259.3 NP„573566.2 Canadian type (Leucine rich
pentatricopeptide repeat
containing)
Leigh syndrome, French LRPPRC P.A354V 607544 NMJ33259.3 NP_573566.2 Canadian type
Leigh syndrome, French LRPPRC c.3900G>T 607544 NM_133259.3 NP„573566.2 Canadian type
Encephalomyopathy FAST D2 .12460T 612322 NM 001136193.1 NP 001129665.1 due to mitochondrial (FAST kinase domains NM 001136194.1 NP 001129666.1 complex IV deficiency 2) NMJI14929.3 NPJ155744.2
Intellectual disability, TACOl .4210T 612958 NM 016360.3 NP 057444.2 extrapyramidal signs, (Trans!ational activator
rigidity, hyperreflexia, of roitochondrially
nystagmus due to encoded cytochrome c
mitochondrial complex oxidase I)
IV deficiency
Leigh syndrome, TACOl c.427insC 612958 NM 016360.3 NP_057444.2 French Canadian type
due to mitochondrial
complex IV deficiency
Degenerative ATPAF2 c.280T>A 608918 NMJ45691.3 NP_663729.1 encephalopathy due to (ATP synthase
mitochondrial complex mitochondrial Fl
V deficiency complex assembly
factor 2)
Hypertrophic T EM70 c.317A>G 612418 NM 001040613.2 NP 001035703.1 cardiomyopathy, lactic (Transmembrane NM_017866.5 NP 060336.3 acidosis, mild protein 70)
psychomotor
retardation due to
mitochondrial complex
V (ΛΤΡ synthase)
deficiency
Multiorgan failure due TMEM70 c.366A>T 612418 NM 001040613.2 NP 001035703.1 to mitochondrial NMJH7866.5 NP 060336.3 complex V (ATP
synthase) deficiency
Hypotonia, TMEM70 .23SOT 612418 NM 001040613.2 NP 001035703.1 hypertrophic NM_017866.5 NP_060336.3 cardiomyopathy,
recurrent
encephalopathic
episodes due to
mitochondrial complex
V (ATP synthase)
deficiency
Mitochondrial complex TMEM70 c.578de!CA 612418 NM 001040613.2 NP 001035703.1 V (ATP synthase) NMJ)17866.5 NP_060336.3 deficiency
Autosomal dominant POLG c.2864A>G 174763 NM 001126131.1 NP 001119603.1 progressive externa! (Polymerase (DNA NMJI02693.2 NP_002684.1 ophthalmoplegia with directed), gamma)
mitochondrial DNA
deletion s-1 (PEOA1)
Mitochondrial DNA POLG c.2899G>T 174763 NM 001126131.1 NP 0011 19603.1 depletion syndrome NM_002693.2 NP 002684.1 4A/ Alpers-
Figure imgf000028_0001
deletions-3 (PEOA3)
Mitochondrial DNA TYMP c.3371A>C 131222 NM 001113755.2 NP 001107227.1 depletion syndrome 1 (Thymidine NM 0011 13756.2 NP 001107228.1 ( NGIE type) phosphorylase) NM 001257988.1 NP 001244917.1
NM 001257989.1 NP 001244918.1 NM 001953.4 NP 001944.1
Mitochondrial DNA TYMP .1504T>C 131222 NM 001113755.2 NP 001 107227.1 depletion syndrome 1 NM 001113756.2 NP 001 107228.1 (MNGIE type) NM 001257988.1 NP 001244917.1
NM 001257989.1 NP 001244918.1 NM 001953.4 NP 001944.1
Mitochondrial DNA TYMP c.i41 A>C 131222 NM 00! 113755.2 NP 001107227.1 depletion syndrome 1 NM 001113756.2 NP 001 107228.1 (MNGIE type) NM 001257988.1 NP 001244917.1
NM 001257989.1 NP 001244918.1 NM 001953.4 NP 001944.1
Mitochondrial DNA DGUOK c.204delA 601465 NM 080916.2 NP 550438.1 depletion syndrome 3 (Deoxyguanosi e NM 080918.2 NP„550440.1 (hepatocerebral type) kinase)
Mitochondrial DNA DGUOK c.763dupGA 601465 NM 080916.2 NP 550438.1 depletion syndrome 3 'IT NM_0809i8.2 NP_550440.1 (hepatocerebral type)
Mitochondrial DNA DGUOK c.313C>T 601465 NM 080916.2 NP 550438.1 depletion syndrome 3 NM_ 08G918.2 NP_550440.1 (hepatocerebral type)
Mitochondrial DNA RRM2B c.580G>A 604712 NM 001172477.1 NP 001165948.1 depletion syndrome 8a (Ribonucleotide NM 001172478.1 NP 001165949.1 (encephalomyopathic reductase M2 B) NM_015713.4 NP_056528.2 type with renal
tubulopathy)
Mitochondrial DNA RM2B c.253delGAG 604712 NM 001172477.1 NP 001165948.1 depletion syndrome 8a NM 001172478.1 NP 001165949.1 (encephalomyopathic NMJH5713.4 NP_056528.2 type with renal
tubulopathy)
Mitochondrial DNA RRM2B .850OT 604712 NM 001172477.1 NP 001165948.1 depletion syndrome 8a NM 001172478.1 NP 001165949,1 (encephalomyopathic NM_015713.4 NP_056528.2 type with renal
tubulopathy)
Mitochondrial DNA SUCLA2 de!43bp and 603921 NM 003850.2 NP_003841.1 depletion syndrome 5 (Succinate-CoA ligase, insSbp
(encephalomyopathic ADP-fonnmg, beta
type without subunit)
methylmalonic
aciduria)
Mitochondrial DNA SUCLA2 c.534G>A 603921 NM_003850.2 NP_003841.1 depletion syndrome 5
(encephalomyopathic
type with
methylmalonic
aciduria)
Mitochondrial DNA SUCLA2 .850OT 603921 NM 03850.2 NP_003841.1 depletion syndrome 5
(encephalomyopathic
type with
methylmalonic
aciduria)
Mitochondrial DNA SUCLG1 c.ll3delAT 61 1224 NM_003849.3 NPJ03840.2
Figure imgf000030_0001
Figure imgf000031_0001
deficiency 5 ribosomal protein S22)
Figure imgf000032_0001
Figure imgf000033_0001
(COQ10D4) containing kinase 3)
Figure imgf000034_0001
Charcot-Marie-Tooth MFN2 c.227T C 608507 NM 001127660.1 NP 001121132.1 Disease-2A2 NMJ14874.3 NP_055689.1
Lethal encephalopathy DNM1L .11840A 603850 NM 001278463.1 NP 001265392.1 due to defective (Dynamin 1-!ike) NM 001278464.1 NP 001265393.1 mitochondrial and NM 001278465.1 NP 001265394.1 peroxisomal fission NM 001278466.1 NP 001265395.1
NM 005690.4 NP 005681.2 NM 012062.4 NP 036192.2 NM 012063.3 NP 036193.2
3-Methylglutaconic TAZ IVS2, G>A, - 300394 NM 000116.4 NP 000107.1 aciduria type 2 (Barth (Tafazzin) 1 NM 001303465.1 NP 001290394.1 Syndrome) NM 181311.3 NP 851828.1
NM 181312.3 NP 851829.1 NMJ81313.3 NPJ!51830.1
3-Methylglutaconic TAZ c.441C>G 300394 NM 000116.4 NP 000107.1 aciduria type 2 (Barth NM 001303465.1 NP 001290394.1 Syndrome) NM 181311.3 NP 851828.1
NM 181312.3 NP 851829.1 NM 181313.3 NP 851830.1
3-Methylglutaconic TAZ c.527G>C 300394 NM 000116.4 NP 000107.1 aciduria type 2 (Barth NM 001303465.1 NP 001290394.1 Syndrome) NM 181311.3 NP 851828.1
NM 181312.3 NP 851829.1 NM 181313.3 NP 851830.1
Pyruvate PDHA1 del4bp 300502 NM 000284.3 NP 000275.1 dehydrogenase El- (Pyruvate NM 001173454.1 NP 001166925.1 alpha deficiency deliydrogenase NM 001173455.1 NP 001166926.1
(Itpoamide) alpha 1) NMJ01173456.1 NPJOl 166927.1
Pyruvate PDHA1 c.l032del7bp 300502 NM 000284.3 NP 000275.1 dehydrogenase El- NM 001173454.1 NP 001166925.1 alpha deficiency NM 001173455.1 NP 001166926.1
NMJ01173456.1 NP_001166927.1
Pyruvate PDHA1 P.R378H 300502 NM 000284.3 NP 000275.1 dehydrogenase El- 001173454.1 NP 001166925.1 alpha deficiency NM 001173455.1 NP 001166926.1
NM_001173456.1 NPJOl 166927.1
Ethyl malonic ETHE1 .4870T 608451 NMJ14297.3 NPJ55112.2 encephalopathy (Ethylmalonic
encephalopathy 1)
Ethylmalonic ETHE1 c.604insG 608451 NMJ14297.3 NPJ55112.2 encephalopathy
Ethylmalonic ETHE1 c.221insA 608451 NMJ14297.3 NPJ55112.2 encephalopathy
Myopathy, lactic PUS1 .6560T 608109 NM 001002019.2 NP-001002019.1 acidosis, and (Pseudouridylate NM 001002020.2 NP 001002020.1 sideroblastic anemia 1 synthase 1 ) NM_025215.5 NPJ79491.2 (MLASA1)
Myopathy, lactic PUS 1 c.658G>T 608109 NM 001002019.2 NP-001002019.1 acidosis, and NM_001002020.2 NPJOl 002020.1 sideroblastic anemia 1 NMJ125215.5 NP_079491.2 (MLASA I)
Mitochondrial complex NDUFS1 c.664deICAT 157655 NM 001199981.1 NP 001186910.1 I deficiency (NADH:ubiquinone NM 001199982.1 NP 001186911.1 oxidoreductase core NM 001199983.1 NP 001186912.1 subunit SI) NM 001199984.! NP 001186913.1
NM„005006.6 NP_004997.4
Mitochondrial complex NDUFS1 .7210T 157655 NM 001199981.1 NP 001186910.1 I deficiency NM 001199982.1 NP 001186911.1
NM 001199983.1 NP 001186912.1 NM 001199984.1 NP 001186913.1 NM_005006.6 NP_004997.4
Mitochondrial complex NDUFS1 c.l783A>G 157655 NM 001199981.1 NP 001186910.1 I deficiency NM 001199982.1 NP 001186911.1
NM 001199983.1 NP 001186912.1 NM 001199984.1 NP 001186913.1 NM_005006.6 NP_004997.4
Mitochondrial complex NDUFS2 c.683G>A 602985 NM 001166159.1 NP 001159631,1 I deficiency (NADH:ubiquinoiie NMJ)Q4550.4 NP_004541.1 oxidoreductase core
subunit S2)
Neonatal lactic acidosis NDUFS2 .686OA 602985 NM 001166159.1 NP 001159631.1 and hypertrophic NMJTO4550.4 NPJH S4 cardiomyopathy due to
mitochondrial complex
I deficiency
Mitochondrial complex NDUFS2 .1237T>C 602985 NM 001166159.1 NP 001159631.1 I deficiency NM_004550.4 NPJD04541.1
Leigh syndrome due to NDUFS3 .4340T 603846 NM 004551.2 NPJJ04542.1 mitochondrial complex (NADH:ubiquinone and
I deficiency oxidoreductase core .5950T
subunit S3)
Leigh syndrome due to NDUFS3 .5320T 603846 NM_004551.2 NP_004542.1 mitochondrial complex
I deficiency
Mitochondrial complex DUFS4 c.466dupAA 602694 NM 001318051.1 NP 001304980.1 1 deficiency (NADH:ubiquinone GTC NM_002495.3 NP_002486.1 oxidoreductase subunit
S4)
Microcephaly and NDUFS4 c.289delG or 602694 NM 001318051.1 NP 001304980.1 lactic academia due to c.290delG NM_002495.3 NP_002486.I mitochondrial complex
I deficiency
Leigh syndrome due to NDUFS4 .3160T 602694 NM 001318051.1 NP 001304980.1 mitochondrial complex NM_002495.3 NP_002486.1 I deficiency
Mitochondrial complex NDUFS6 IVS2DS, 603848 NM_004553.4 NP_004544.1 I deficiency (NADH:ubiqumone T>A, +2 oxidoreductase subunit
S6)
Mitochondrial complex NDUFS6 del4.175-kbp 603848 NM_004553.4 NP_004544.1 I deficiency
Fatal infantile lactic NDUFS6 c.344G>A 603848 NM 004553.4 NPJ04544.1 acidosis due to
mitochondrial complex
I deficiency
Leigh syndrome due to NDUFS7 P.V122M 601825 NMJ&4407.4 NP_077718.3 mitochondrial complex (NADH:ubiquinone
1 deficiency oxidoreductase subunit
S7)
Leigh syndrome due to NDUFS7 c.434G>A 601825 NM_024407.4 NP_077718.3 mitochondrial complex
I deficiency
Leigh syndrome due to NDUFS7 C.U670G 601825 NM 024407.4 NPJ)77718.3 mitochondrial complex
I deficiency
Leigh syndrome due to NDUFS8 c.305G>A 602141 NM_002496.3 NPJ)02487.1 mitochondrial complex (NADH:ubiquinoiie or
I deficiency oxidoreductase subunit 0.236OT
S8)
Leigh syndrome due to NDUFS8 P.R102H 602141 NM_002496.3 NPJ)02487.1 mitochondrial complex
1 deficiency
Leigh syndrome due to DTJFS8 c.2540T or 602141 NMJ102496.3 NP_002487.1 mitochondrial complex c.413G>A
1 deficiency
Mitochondrial complex NDUFB3 c.64T>C 603839 NM 001257102. 1 NP 001244031.1 1 deficiency (NADHiubiquinone NM 002491.2 NPJW2482.1 oxidoreductase subunit
B3)
Encephalopathy, NDUFB3 c.208G>T 603839 NM 001257102. 1 NP 001244031.1 myopathy, hypotonia, or NM_G0249 L2 NPJ)02482.1 developmental delay, d c.64T>C
and lactic acidosis due
to mitochondrial
complex 1 deficiency
Mitochondrial complex NDUFVi C.126SOT 161015 NM 001166102.1 NP 001159574.1 I deficiency (NADHuibiquinone or NM_007103.3 NP_009034.2 oxidoreductase core C.1750T
subunit VI)
Mitochondrial complex NDUFVI c.640G>A 161015 NM 001166102.1 NP 001159574.1 I deficiency NM„007103.3 NP_009034.2
Mitochondrial complex NDUFVI C.1022OT 161015 NM 001166102.1 NP 001159574.1 I deficiency NM_007103.3 NP_009034.2
Hypertrophic NDUFV2 de!4bp 600532 NM_021074.4 NP„066552.2 cardiomyopathy, (NADHuibiquinone
truncal hypotonia, and oxidoreductase core
encephalopathy due to subunit V2)
mitochondrial complex
Figure imgf000038_0001
Figure imgf000039_0001
(CPT II) deficiency Carnitine CPT2 c.665C>A 600650 NM_O0OO98.2 NP_000089.i palmitoyltransferase 2
(CPT H) deficiency
Carnitine CPT1A (Carnitine P.D454G 600528 NM 001031847.2 NP 001027017.1 palmitoyltransferase 1 palmitoyltransferase 1A NM„001876.3 NP_001867.2 (CPT I) deficiency (liver))
Carnitine CPTIA c.l079A>G 600528 NM 001031847.2 NP 001027017.1 palmitoyltransferase 1 NM_001876.3 NPJ)01 67.2 (CPT I) deficiency
Carnitine CPT1A c.298C>T 600528 NM 001031847.2 NP 001027017.1 palmitoyltransferase 1 NM_001876.3 NP 001867.2 (CPT 1) deficiency
Cerebral creatine SLC6A8 (Solute carrier P.R514X 300036 NM 001142805.1 NP 001136277.1 deficiency syndrome- 1 family 6 NM 001142806.1 NP 001136278.1
(neurotransmitter NM_005629.3 NP_005620.1 transporter), member 8)
Cerebral creatine SLC6A8 .1141G>C 300036 NM 001142805.1 NP 001136277.1 deficiency syndrome- 1 NM 001142806.1 NP 001136278.1
NM 005629.3 NP 005620.1
Cerebral creatine SLC6A8 c.l221delTT 300036 NM 001142805.1 NP 001136277.1 deficiency syndrome- 1 C NM 001142806.1 NP 001136278.1
NM_005629.3 NP_005620.1
Cerebral creatine GAMT c.327G>A 601240 NM 000156.5 NP 000147.1 deficiency syndrome-2 (Guanidinoacetate N- NM_138924.2 NP_620279.1 methyltratisferase)
Cerebral creatine GAMT c.59G>C 601240 NM 000156.5 NP 000147.1 deficiency syndrome-2 N J 38924.2 NP_620279.1
Cerebral creatine GAMT c.506G>A 601240 NM 000156.5 NP 000147.1 deficiency syndrome-2 NM 138924.2 NP 620279.1
Cerebral creatine GATM (Glycine c.9297G>A 602360 NM 001482.2 NP 001473.1 deficiency syndrome-3 amidinotransferase)
Cerebral creatine GATM c.ll linsA 602360 NM 001482.2 NP_001473.1 deficiency syndrome-3
Cerebral creatine GATM P.R169X 602360 NM_ 001482.2 NP_001473.1 deficiency syndrome-3
Pyruvate carboxylase PC (Pyruvate c.l828G>A 608786 NM 000920.3 NP 000911.2 deficiency carboxylase) NM 001040716.1 NP 001035806.1
NM 022172.2 NP 071504.2
Pyruvate carboxylase PC c.2229G>T 608786 NM 000920.3 NP 000911.2 deficiency NM 001040716.1 NP 001035806.1
NM 022172.2 NP 071504.2
Pyruvate carboxylase PC c.434T>C 608786 NM 000920.3 NP 000911.2 deficiency NM 001040716.1 NP 001035806.1
NM 022172.2 NP 071504.2
Long-chain 3- HADHA (hydroxyacyl- P.E474Q 600890 NM_000182.4 NPJS00173.2 hydroxyl-CoA CoA dehydrogeiiase/3- dehydrogenase ketoacyl-CoA
(LCHAD) deficiency thiolase/enoyl-CoA
hydratase (trifunctkmai
protein), alpha subunit)
(LCHAD)
Long-chain 3- HADHA (LCHAD) .11320T 600890 NM_000182.4 NPJ)00173.2 hydroxyl-CoA
dehydrogenase
(LCHAD) deficiency
Long-chain 3- HADHA (LCHAD) .167SOT 600890 NMJ)00182.4 NP_000173.2 hydroxyl-CoA
dehydrogenase
Figure imgf000041_0001
Figure imgf000042_0001
{cardiomyopathic type)
[0075] Examples of mitochondrial diseases resulting from mutations in mitochondrial genes expressed in the mitochondria (mitochondria-encoded) that may be treated using the present engineered exosomes are set out in Table 2 below. Table 2 identifies the mitochondrial gene product involved in each disease, and mutations thereof, as well as providing gene sequence information for the mitochondrial products useful to treat each disease by reference to the nucleotide position range of the gene for said mitochondrial product in the complete genome of the Homo sapiens mitochondrion (NCBI Genbank Reference Sequence: NC 012920.1) and the Online Mendelian Inheritance in Man (OMIM) reference number.
Table 2.
Figure imgf000043_0001
Figure imgf000044_0001
encoded tRNA glutamine)
MELAS syndrome MT-TQ m.4332G>A 590030 4329..4400
Mitochondrial myopathy MT-TM (Mitochondrially m.4409T C 590065 4402..4469 encoded tRNA Methionine)
Mitocliondrial encephalopathy MT-TW (Mitocliondrial ly m.5549G>A 590095 5512..5579 encoded tRNA tryptophan)
Mitochondrial encephalopathy MT-TW m.5537insT 590095 5512..5579 and Leigh syndrome
Mitochondrial encephalopathy MT-TW m.552 !G>A 590095 5512..5579
Myotonic dystrophy-like myopathy MT-TA (Mitochondrially m.5650G>A 590000 5587..5655 encoded tRNA alanine)
Myotonic myopathy MT-TA m.3591G>A 590000 55S7..5655
Isolated ophthalmoplegia MT-TN (Mitochondrially m.5703G>A 590010 56S7..5729 encoded fRNA asparagtne)
Isolated ophthalmoplegia MT-TN m.5692A>G 590010 56S7..5729
Mitochondrial complex 1 deficiency MT-TN m.5728A>G 590010 56S7..5729 and
Mitocliondrial complex IV deficiency
MELAS syndrome MT-TC (Mitochondrially m.5814A>G 590020 5761..5826 encoded tRNA cysteine)
Mitochondrial dystonia MT-TC m.5816A>G 590020 5761..5826
Exercise intolerance and complex III MT-TY (Mitochondrially m.5874A>G 590100 5826..5891 deficiency encoded tRNA tyrosine)
Exercise intolerance and complex III MT-TY m.5885delT 590100 5826..58 1 deficiency
Chronic progressive external MT-TY m.5877G>A 590100 5826..5891 ophthalmoplegia with myopathy
MERRF/MELAS overlap syndrome MT-TS1 (Mitochondrially m.7512T C 590080 7446..7514 and encoded tRNA serine 1 (UCN))
Mitochondrial cytochrome c oxidase
deficiency
Palmoplantar keratoderma with MT-TS1 m.7445A>G 590080 7446..7514 deafness
and nonsyndromic sensorineural
deafness,
Mitochondrial cytochrome c oxidase MT-TS1 m.7472insC 590080 7446..7514 deficiency and Sensorineural deafness
with neurologic features and
Nonsyndromic sensorineural deafness
Isolated mitochondrial myopathy MT-TD (Mitochondrially m.7526A>G 590015 7518..7585 encoded tRNA asparlic acid)
MERRF syndrome and MT-TK (Mitochondrially m.8344A>G 590060 S295..8364 Leigh syndrome and encoded tRNA lysine)
Parkinson's disease
MERRF syndrome and MT-TK m.8356T>C 590060 8295..S364 MERRF/MELAS overlap syndrome
Cardiomyopathy and deafness MT-TK m.8363G>A 590060 8295..S364
Hypertrophic cardiomyopathy MT-TG (Mitochondrially m.9997T>C 590035 9991..10058 encoded tRNA glycine)
Exercise intolerance MT-TG m. l0010T C 590035 9991..10058
Sudden death MT-TG m. l0044A>G 590035 9991..10058
Mitochondrial encepha!omyopathy MT-TR (Mitochondrially m. i 0438A>G 590005 10405..10469 encoded tRNA avginine)
Mitochondrial encephalomyopathy MT-TR m. l0450A>G 590005 10405..10469
Hypertrophic cardiomyopathy MT-TH {Mitochondrially m.l2192G>A 590040 12138..12206 encoded tRNA histidine)
MERRF/MELAS overlap syndrome MT-TH m.l2147G>A 590040 12138..12206
Pigmentary retinopathy and MT-TH m. l2183G>A 590040 12138..12206 Sensorineural deafness Cerebellar ataxia, cataract, and diabetes MT-TS2 (Mitochondrial^ m. l2258C>A 590085 12207..12265 mellitus and encoded tRNA Serine 2 (AGY))
Retinitis pigmentosa-deafhess
syndrome
MERRF MELAS overlap syndrome MT-TS2 m. l2207G>A 590085 12207..12265
Mitochondrial encepha!omyopathy MT-TL2 (Mitochondrial ly m, 12315G>A 590055 12266..12336 encoded tRNA Leucine 2 (CUN))
Mitochondrial myopathy MT-TL2 m. l2320A>G 590055 12266..12336
Mitochondrial cardiomyopathy MT-TL2 m. l2297T>C 590055 12266..12336
Mitochondrial myopathy with diabetes MT-TE (Mitochondrially m.l4709T>C 590025 14674..14742 mellitus encoded tRNA glutamic acid)
and Deafness
Transient infantile mitochondrial MT-TE m.l4674T>C 590025 14674..14742 myopathy
[0076] Thus, in one embodiment, exosomes are used to deliver to a mammal one or more mitochondrial products selected from the group consisting of nuclear-encoded mitochondrial products such as Lon peptidase 1, mitochondrial (LONPl), NADH ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADH ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH:ubiquinone oxidoreductase complex assembly factor 3 (NDUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH: ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl- CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1), Succinate dehydrogenase complex assembly factor 2 (SDHAF2), BCS1 homolog, ubiquinol- cytochrome c reductase complex chaperone (BCS1L), Surfeit 1 (SURF1), SCOl cytochrome c oxidase assembly protein (SCOl), COX10 heme A;farnesyltransferase cytochrome c oxidase assembly factor (COX10), Cytochrome c oxidase assembly homolog 15 (COX 15), Leucine rich pentatricopeptide repeat containing (LRPPRC), FAST kinase domains 2 (FASTKD2), Translational activator of mitochondrially encoded cytochrome c oxidase I (TACOl), ATP synthase mitochondrial Fl , complex assembly factor 2 (ATPAF2), Transmembrane protein 70 (TMEM70), Polymerase (DNA directed), gamma or DNA polymerase gamma (POLG), Polymerase (DNA directed), gamma 2, accessory subunit (POLG2), MpV17 mitochondrial inner membrane protein (MPV17), Chromosome 10 open reading frame 2 (C10orf2), Thymidine phosphorylase (TYMP), Deoxyguanosine kinase (DGUOK), Ribonucleotide reductase M2 B (RRM2B), Succinate-CoA iigase, ADP-forming, beta subunit (SUCLA2), Succinate-CoA ligase, alpha subunit (SUCLG1), Thymidine kinase 2, mitochondrial (TK2), Translocase of inner mitochondrial membrane 8 homolog A (TIMM8A), DnaJ heat shock protein family (Hsp40) member CI 9 (DNAJC19), G elongation factor, mitochondrial 1 (GFMl), Aminoacyl tRNA synthetase 2 (e.g. Leucyl-tRNA synthetase 2 (LARS2), TyrosyL-tRNA synthetase 2 (YARS2), Seryl-tRNA synthetase 2, mitochondrial (SARS2), Aspartyl-tRNA synthetase 2, mitochondrial (DARS2), Arginyl-tRNA synthetase 2, mitochondrial (RARS2)), Mitochondrial ribosomal protein S16 (MRPS16), Mitochondrial ribosomal protein S22 (MRPS22), Ts translation elongation factor, mitochondrial (TSFM), Tu translation elongation factor, mitochondrial (TUFM), Frataxin (FX ), ATP-binding cassette subfamily B member 7 (ABCB7), Solute earner family 25 member 38 (SLC25A38), Iron-sulfur cluster assembly enzyme (ISCU), BolA family member 3 (BOLA3), NFU1 iron-sulfur cluster scaffold (NFU3), Coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (COQ2), Coenzyme Q4 (COQ4), Coenzyme Q9 (COQ9), Aprataxin (APTX), Prenyl (decaprenyl) diphosphate synthase, sub nit 1 (PDSS1), Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2), AarF domain containing kinase 3 (ADCK3), Paraplegin (SPG7), Heat shock protein family D (Hsp60) member 1 (HSPD1), Optic atrophy 1 (autosomal dominant) (OPA1), Mitofusin 2 (MFN2), Dynamin 1-like (DNMIL), Tafazzin (TAZ), Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Ethylmalonic encephalopathy 1 (ETHE1), Pseudouridylate synthase 1 (PUS1), NADH:ubiquinone oxidoreductase core subunit SI (NDUFS1), NADH: ubiquinone oxidoreductase core subunit S2 (NDUFS2), NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), NADH: ubiquinone oxidoreductase subunit S6 (NDUFS6), NADH:ubiquinone oxidoreductase core subunit S7 (NDUFS7), NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8), NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3), NADH:ubiquinone oxidoreductase core subunit VI (NDUFV1), NADH:ubiquinone oxidoreductase core subunit V2 (NDUFV2), NADH:ubiquinone oxidoreductase subunit Al (NDUFA1), NADH:ubiquinone oxidoreductase subunit A2 ( DUFA2), NADH:ubiquinone oxidoreductase subunit A10 (NDUFA10), NADH:ubiquinone oxidoreductase subunit Al l (NDUFA11), Succinate dehydrogenase complex subunit A, flavoprotein (Fp) (SDHA), Succinate dehydrogenase complex subunit B, iron sulfur (Ip) (SDHB), Succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa (SDHC), Succinate dehydrogenase complex subunit D, integral membrane protein (SDHD), Ubiquinol-cytochrome c reductase binding protein (UQCRB), Ubiquinol-cytochrome c reductase complex III subunit VII (UQCRQ), Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) (COX6B1), and ATP synthase, H+ transporting, mitochondrial Fl complex, epsilon subunit (ATP5E), Solute carrier family 22 (organic cation/camitine transporter), member 5 (SLC22A5), Carnitine palmitoyltransferase 2 (CPT2), Carnitine palmitoyltransferase 1A (liver) (CPT1A), Solute carrier family 6 (neurotransmitter transporter), member 8 (SLC6A8), Guanidinoacetate N-methyltransferase (GAMT), Glycine amidinot ansferase (GATM), Pyruvate carboxylase (PC), hydroxyacyl-CoA dehydrogenase/3 -ketoacyl-Co A thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA)/(LCHAD), Acyl-CoA dehydrogenase, very long chain (ACADVL), Acyl-CoA dehydrogenase, C-2 to C-3 short chain (ACADS), Hydroxyacyl-CoA dehydrogenase (HADH)/(SCHAD), Electron transfer flavoprotein alpha subunit (ETFA), Electron transfer flavoprotein beta subunit (ETFB), Electron transfer flavoprotein dehydrogenase (ETFDH), Solute carrier family 25 (SLC25A20), Pyruvate dehyrogenase phosphatase catalytic subunit 1 (PDP1), Pyruvate dehydrogenase complex component X (PDHXD), Dihydrolipoamide S-acetyltransferase (DLAT), Pyruvate dehydrogenase (lipoamide) beta (PDHB), Solute carrier family 25 (mitochondrial earner; adenine nucleotide translocator), member (4SLC25A4)/(ANT), or nucleic acid encoding one or more of these mitochondrial products; and mitochondria-encoded mitochondrial products such as Mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), Mitochondrially encoded NADH dehydrogenase 2 (MT-ND2), Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3), Mitochondrially encoded NADH dehydrogenase 4 (MT-ND4 or ND4)5 Mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), Mitochondrially encoded NADH dehydrogenase 5 (MT- ND5), Mitochondrially encoded NADH dehydrogenase 6 (MT-ND6 or ND6), Mitochondrially encoded cytochrome b (MT-CYB), Mitochondrially encoded cytochrome c oxidase I (MT-COI), Mitochondrially encoded cytochrome c oxidase II (MT-COII), Mitochondrially encoded cytochrome c oxidase III (MT-COIII), ATPase6 (MT-ATP6), ATPase8 (MT-ATP8), 12S rRNA (MT-RNRl), 16S rRNA (MT-RNR2), Mitochondrially encoded tRNA threonine (MT-TT), Mitochondrially encoded tRNA proline (MT-TP), Mitochondrially encoded tRNA phenylalanine (MT-TF), Mitochondrially encoded tRNA valine (MT-TV), Mitochondrially encoded tRNA leucine 1 (UUA/G) or tRNALeu^"^ (MT-TL1), Mitochondrially encoded tRNA isoleucine (MT-Tl), Mitochondrially encoded tRNA glutamine (MT-TQ), Mitochondrially encoded tRNA Methionine (MT-TM), Mitochondrially encoded tRNA tryptophan (MT-TW), Mitochondrially encoded tRNA alanine (MT-TA), Mitochondrially encoded tRNA asparagine (MT-TN), Mitochondrially encoded tRNA cysteine (MT-TC), Mitochondrially encoded tRNA tyrosine (MT-TY), Mitochondrially encoded tRNA serine 1 (UCN) (MT-TS1), Mitochondrially encoded tRNA aspartic acid (MT-TD), Mitochondrially encoded tRNA lysine (MT-TK), Mitochondrially encoded tRNA glycine (MT-TG), Mitochondrially encoded tRNA arginine (MT-TR), Mitochondrially encoded tRNA histidine (MT-TH), Mitochondrially encoded tRNA Serine 2 (AGY) (MT-TS2), Mitochondrially encoded tRNA Leucine 2 (CUN) (MT-TL2) and Mitochondrially encoded tRNA glutamic acid (MT-TE), or nucleic acid encoding one or more of these mitochondrial products.
[0077] Accordingly, the present method is useful to treat a mitochondrial disease selected from the group consisting of Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQ10D), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG (e.g. Chronic Progressive External Ophthalmoplegia syndrome (CPEO), Alpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS) (including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO)), adPEO due to mutations in ANT or due to C10orf2 (twinkle) mutations, Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1/MyoNeurogenic Gastrointestinal Sideroblastic Encephalopathy (MNGIE), Mohr-Tranebjaerg syndrome, 3-methylglutaconic aciduria, Combined oxidative phosphoiyiation deficiency (COXPD), Myopathy, Lactic Acidosis, and Sideroblastic Anemia (MLASA), Hyperuricemia, Pulmonary hypertension, Renal failure, and Alkalosis (HUPRA) syndrome, Leigh Syndrome, Leigh syndrome, French Canadian type, Friedreich ataxia, Gracile syndrome, Bjornstad syndrome, Multiple Mitochondrial Dysfunctions Syndrome (MMDS), Early-onset Ataxia with Ocular motor apraxia and Hypoalbuminemia (EAOH), Charcot-Marie-Tooth Disease-2A2, Leber Hereditary Optic Neuropathy (LHON), Sudden Infant Death Syndrome, Myoclonic Epilepsy with Ragged Red Fibers (MERRF), MERRF/MELAS overlap syndrome, Neuropathy, Ataxia, Retinitis Pigmentosa (NARP), Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS), Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL), Mitochondrial disease resulting from SDH mutations (e.g. Pheochromocytoma and Paragangliomas), Optic atrophy type 1, Ethylmalonic encephalopathy, Carnitine-acylcamitine translocase deficiency, Primary systemic carnitine deficiency, Creatine deficiency syndromes (e.g. Cerebral creatine deficiency syndrome- 1, Cerebral creatine deficiency syndrome-2 or Cerebral creatine deficiency syndrome-3), Carnitine palmitoyltransferase 1 (CPT I) deficiency, Carnitine palmitoyltransferase 2 (CPT II) deficiency, Short-chain acyl-CoA dehydrogenase deficiency, Very long chain acyl- CoA dehydrogenase deficiency, Long-chain 3-hydroxyl-CoA dehydrogenase (LCHAD) deficiency, Pyruvate carboxylase deficiency, Multiple acyl-CoA dehydrogenase deficiency (e.g. Glutaric acidemia IIA, Glutaric acidemia IIB or Glutaric acidemia IIC), Pyruvate dehydrogenase deficiency (e.g. Pyruvate dehydrogenase El-alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3 -binding protein deficiency, Pyruvate dehydrogenase E2 deficiency or Pyruvate dehydrogenase El -beta deficiency), 3-hydroxyacyl- CoA dehydrogenase (SCHAD) deficiency/(HADH) deficiency, and Perrault syndrome.
[0078] As one of skill in the art will appreciate, the present method may also be effective to treat pathologies that result from secondary mitochondrial dysfunction including, but not limited to, metabolic syndrome, obesity, diabetes, blindness, deafness, cardiovascular diseases, movement disorders, neurometabolic and neuromuscular pathologies, epilepsy, autoimmune diseases, dementia, schizophrenia, bipolar disorder, neurodegenerative disease such as Alzheimer's disease and Parkinson's disease, sarcopenia, cancer, stroke, cachexia, cataracts, infertility, skin aging, and other aging-associated co -morbidities.
[0079] As one of skill in the art will appreciate, the mitochondrial products for incorporation into exosomes according to the invention may be. a functional native mammalian mitochondrial product, including for example, a mitochondrial product from human and non- human mammals, or a functionally equivalent mitochondrial product. The term "functionally equivalent" is used herein to refer to a protein which exhibits the same or similar function to the native protein (e.g. retains at least about 30% of the activity of the native protein), and includes all isoforms, variants, recombinant produced forms, and naturally-occurring or artificially modified forms, i.e. including modifications that do not adversely affect activity and which may increase cell uptake, stability, activity and/or therapeutic efficacy. The term "functionally equivalent" also refers to nucleic acid, e.g. mRNA, rRNA, tRNA, DNA, or cDNA, encoding a mitochondrial product, and is meant to include any nucleic acid sequence which encodes a functional mitochondrial product, including all transcript variants, valiants that encode mitochondrial product isoforms, variants due to degeneracy of the genetic code, artificially modified variants, and the like. Protein modifications may include, but are not limited to, one or more amino acid substitutions (for example, with a similarly charged amino acid, e.g.
substitution of one amino acid with another each having non-polar side chains such as valine, leucine, alanine, isoleucine, glycine, methionine, phenylalanine, tryptophan, proline; substitution of one amino acid with another each having basic side chains such as histidine, lysine, arginine; substitution of one amino acid with another each having acidic side chains such as aspartic acid and glutamic acid; and substitution of one amino acid with another each having polar side chains such as cysteine, serine, threonine, tyrosine, asparagine, glutamine), additions or deletions;
modifications to amino acid side chains, addition of a protecting group at the N- or C- terminal ends of the protein, addition of a mitochondrial targeting sequence such as
MLSARSAI RPIVRGLATV (SEQ ID NO: 1), MLRFTNCSCKTF V S SYKL I RRMNTV (SEQ ID NO: 2), MLRS S WRS RATLRPLLRRAYSS SFRT (SEQ ID NO: 3),
MLSARSAIKRPIVRGLATVSSFRT (SEQ ID NO: 4), MLRFTNCS CKTFVKSS YKLNIRRM SSFRT (SEQ ID NO: 5), MLSRAVCGTSRQLAPV (SEQ ID NO: 6), or targeting fragments thereof, at the N-terminat end of the protein and the like. Suitable modifications will generally maintain at least about 70% sequence similarity with the active site and other conserved domains of a native mitochondrial protein, and preferably at least about 80%, 90%, 95% or greater sequence similarity. Nucleic acid modifications may include one or more base substitutions or alterations, addition of 5' or 3' protecting groups, and the like, preferably maintaining significant sequence similarity, e.g. at least about 70%, and preferably, 80%, 90%, 95% or greater.
[0080] Engineered exosomes incorporating a mitochondrial product, and/or nucleic acid encoding the mitochondrial product, in accordance with the invention, may be formulated for therapeutic use by combination with a pharmaceutically or physiologically acceptable earner. The expressions "pharmaceutically acceptable" or "physiologically acceptable" means acceptable for use in the pharmaceutical and veterinary arts, i.e. not being unacceptably toxic or otherwise unsuitable for physiological use. As one of skill in the art will appreciate, the selected carrier will vary with intended utility of the exosome formulation. In one embodiment, exosomes are formulated for administration by infusion or injection, e.g. subcutaneously, intraperitoneally, intramuscularly or intravenously, and thus, are formulated as a suspension in a medical-grade, physiologically acceptable carrier, such as an aqueous solution in sterile and pyrogen-free form, optionally, buffered or made isotonic. The carrier may be distilled water (DNase- and RNase-free), a sterile carbohydrate-containing solution (e.g. sucrose or dextrose) or a sterile saline solution comprising sodium chloride and optionally buffered. Suitable sterile saline solutions may include varying concentrations of sodium chloride, for example, normal saline (0.9%), half-normal saline (0,45%), quarter-normal saline (0.22%), and solutions comprising greater amounts of sodium chloride (e.g. 3%-7%, or greater). Saline solutions may optionally include additional components, e.g. carbohydrates such as dextrose and the like. Examples of saline solutions including additional components, include Ringer's solution, e.g. lactated or acetated Ringer's solution, phosphate buffered saline (PBS), TRIS (hydroxymethyl) aminomethane hydroxymethyl) aminomethane)-buffered saline (TBS), Hank's balanced salt solution (HBSS), Earle's balanced solution (EBSS), standard saline citrate (SSC), HEPES- buffered saline (HBS) and Gey's balanced salt solution (GBSS).
[0081] In other embodiments, the present exosomes are formulated for administration by routes including, but not limited to, oral, intranasal, enteral, topical, sublingual, intra-arterial, intramedullary, intrauterine, intrathecal, inhalation, ocular, transdermal, vaginal or rectal routes, and will include appropriate carriers in each case. For oral administration, exosomes may be formulated in normal saline, complexed with food, in a capsule or in a liquid formulation with an emulsifying agent (honey, egg yolk, soy lecithin, and the like). Oral compositions may additionally include adjuvants including sugars, such as lactose, trehalose, glucose and sucrose; starches such as com starch and potato starch; cellulose and derivatives thereof, including sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil and com oil; polyols such as propylene glycol, glycerine, sorbital, mannitoi and polyethylene glycol; agar; alginic acids; water; isotonic saline and phosphate buffer solutions. Wetting agents, lubricants such as sodium lauryl sulfate, stabilizers, colouring agents and flavouring agents may also be present. Exosome compositions for topical application may be prepared including appropriate carriers. Creams, lotions and ointments may be prepared for topical application using an appropriate base such as a triglyceride base. Such creams, lotions and ointments may also contain a surface active agent. Aerosol formulations may also be prepared in which suitable propellant adjuvants are used. Other adjuvants may also be added to the composition regardless of how it is to be administered, for example, anti-microbial agents, anti-oxidants and other preservatives may be added to the composition to prevent microbial growth and/or degradation over prolonged storage periods.
[0082] The present engineered exosomes are useful in a method to treat a pathological condition involving a defective mitochondrial product, or a condition involving lack of expression of a mitochondrial product, e.g. a mitochondrial disease. The terms "treat", "treating" or "treatment" are used herein to refer to methods that favourably alter a mitochondrial disease or disorder, including those that moderate, reverse, reduce the severity of, or protect against, the progression of a mitochondrial disease or disorder. Thus, for use to treat such a disease, a therapeutically effective amount of exosomes engineered to incorporate a functional mitochondrial product, and/or nucleic acid encoding the functional mitochondrial product, useful to treat the disease, are administered to a mammal. The term "therapeutically effective amount" is an amount of exosome required to treat the disease, while not exceeding an amount that may cause significant adverse effects, Exosome dosages that are therapeutically effective will vary on many factors including the nature of the condition to be treated as well as the particular individual being treated. Appropriate exosome dosages for use include dosages sufficient to result in an increase in the amount or activity of the target mitochondrial product in the patient by at least about 10%, and preferably an increase in activity of the target mitochondrial product of greater than 10%, for example, at least 20%, 30%, 40%, 50% or greater. For example, in one embodiment, the dosage may be a dosage in an amount in the range of about 1 ug to about 500 mg of total exosomal protein for the delivery of a mitochondrial protein, or an amount in the range of about 20 ng to about 200 mg of total exosomal protein for the delivery of RNA species such as mRNA, tRNA, rRNA, miRNA, SRP RNA, snR A, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, piRNA or total mitochondrial RNA. In another exemplary embodiment, a dosage of exosomes sufficient to deliver about 0.1 mg/kg to about 100 mg/kg of a protein mitochondrial product, or a dosage of exosomes sufficient to deliver about 1 ng/kg to about 100 ug kg of a nucleic acid mitochondrial product (e.g. an RNA species), is administered to the mammal in the treatment of a target mitochondrial disease. The term "about" is used herein to mean an amount that may differ somewhat from the given value, by an amount that would not be expected to significantly affect activity or outcome as appreciated by one of skill in the art, for example, a variance of from 1-10% from the given value.
[0083] As will be appreciated by one of skill in the art, exosomes comprising a mitochondrial product, and/or nucleic acid encoding the mitochondrial product, for example, to treat a mitochondrial disease, may be used in conjunction with (at different times or simultaneously, either in combination or separately) one or more additional therapies to facilitate treatment, including but not limited to; anti-oxidants (i.e., coenzyme Q10, alpha lipoic acid, vitamin E, synthetic coenzyme Q10 analogues, resveratrol, N-acetylcysteine, etc.), creatine monohydrate, exercise (endurance, resistance or sprint-interval), mitochondrial targeting sequence motifs, or AMPK activators.
[0084] The use of engineered exosomes in a therapy to treat mitochondrial disease advantageously results in delivery of nucleic acid (mRNA, lRNA and tRNA) or protein safely to a cell to treat either mitochondrial or nuclear defects. Thus, the use of exosomes overcomes the challenges of delivery to the mitochondria, including the double mitochondrial membrane and the elaborate system of import that includes the TIMM and TOMM complex as well as various chaperone proteins.
[0085] Embodiments of the invention are described in the following examples which are not to be construed as limiting.
Example 1 - Treatment of Mitochondrial Disease due to Mutations in Mitochondrial Genes
[0086] To determine the efficacy of the present engineered exosomes to treat mitochondrial disease resulting from mutations in mitochondrial genes expressed in the mitochondria, exosomes were engineered to treat two of the most common and representative examples of mitochondrial DNA-based disease: LHON m.l l778G>A and MELAS m.3243A>G.
[0087] Exosome isolation methodology- Immature dendritic cells from human and mice are grown to 65-70% confluency in alpha minimum essential medium supplemented with ribonucleosides, deoxyribonucleosides, 4 mM L-glutamine, 1 niM sodium pyruvate and 5 ng/ml murine GM-CSF, and 20% fetal bovine serum. For conditioned media collection, cells were washed twice with sterile PBS (pH 7.4, Life Technologies) and aforementioned media (with exosome depleted fetal bovine serum) was added. Conditioned media from human and mouse immature dendritic cell culture was collected after 48 hours of induction. The media (10 mL) was spun at 2,000x g for 15 min at 4°C to remove any cellular debris. This is followed by 2000x g spin for 60 min at 4°C to further remove any contammating non-adherent cells. The supernatant was then spun at 14,000x g for 60 min at 4°C. The resulting supernatant was then filtered through a 40 μηι filter, followed by filtration through a 0.22 μηι syringe filter (twice) and finally filtered with a 0.10 μηι syringe filter. The resultant filtered supernatant was spun at 50,000x g for 60 min at 4°C, The supernatant was then carefully transferred into ultracentrifuge tubes and diluted with an equal amount of sterile PBS (pH 7.4, Life Technologies). This mixture was then subjected to ultracentrifugation at 100,000x-170,000x g for 2 hours at 4°C using a fixed-angle rotor. The resulting pellet was re-suspended in PBS and re-centrifuged at 100,000x- 170,000x g for 2 hours at 4°C. The pellet was resuspended carefully with 600 μ]^ of sterile PBS (pH 7.4, Life Technologies) and then gently added on top of 300 L of 30% Percoll cushion in an ultracentrifuge tube. This mixture was spun at 100,000x-170,000x g for 90 minutes at 4°C. With a syringe/pipette, the exosomal fraction (pellet-containing) was isolated carefully followed by a final spin for 90 minutes at 100,000x-170,000x g at 4°C to obtain purified exosomes. The resulting exosomes were resuspended in sterile PBS or sterile 0.9% saline for downstream use. Exosomal fraction purity was confirmed by sizing using a NanoSight LM10 instrument, and by immuno-gold labelling/Western blotting using the exosome membrane markers, CD9, CD63, TSG101 and ALIX.
[0088] Production of Hitman ND4 (mRNA and protein) and tRNAteu{VVK) - Human
ND4 (NCBI Reference Sequence: NC Ol 1137.1) cDNA from skeletal muscle (healthy human subject muscle biopsy obtained with prior consent and according to relevant Guidelines) was sub-cloned into the mammalian vector, pGEX GST-fusion vector (GE Healthcare Life Sciences) to form an ND4-pGEX vector. The vector was maintained using the competent E. coll DHSalpha cell-line (Life Technologies). The pGEX mammalian vector was then transfected into Chinese Hamster Ovary Cells (CHO; ATCC Cat. CCL-661) for mass production of recombinant human ND4 protein. To isolate recombinant ND4 protein, CHO cells transfected with ND4- pGEX vector were lysed using known techniques and CHO cell iysate was cleared using ultra- performance resins for GST-tagged fusion protein purification (GE Healthcare Life Sciences). Over 75% of the recombinant protein was eluted after 3 washes. Elution #1 and Elution #2 were combined to obtain a high yield of protein. GST tag was removed from recombinant ND4 using PreScission Protease (GE Healthcare Life Sciences).
[0089] To synthesize ND4 mRNA, ND4 cDNA from skeletal muscle was sub-cloned into pCMV6 entry vector (Origene) and amplified. Using conventional PCR, start codon (ATG) and Kozak sequence (GCCACC) were introduced. This cDNA was then cloned into the pMRNA p plasmid (System Biosciences) using EcoRI and BamHI restriction enzymes sites and the plasmids were used to transform competent E. coli DH5alpha cell-line (Life Technologies). Colonies containing the ND4 vector were amplified. The vector was isolated from these colonies (Qiagen) and T7 RNA polymerase-based in vitro transcription reaction was carried out to yield ND4 mRNA. An anti-reverse cap analog (ARCA), modified nucleotides (5-Methylcytidine-5'- Triphosphate and Pseudouridine-5'-Triphosphate) and poly-A tail were incorporated within the mRNAs to enhance their stability and to reduce the immune response of host cells. DNase I digest and phosphatase treatment was carried out to remove any DNA contamination and to remove the 5' triphosphates at the end of the RNA to further reduce innate immune responses in mammalian cells, respectively. The clean-up spin columns were used to recover ND4 mRNA for downstream encapsulation in engineered exosomes.
[0090] To synthesize mitochondrial tRNALeu(UUR) , tRNALeu^ from human muscle
Percoll-pmified mitochondrial fraction (from healthy human subject muscle biopsy) was sub- cloned into pCMV6 entry vector (Origene) and amplified. T7 RNA polymerase-based in vitro transcription reaction was carried out followed by charging of tRNA eu^m) with Leu amino acid using aminoacyl tRNA synthetase, LARS2, which was over- expressed and isolated from CHO cells. [0091] Introduction of ND4 mRNA or protem and tRNALeu(UUR) into exosomes -
Electroporation mixture was prepared by carefully mixing exosomes and ND4 mRNA, protein or tRNALeu^ in 1 :1 ratio (for example, 150 \x exosome suspension (10-15 £/μ3_, of total protein concentration as determined by BCA) to 150 \i of mRNA, tRNA, or protein suspension (at a concentration of 100-1000
Figure imgf000057_0001
in electroporation buffer. Electroporation was earned out in 0,4 mm electroporation cuvettes at 400 mV and 125
Figure imgf000057_0002
capacitance (pulse time 14 ms for mRNA and 24 ms for protein) using Gene Pulse XCell electroporation system (BioRad). After electroporation, exosomes were resuspended in 20 mL of 0.9% saline solution followed by ultracentrifugation for 2 hours at 170,000x g at 4°C. For in vitro and in vivo exosome administration, ND4 (mRNA or protein) or tRNA-Leu-loaded exosomes were re-suspended in 5% (wt/vol) glucose in 0.9% saline solution.
[0092] Mitochondrial fraction isolation- The mitochondrial fraction was isolated from
WT mice using differential centrifugation as previously described (Safdar et al., PNAS 108(10), 4135-40, 2011). In brief, skeletal muscle (quadriceps femoris) from WT mice was obtained, finely minced and homogenized on ice in 1 :10 (wt vol) ice-cold isolation buffer A (10 mM sucrose, 10 mM Tris/HCl, 50mM KC1, ImM EDTA, and 0.2% fatty acid free BSA (pH 7.4), supplemented with Complete EDTA-free protease inhibitor mixture (Roche Applied Science)) using a Potter-Elvehjem glass homogenizer. The resulting homogenate was centrifuged for 15 min at 700x g at 4°C, and the resulting supernatants were centrifuged for 20 min at 12,000x g at 4°C. The mitochondrial pellets from the 12,000x g spin were washed and then resuspended in a small volume of ice-cold isolation buffer B (10 mM sucrose, 0.1 mM EGTA/Tris, and 10 M Tris/HCl (pH 7.4), supplemented with Complete ETDA-free protease inhibitor mixture (Roche Applied Science)). All centrifugation steps were carried out at 4 °C. The mitochondrial pellets were then immediately flash frozen with liquid nitrogen and thawed 3 times to break open the mitochondria. The mitochondrial pellet was then treated with Proteinase K (20 pg^iL) to remove protein contamination. Mitochondrial total RNA (including mRNA, rRNA, tRNA, and miRNA species) was isolated using Qiagen total RNeasy kit and packaged into exosomes as described above.
[0093] Exosome studies in vitro with ND4 loaded exosomes - Human primary dermal fibroblasts were isolated from skin biopsies of healthy subjects (referred to as Control/CON) and patients with LHON (ND4 m.H778G>A mutation) (LHON) (n - 3 age/sex-matched per group) were treated with vehicle/saline (Vehicle), 100 ng of naked ND4 protein (naked ND4)), empty exosomes as an exosome control (Empty EXO), ND4 mRNA-loaded exosomes (EXO + ND4 mRNA), or ND4 protein-loaded exosomes (EXO + ND4 protein) (100 ng ND4 protein/mRNA in ~5-^g of total exosomal protein) for 48 hours. Cells were .harvested after 48 hours to assess mitochondrial complex I/citrate synthase (CS) enzyme activity, and oxygen consumption rate (OCR) using XFe24 Extracellular Flux Analyzer (Seahorse Bioscience).
[0094] Exosome studies in vitro with tRNALeu(UVRj 1 -loaded exosomes - Human primary dermal fibroblasts were isolated from skin biopsies of healthy subjects and patients with MELAS (tRNALeu(UUR), m.3243A>G mutation) (n = 3 age/sex-matched per group) using standard procedures. Healthy (Referred to as Control) and MELAS fibroblasts were treated with vehicle/saline (Vehicle), naked tRNALeu^^lOO ng) (naked tRNALeu), empty exosomes as exosome control (Empty EXO), and tRNALeu(UUR)-loaded exosomes (100 ng of tRNALeu(UUR) in -5-10 g of total exosomal protein) (EXO + tRNALeu) for 48 hours. Cells were harvested after 48 hours to assess mitochondrial complex IV (cytochrome c oxidase, COX)/CS enzyme activity.
RESULTS
[0095] Human ND4 mRNA or ND4 protein-loaded exosomes rescue mitochondrial dysfunction in LHON patients' fibroblasts. The treatment of LHON fibroblasts (from 3 independent patients with m.H778G>A ND4 mutation) using exosomes transfected with ND4 mRNA or ND4 protein was shown to reverse complex I deficiency and to result in complex I/citrate synthase (CS) activity levels comparable to fibroblasts from healthy subjects (Figure 1). The treatment also resulted in normalized oxygen consumption rates of LHON fibroblasts, indicating improved mitochondrial function (Figure 2).
[0096] Human tRNALeu(UUR) -loaded exosomes rescue dysfunction in MELAS patients' fibroblasts. Mitochondrial myopathy, encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a complex mitochondrial myopathy that presents with heterogeneous clinical symptoms, particularly affecting the brain, nervous system (encephalo-) and muscles (myopathy). The treatment of MELAS fibroblasts (3 independent patients with tRNALeu(UUR) m.3243A>G mutation) using exosomes loaded with tRNALeu^ reversed/mitigated complex IV deficiency. Complex IV/citrate synthase (CS) activity levels in treated MELAS fibroblasts were comparable to that in fibroblasts from healthy subjects (Figure 4).
Example 2 - Treatment of Mitochondrial Disease due to Mutations in Nuclear-encoded Mitochondrial Genes
[0097] To determine the efficacy of the present engineered exosomes to treat mitochondrial disease resulting from mutations in mitochondrial genes expressed in the nucleus, exosomes engineered to treat LONP1 deficiency, PolG deficiency and Friedreich ataxia (representative of such mitochondrial disease) were prepared and tested as described in the foregoing and further below.
[0098] Exoso e studies in vivo - Heterozygous mice (C57B1/6J, PolgA+ D257A) for the mitochondrial DNA polymerase gamma knock-in mutation were donated by Dr. Tomas A. Prolla, University of Wisconsin-Madison, USA. Homozygous knock-in mtDNA mutator mice (PolG; PolgAD257A/D257A) and littermate wildtype (WT; PolgA+/+) were generated from the heterozygous PolgA mice. During breeding, all animals were housed three to five per cage in a 12-h light/dark cycle and were fed ad libitum (Harlan-Teklad 8640 22/5 rodent diet) after weaning. The presence of the polymerase gamma homozygous knock-in mutation was confirmed as previously described (Safdar et al., PNAS 108(10), 4135-40, 2011). PolG mice were injected intravenously with empty exosomes (POLG1 mice + Empty Exosomes) (n = 5 mice) or exosomes packaged with the mitochondrial total RNA (10 g kg per dose in sterile saline in -10-20 μg of total exosomal protein) fraction from healthy mice (POLG1 mice + Mitochondrial mRNA cocktail healthy Exosomes) (n = 5) intravenously 5x a week for 4 weeks. Quadriceps femoris were harvested from all mice for Western blotting using mito-cocktail antibody (Mitosciences) to probe for subunits of mitochondrial electron transport chain (mitochondrial OXPHOS subunits) as previously described (Safdar et al., PNAS 108(10), 4135- 40, 2011). [0099] Production of Human LONPl mRNA - To synthesize LONPl mRNA, LONPl cDNA
(NCBI Reference Sequence: NM 004793), from skeletal muscle was sub-cloned into pCMV6 entry vector (Origene) and amplified as described for ND4 mRNA above. The cDNA was then cloned separately into the pMRNA*p plasmid (System Biosciences) using EcoRl and BamHI restriction enzymes sites and the plasmids were used to transform competent E. coli DH5alpha cell-line (Life Technologies). Colonies containing the LONPl vectors were amplified, isolated (Qiagen) and a T7 RNA polymerase- based in vitro transcription reaction was carried out to yield mRNA. Further processing was as described for ND4 mRNA.
[00100] Exosomes were prepared and loaded with LONP 1 as described above.
[00101 ] Exosome studies in vitro with LONPl mRNA loaded exosomes - Human primary dermal fibroblasts were isolated from skin biopsies of healthy subjects (referred to as Healthy) and patients with LONPl (LONPl Patients) (n = 3 age/sex-matched per group) and were treated with vehicle (saline), empty exosomes as exosome control (EMPTY EXO), and LONPl mRNA-loaded exosomes (EXO mRNA) (100 ng LONPl mRNA in l O^tg of total exosomal protein) for 48 hours. Cells were harvested after 48 hours to assess mitochondrial complex IV activity using standard photometric methods.
RESULTS
[00102] Mouse mitochondrial total RNA cocktail rescues mitochondrial dysfunction in PoIG mutator mouse model of systemic mitochondrial disease. The PoIG mtDNA mutator mice demonstrate how a primary genetic mitochondrial mutation can result in systemic mitochondrial dysfunction, and multisystem pathology. Eight month old PoIG mutator mice show early signs of aging, display vast array of systemic pathologies, and are exercise intolerant. After treatment as described above with exosomes loaded with total mitochondrial RNA from healthy mice showed a significant up-regulation of mitochondrial OXPHOS summits in muscle of PoIG mutator mice (Figure 3).
[00103] Human LONPl mRNA-loaded exosomes rescue mitochondrial dysfunction in LONPl patients* fibroblasts. The treatment of LONPl fibroblasts (from 4 independent patients) using exosomes transfected with LONPl mRNA was shown to mitigate mitochondrial complex IV deficiency and resulted in complex IV activity levels comparable to fibroblasts from healthy subjects (Figure 5).

Claims

1. Exosomes which are genetically modified to incorporate at least one functional mitochondrial product or precursor thereof, and/or nucleic acid encoding a functional mitochondrial product or precursor thereof.
2. The exosomes of claim 1, essentially free from particles having a diameter less than 20 nm or greater than 120 nm.
3. The exosomes of claim 1, which exhibit a zeta potential having a magnitude of at least about 30 mV, and preferably 40 mV or greater.
4. The exosomes of claim 1, wherein the mitochondrial product is a native mitochondrial product or a modified mitochondrial product.
5. The exosomes of claim 1 , which are mammalian exosomes.
6. The exosomes of claim 1, wherein the functional mitochondrial product is a protein, or RNA selected from the group consisting of mRNA, tRNA, rRNA, miRNA, SRP RNA, snRNA, scRNA, snoRNA, gRNA, RNase P, RNase MRP, yRNA, TERC, SLRNA, IncRNA, piRNA or total mitochondrial RNA.
7. The exosomes of claim 6, wherein the mitochondrial product is expressed in the mitochondria.
8. The exosomes of claim 7, wherein the mitochondrial product is selected from the group consisting of: Mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), Mitochondrially encoded NADH dehydrogenase 2 (MT-ND2), Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3), Mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), Mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), Mitochondrially encoded NADH dehydrogenase 5 (MT-ND5), Mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), Mitochondrially encoded cytochrome b (MT-CYB), Mitochondrially encoded cytochrome c oxidase I (MT-COI), Mitochondrially encoded cytochrome c oxidase II (MT-COII), Mitochondrially encoded cytochrome c oxidase III (MT-COIII), ATPase6 (MT-ATP6), ATPase8 (MT-ATP8), 12S rRNA (MT-RNRl), 16S rRNA (MT-RNR2), Mitochondrially encoded tRNA threonine (MT-TT), Mitochondrially encoded tRNA proline (MT-TP), Mitochondrially encoded tRNA phenylalanine (MT-TF), Mitochondrially encoded tRNA valine (MT-TV), Mitochondrially encoded tRNA leucine 1 (UUA/G) (MT-TLl), Mitochondrially encoded tRNA isoleucine (MT-TI), Mitochondrially encoded tRNA glutamine (MT-TQ), Mitochondrially encoded tRNA Methionine (MT-TM), Mitochondrially encoded tRNA tryptophan (MT-TW), Mitochondrially encoded tRNA alanine (MT-TA), Mitochondrially encoded tRNA asparagine (MT-TN), Mitochondrially encoded tRNA cysteine (MT-TC), Mitochondrially encoded tRNA tyrosine (MT-TY), Mitochondrially encoded tRNA serine 1 (UCN) (MT-TS1), Mitochondrially encoded tRNA aspartic acid (MT-TD), Mitochondrially encoded tRNA lysine (MT-TK), Mitochondrially encoded tRNA glycine (MT-TG), Mitochondrially encoded tRNA arginine (MT-TR), Mitochondrially encoded tRNA histidine (MT-TH), Mitochondrially encoded tRNA Serine 2 (AGY) (MT-TS2), Mitochondrially encoded tRNA Leucine 2 (CUN) (MT-TL2), Mitochondrially encoded tRNA glutamic acid (MT-TE).
9. The exosomes of claim 6, wherein the mitochondrial product is expressed in the nucleus,
10. The exosomes of claim 9, wherein the mitochondrial product is selected from the group consisting of: Lon peptidase 1, mitochondrial (LONP1), NADHrubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADFLubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADFLubiquinone oxidoreductase complex assembly factor 3 (NDUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADFLubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl- CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1), Succinate dehydrogenase complex assembly factor 2 (SDHAF2), BCS1 homolog, ubiquinol-cytochrome c reductase complex chaperone (BCS1L), Surfeit 1 (SURF1), SCOl cytochrome c oxidase assembly protein (SCOl), COX10 heme A:farnesyltransferase cytochrome c oxidase assembly factor (COX10), Cytochrome c oxidase assembly homolog 15 (COX15), Leucine rich pentatricopeptide repeat containing (LRPPRC), FAST kinase domains 2 (FASTKD2), Translational activator of mitochondrially encoded cytochrome c oxidase I (TACOl), ATP synthase mitochondrial Fl complex assembly factor 2 (ATPAF2), Transmembrane protein 70 (TMEM70), Polymerase (DNA directed), gamma (POLG), Polymerase (DNA directed), gamma 2, accessory subunit (POLG2), MpV17 mitochondrial inner membrane protein (MPV17), ChiOmosome 10 open reading frame 2 (C10orf2), Thymidine phosphorylase (TYMP), Deoxyguanosine kinase (DGUOK), Ribonucleotide reductase M2 B (RRM2B), Succinate-CoA ligase, ADP-forming, beta subunit (SUCLA2), Succinate-CoA ligase, alpha subunit (SUCLG1), Thymidine kinase 2, mitochondrial (TK2), Translocase of inner mitochondrial membrane 8 homolog A (TIMM8A), DnaJ heat shock protein family (Hsp40) member C19 (DNAJC19), G elongation factor, mitochondrial 1 (GFM1), Leucyl-tRNA synthetase 2 (LARS 2), TyrosyL-tRNA synthetase 2 (YARS2), Seryl-tRNA synthetase 2, mitochondrial (SARS2), Aspartyl-tRNA synthetase 2, mitochondrial (DARS2), Arginyl-tRNA synthetase 2, mitochondrial (RARS2), Mitochondrial ribosomal protein SI 6 (MRPS16), Mitochondrial ribosomal protein S22 (MRPS22), Ts translation elongation factor, mitochondrial (TSFM), Tu translation elongation factor, mitochondrial (TUFM), Frataxin (FXN), ATP-binding cassette subfamily B member 7 (ABCB7), Solute carrier family 25 member 38 (SLC25A38), Iron-sulfur cluster assembly enzyme (ISCU), BolA family member 3 (BOLA3), NFU1 iron- sulfur cluster scaffold (NFU3), Coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (COQ2), Coenzyme Q4 (COQ4), Coenzyme Q9 (COQ9), Aprataxin (APTX), Prenyl (decaprenyl) diphosphate synthase, subunit 1 (PDSS1), Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2), AarF domain containing kinase 3 (ADC 3), Paraplegin (SPG7), Heat shock protein family D (Hsp60) member 1 (HSPD1), Optic atrophy 1 (autosomal dominant) (OPA1), Mitofusin 2 (MFN2), Dynamin 1-like (DNM1L), Tafazzin (TAZ), Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Ethylmalonic encephalopathy 1 (ETHE1), Pseudouridylate synthase 1 (PUS1), NADH:ubiquinone oxidoreductase core subunit SI (NDUFS1), NADH:ubiquinone oxidoreductase core subunit S2 (NDUFS2), NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), NADH:ubiquinone oxidoreductase subunit S6 (NDUFS6), NADH: ubiquinone oxidoreductase core subunit S7 (NDUFS7), NADH:ubiquinone oxidoreductase core subunit S8 ( DUFS8), NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3), NADH:ubiquinone oxidoreductase core subunit VI ( DUFV1), NADH:ubiquinone oxidoreductase core subunit V2 (NDUFV2), NADH:ubiquinone oxidoreductase subunit Al (NDUFA1), NADH:ubiquinone oxidoreductase subunit A2 (NDUFA2), NADH:ubiquinone oxidoreductase subunit A10 (NDUFA10), NADH:ubiquinone oxidoreductase subunit Al l (NDUFA11), Succinate dehydrogenase complex subunit A, flavoprotein (Fp) (SDHA), Succinate dehydrogenase complex subunit B, iron sulfur (Ip) (SDHB), Succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa (SDHC), Succinate dehydrogenase complex subunit D} integral membrane protein (SDHD), Ubiquinol-cytochrome c reductase binding protein (UQCRB), Ubiquinol-cytochronie c reductase complex III subunit VII (UQCRQ), Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) (COX6B1), ATP synthase, H+ transporting, mitochondrial Fl complex, epsilon subunit (ATP5E), Solute earner family 22 (organic cation/carnitine transporter), member 5 (SLC22A5), Carnitine palmitoyltransferase 2 (CPT2), Carnitine palmitoyltransferase 1A (liver) (CPT1A), Solute carrier family 6 (neurotransmitter transporter), member 8 (SLC6A8), Guanidinoacetate N-methyitransferase (GAMT), Glycine amidinotransferase (GATSVI), Pyruvate carboxylase (PC), hydroxyacyl-CoA dehydrogenase/3 - ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (H ADH A)/ (LCH AD) , Acyl-CoA dehydrogenase, very long chain (ACADVL), Acyl-CoA dehydrogenase, C-2 to C-3 short chain (ACADS), Hydroxyacyl-CoA dehydrogenase (HADH)/(SCHAD), Electron transfer flavoprotein alpha subunit (ETFA), Electron transfer flavoprotein beta subunit (ETFB), Electron transfer flavoprotein dehydrogenase (ETFDH), Solute carrier family 25 (SLC25A20), Pyruvate dehyrogenase phosphatase catalytic subunit 1 (PDP1), Pyruvate dehydrogenase complex component X (PDHXD), Dihydrolipoamide S- acetyltransferase (DLAT), Pyruvate dehydrogenase (lipoamide) beta (PDHB), and Solute carrier family 25 (mitochondrial carder; adenine nucleotide translocator), member (4SLC25A4)/(ANT).
11. The exosomes of claim 1, wherein the mitochondrial product is selected from the group consisting of Lo peptidase 1, mitochondrial (LONP1), NADH:ubiquinone oxidoreductase complex assembly factor 1 (NDUFAFl), NADH:ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH biquinone oxidoreductase complex assembly factor 3 (NDUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH '.ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl- CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1), Succinate dehydrogenase complex assembly factor 2 (SDHAF2), BCS1 homolog, ubiquinol-cytochrome c reductase complex chaperone (BCS1L), Surfeit 1 (SURF1), SCOl cytochrome c oxidase assembly protein (SCOl), COX10 heme A:farnesyltransferase cytochrome c oxidase assembly factor (COX10), Cytochrome c oxidase assembly homolog 15 (COX15), Leucine rich pentatricopeptide repeat containing (LRPPRC), FAST kinase domains 2 (FASTKD2), Translational activator of mitochondrially encoded cytochrome c oxidase I (TACOl), ATP synthase mitochondrial Fl complex assembly factor 2 (ATPAF2), Transmembrane protein 70 (TMEM70), Polymerase (DNA directed), gamma (POLG), Polymerase (DNA directed), gamma 2, accessory subunit (POLG2), MpV17 mitochondrial inner membrane protein (MPV17), Chromosome 10 open reading frame 2 (C10orf2), Thymidine phosphorylase (TYMP), Deoxyguanosine kinase (DGUOK), Ribonucleotide reductase M2 B (RRM2B), Succinate-CoA ligase, ADP-forming, beta subunit (SUCLA2), Succinate-CoA ligase, alpha subunit (SUCLG1), Thymidine kinase 2, mitochondrial (TK2), Translocase of inner mitochondrial membrane 8 homolog A (TIMM8A), DnaJ heat shock protein family (Hsp40) member C19 (DNAJC19), G elongation factor, mitochondrial 1 (GFM1), TyrosyL-tRNA synthetase 2 (YARS2), Seryl-tRNA synthetase 2, mitochondrial (SARS2), Aspartyl-tRNA synthetase 2, mitochondrial (DARS2), Arginyl-tRNA synthetase 2, mitochondrial (RARS2), Mitochondrial ribosomal protein S16 (MRPS16), Mitochondrial ribosomal protein S22 (MRPS22), Ts translation elongation factor, mitochondrial (TSFM), Tu translation elongation factor, mitochondrial (TUFM), Frataxin (FXN), ATP-binding cassette subfamily B member 7 (ABCB7), Solute carrier family 25 member 38 (SLC25A38), Iron-sulfur cluster assembly enzyme (ISCU), BolA family member 3 (BOLA3), NFU1 iron-sulfur cluster scaffold (NFU3), Coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (COQ2), Coenzyme Q4 (COQ4), Coenzyme Q9 (COQ9), Aprataxin (APTX), Prenyl (decaprenyl) diphosphate synthase, subunit 1 (PDSS1), Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2), AarF domain containing kinase 3 (ADCK3), Paraplegin (SPG7), Heat shock protein family D (Hsp60) member 1 (HSPD1), Optic atrophy 1 (autosomal dominant) (OPA1), Mitofusin 2 (MFN2), Dynamin 1- like (DNM1L), Tafazzin (TAZ), Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Ethylmalonic encephalopathy 1 (ETHE1), Pseudouridylate synthase 1 (PUS1), NADH:ubiquinone oxidoreductase core subunit SI (NDUFS1), NADH: ubiquinone oxidoreductase core subunit S2 (NDUFS2), NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), NADH ubiquinone oxidoreductase subunit S6 (NDUFS6), NADH:ubiquinone oxidoreductase core subunit S7 (NDUFS7), NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8), NADH ubiquinone oxidoreductase subunit B3 (NDUFB3), NADH:ubiquinone oxidoreductase core subunit VI (NDUFV1), NADH:ubiquinone oxidoreductase core subunit V2 NDUFV2), NADH:ubiquinone oxidoreductase subunit Al (NDUFA1), NADH: ubiquinone oxidoreductase subunit A2 (NDUFA2), NADH:ubiquinone oxidoreductase subunit A10 (NDUFA10), NADH:ubiquinone oxidoreductase subunit Al l (NDUFA11), Succinate dehydrogenase complex subunit A, flavoprotein (Fp) (SDHA), Succinate dehydrogenase complex subunit B, iron sulfur (Ip) (SDHB), Succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa (SDHC), Succinate dehydrogenase complex subunit D, integral membrane protein (SDHD), Ubiquinol-cytochrome c reductase binding protein (UQCRB), Ubiquinol-py to chrome c reductase complex III subunit VII (UQCRQ), Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) (COX6B1), ATP synthase, H+ transporting, mitochondrial Fl complex, epsilon subunit (ATP5E), Solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5), Carnitine palmitoyltransferase 2 (CPT2), Carnitine palmitoyltransferase 1A (liver) (CPT1A), Solute carrier family 6 (neurotransmitter transporter), member 8 (SLC6A8), Guanidinoacetate N-methyltransferase (GAMT), Glycine amidinotransferase (GATM), Pyruvate carboxylase (PC), hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA)/(LCHAD), Acyl-CoA dehydrogenase, very long chain (ACADVL), Acyl-CoA dehydrogenase, C-2 to C-3 short chain (ACADS), Hydroxyacyl-CoA dehydrogenase (HADH)/(SCHAD), Electron transfer flavoprotein alpha subunit (ETFA), Electron transfer flavoprotein beta subunit (ETFB), Electron transfer flavoprotein dehydrogenase (ETFDH), Solute carrier family 25 (SLC25A20), Pyruvate dehyrogenase phosphatase catalytic subunit 1 (PDP1), Pyruvate dehydrogenase complex component X (PDHXD), Dihydrohpoamide S-acetyltiansferase (DLAT), Pyruvate dehydrogenase (lipoamide) beta (PDHB), Solute carrier family 25 (mitochondrial earner; adenine nucleotide translocator), member (4SLC25A4)/(ANT), Mitochondrially encoded NADH dehydrogenase 1 (MT-ND1), Mitochondrially encoded NADH dehydrogenase 2 (MT-ND2), Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3), Mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), Mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), Mitochondrially encoded NADH dehydrogenase 5 (MT-ND5), Mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), Mitochondrially encoded cytochrome b (MT-CYB), Mitochondrially encoded cytochrome c oxidase I (MT-COI), Mitochondrially encoded cytochrome c oxidase II (MT-COII), Mitochondrially encoded cytochrome c oxidase III (MT- COIII), ATPase6 (MT-ATP6), ATPase8 (MT-ATP8), 12S lRNA (MT-RNR1), 16S lRNA (MT- RNR2), Mitochondrially encoded tRNA threonine (MT-TT), Mitochondrially encoded tRNA proline (MT-TP), Mitochondrially encoded tRNA phenylalanine (MT-TF), Mitochondrially encoded tRNA valine (MT-TV), Mitochondrially encoded tRNA leucine 1 (UUA/G) (MT-TL1), Mitochondrially encoded tRNA isoleucine (MT-TI), Mitochondrially encoded tRNA glutamine (MT-TQ), Mitochondrially encoded tRNA Methionine (MT-TM), Mitochondrially encoded tRNA tryptophan (MT-TW), Mitochondrially encoded tRNA alanine (MT-TA), Mitochondrially encoded tRNA asparagine (MT-TN), Mitochondrially encoded tRNA cysteine (MT-TC), Mitochondrially encoded tRNA tyrosine (MT-TY), Mitochondrially encoded tRNA serine 1 (UCN) (MT-TS1), Mitochondrially encoded tRNA aspartic acid (MT-TD), Mitochondrially encoded tRNA lysine (MT-TK), Mitochondrially encoded tRNA glycine (MT-TG), Mitochondrially encoded tRNA arginine (MT-TR), Mitochondrially encoded tRNA histidine (MT-TH), Mitochondrially encoded tRNA Serine 2 (AGY) (MT-TS2), Mitochondrially encoded tRNA Leucine 2 (CUN) (MT-TL2), and Mitochondrially encoded tRNA glutamic acid (MT-TE).
12. The exosomes of claim 1, wherein the mitochondrial product is exogenous.
13. The exosomes of claim 1, further modified to incorporate or express a target-specific fusion product comprising a mitochondrial-targeting sequence linked to an exosomal membrane marker.
14. The exosomes of claim 13, wherein the exosomal membrane marker is selected from the group consisting of CD9, CD37, CD53, CD63, CD81, CD82, CD151, an integrin, ICAM-1, CDD31, an annexin, TSG101, ALIX, lysosome-associated membrane protein 1, lysosome- associated membrane protein 2, lysosomal integral membrane protein and a fragment of any exosomal membrane marker that comprises at least one intact transmembrane domain.
15. The exosomes of claim 13, wherein the mitochondrial-targeting sequence is selected from the group consisting of aconitase, superoxide dismutase 2, MLSARSAIKRPIVRGLATV (SEQ ID NO: 1), LRFTNCSCKTFVKSSYKLNIRRM TV (SEQ ID NO: 2),
MLRSSVVRSRATLRPLLRRAYSSSFRT (SEQ ID NO: 3), M LS ARS AIKRPI V GL AT V S SFRT (SEQ ID NO: 4), MLRFTNCSCKTF VKSS YKLNIRRMNS SFRT (SEQ ID NO: 5), ML SRA VCGTSRQL AP V (SEQ ID NO: 6), or a targeting fragment of any one of these.
16. A composition comprising genetically modified exosomes as defined in claim 1 combined with a pharmaceutically acceptable carrier.
17. The composition of claim 16, comprising exosomal protein in an amount of about 100- 2000
18. A method of increasing the amount or activity of a mitochondrial product in a mammal, comprising administering to the mammal a composition comprising exosomes that are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
1 . The method of claim 18, wherein the exosomes are essentially free from particles having a diameter less than 20 nm or greater than 120 nm.
20. The method of claim 18, wherein the exosomes exhibit a zeta potential having a magnitude of at least about 30 mV, and preferably 40 mV or greater.
21. A method of treating a mitochondrial disease in a mammal comprising administering to the mammal a composition comprising exosomes which are genetically modified to incorporate a functional mitochondrial product and/or nucleic acid encoding a functional mitochondrial product.
22. The method of claim 21, wherein the exosomes are essentially free from particles having a diameter less than 20 nm or greater than 120 nm.
23. The method of claim 21, wherein the exosomes exhibit a zeta potential having a magnitude of at least about 30 mV, and preferably 40 mV or greater.
24. The method of claim 21, wherein the mitochondrial product is expressed in the mitochondria.
25. The method of claim 24, wherein the mitochondrial product is selected from the group consisting of: Mitochondrially encoded NADH dehydrogenase 1 (MT-NDl), Mitochondrially encoded NADH dehydrogenase 2 (MT-ND2), Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3), Mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), Mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), Mitochondrially encoded NADH dehydrogenase 5 (MT-ND5), Mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), Mitochondrially encoded cytochrome b (MT-CYB), Mitochondrially encoded cytochrome c oxidase I (MT-COl), Mitochondrially encoded cytochrome c oxidase II (MT-COII), Mitochondrially encoded cytochrome c oxidase III (MT-COIII), ATPase6 (MT-ATP6), ATPaseS (MT-ATP8), 12S rRNA (MT-RNR1), 16S rRNA (MT-RNR2), Mitochondrially encoded tRNA threonine (MT-TT), Mitochondrially encoded tRNA proline (MT-TP), Mitochondrially encoded tRNA phenylalanine (MT-TF), Mitochondrially encoded tRNA valine (MT-TV), Mitochondrially encoded tRNA leucine 1 (UUA G) (MT-TL1), Mitochondrially encoded tRNA isoleucine (MT-TI), Mitochondrially encoded tRNA glutamine (MT-TQ), Mitochondrially encoded tRNA Methionine (MT-TM), Mitochondrially encoded tRNA tryptophan (MT-TW), Mitochondrially encoded tRNA alanine (MT-TA), Mitochondrially encoded tRNA asparagine (MT-TN), Mitochondrially encoded tRNA cysteine (MT-TC), Mitochondrially encoded tRNA tyrosine (MT-TY), Mitochondrially encoded tRNA serine 1 (UCN) (MT-TS1), Mitochondrially encoded tRNA aspartic acid (MT-TD), Mitochondrially encoded tRNA lysine (MT-TK), Mitochondrially encoded tRNA glycine (MT-TG), Mitochondrially encoded tRNA arginine (MT-TR), Mitochondrially encoded tRNA histidine (MT-TH), Mitochondrially encoded tRNA Serine 2 (AGY) (MT-TS2), Mitochondrially encoded tRNA Leucine 2 (CUN) (MT-TL2), and Mitochondrially encoded tRNA glutamic acid (MT-TE).
26. The method of claim 21 , wherein the mitochondrial product is expressed in the nucleus.
27. The method of claim 26, wherein the mitochondrial product is selected from the group consisting of: Lon peptidase 1, mitochondrial (LONP1), NADH:ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADH:ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH:ubiquinone oxidoreductase complex assembly factor 3 (NDUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH -.ubiquinone oxidoreductase complex assembly factor 5 (NDUFAF5), Nucleotide-binding protein-like ( UBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl- CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1), Succinate dehydrogenase complex assembly factor 2 (SDHAF2), BCS1 homolog, ubiquinol-cytochrome c reductase complex chaperone (BCS1L), Surfeit 1 (SURF1), SCOl cytochrome c oxidase assembly protein (SCOl), COX10 heme A:farnesyltransferase cytochrome c oxidase assembly factor (COX 10), Cytochrome c oxidase assembly homolog 15 (COX15), Leucine rich pentatricopeptide repeat containing (LRPPRC), FAST kinase domains 2 (FASTKD2), Translational activator of mitochondrially encoded cytochrome c oxidase I (TACOl), ATP synthase mitochondrial Fl complex assembly factor 2 (ATPAF2), Transmembrane protein 70 (TMEM70), Polymerase (DNA directed), gamma (POLG), Polymerase (DNA directed), gamma 2, accessory subunit (POLG2), MpV17 mitochondrial inner membrane protein (MPV17), Chromosome 10 open reading frame 2 (C10orf2), Thymidine phosphorylase (TYMP), Deoxyguanosine kinase (DGUOK), Ribonucleotide reductase M2 B (RRM2B), Succinate-CoA ligase, ADP -forming, beta subunit (SUCLA2), Succinate-CoA Hgase, alpha subunit (SUCLGl), Thymidine kinase 2, mitochondrial (TK2), Translocase of inner mitochondrial membrane 8 homolog A (TIMM8A), DnaJ heat shock protein family (Hsp40) member C19 (DNAJC19), G elongation factor, mitochondrial 1 (GFMl), Leucyl-tRNA synthetase 2 (LARS 2), TyrosyL-tRNA synthetase 2 (YARS2), Seryl-tRNA synthetase 2, mitochondrial (SARS2), Aspartyl-tRNA synthetase 2, mitochondrial (DARS2), Arginyl-tRNA synthetase 2, mitochondrial (RARS2), Mitochondrial ribosomal protein SI 6 (MRPS16), Mitochondrial ribosomal protein S22 (MRPS22), Ts translation elongation factor, mitochondrial (TSFM), Tu translation elongation factor, mitochondrial (TUFM), Frataxin (FXN), ATP-binding cassette subfamily B member 7 (ABCB7), Solute carrier family 25 member 38 (SLC25A38), Iron-sulfur cluster assembly enzyme (ISCU), BolA family member 3 (BOLA3), NFU1 iron- sulfur cluster scaffold (NFU3), Coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (COQ2), Coenzyme Q4 (COQ4), Coenzyme Q9 (COQ9), Aprataxin (APTX), Prenyl (decaprenyl) diphosphate synthase, subunit 1 (PDSS1), Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2), AarF domain containing kinase 3 (ADCK3), Paraplegin (SPG7), Heat shock protein family D (Hsp60) member 1 (HSPD1), Optic atrophy 1 (autosomal dominant) (OPA1), Mitofusin 2 (MFN2), Dynamin 1-like (DNM1L), Tafazzin (TAZ), Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Ethylmalonic encephalopathy 1 (ETHE1), Pseudouridylate synthase 1 (PUS1), NADH:ubiquinone oxidoreductase core subunit SI (NDUFS1), NADH ubiquinone oxidoreductase core subunit S2 (NDUFS2), NADH:ubiquinone oxidoreductase core subunit S3 (NDUFS3), NADH:ubiquinone oxidoreductase subunit S4 ( DUFS4), NADH:ubiquinone oxidoreductase subunit S6 ( DUFS6), NADH: ubiquinone oxidoreductase core subunit S7 (NDUFS7), NADH:ubiquinone oxidoreductase core subunit S8 (NDUFS8), NADHmbiquinone oxidoreductase subunit B3 (NDUFB3), NADH:ubiquinone oxidoreductase core subunit VI (NDUFV1), NADH:ubiquinone oxidoreductase core subunit V2 (NDUFV2), NADH:ubiquinone oxidoreductase subunit Al (NDUFA1), NADH:ubiquinone oxidoreductase subunit A2 (NDUFA2), NADH:ubiquinone oxidoreductase subunit A10 (NDUFA10), NADH ubiquinone oxidoreductase subunit Al l (NDUFA11), Succinate dehydrogenase complex subunit A, flavoprotein (Fp) (SDHA), Succinate dehydrogenase complex subunit B, iron sulfur (Ip) (SDHB), Succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa (SDHC), Succinate dehydrogenase complex subunit D, integral membrane protein (SDHD), Ubiquinol-cytochrome c reductase binding protein (UQCRB), Ubiquinol-cytochrome c reductase complex HI subunit VII (UQCRQ), Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) (COX6B1), ATP synthase, H+ transporting, mitochondrial Fl complex, epsilon subunit (ATP5E), Solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5), Carnitine palmitoyltransferase 2 (CPT2), Carnitine palmitoyltransferase 1A (liver) (CPT1A), Solute carrier family 6 (neurotransmitter transporter), member 8 (SLC6A8), Guanidinoacetate N-methyltransferase (GAMT), Glycine amidinotransferase (GATM), Pyruvate carboxylase (PC), hydroxyacyl-CoA dehydrogenase/3 - ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (HADHA)/(LCHAD), Acyl-CoA dehydrogenase, very long chain (ACADVL), Acyl-CoA dehydrogenase, C-2 to C-3 short chain (ACADS), Hydroxyacyl-CoA dehydrogenase (H ADH)/ (S CHAD), Electron transfer flavoprotein alpha subunit (ETFA), Electron transfer flavoprotein beta subunit (ETFB), Electron transfer flavoprotein dehydrogenase (ETFDH), Solute carrier family 25 (SLC25A20), Pyruvate dehyrogenase phosphatase catalytic subunit 1 (PDP1), Pyruvate dehydrogenase complex component X (PDHXD), Dihydrolipoamide S- acetyltransferase (DLAT), Pyruvate dehydrogenase (lipoamide) beta (PDHB), and Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member (4SLC25A4)/(ANT),
28. The method of claim 21 , wherein the mitochondrial disease is selected from the group consisting of Mitochondrial complex I deficiency, Mitochondrial complex II deficiency, Mitochondrial complex III deficiency, Mitochondrial complex IV deficiency, Mitochondrial complex V (ATP synthase) deficiency, Primary coenzyme Q10 deficiency (COQ10D), Cerebral, Ocular, Dental, Auricular, and Skeletal anomalies (CODAS) syndrome, Mitochondrial disease resulting from mutations in PolG, Chronic Progressive External Ophthalmoplegia syndrome (CPEO), Alpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS), mitochondrial recessive ataxia syndrome (MIRAS), sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO), Mitochondrial DNA depletion syndrome, Mitochondrial DNA depletion syndrome 1 MyoNeuro genie Gastrointestinal Encephalopathy (MNGIE), Mohr-Tranebjaerg syndrome, 3- methylglutaconic aciduria, Combined oxidative phosphorylation deficiency, Myopathy, Lactic Acidosis, and Sideroblastic Anemia (MLASA), Hyperuricemia, Pulmonary hypertension, Renal failure, and Alkalosis (HUPRA) syndrome, Leigh Syndrome, Leigh syndrome, French Canadian type, Friedreich ataxia, Gracile syndrome, Bjornstad syndrome, Multiple Mitochondrial Dysfunctions Syndrome (MMDS), Early-onset Ataxia with Ocular motor apraxia and Hypoalbuminemia (EAOH), Charcot-Marie-Tooth Disease-2A2, Leber Hereditary Optic Neuropathy (LHON), Sudden Infant Death Syndrome, Myoclonic Epilepsy with Ragged Red Fibers (MERRF), MERRF/MELAS overlap syndrome, Neuropathy, Ataxia, Retinitis Pigmentosa (NARP), Mitochondrial myopathy, Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS), Leukoencephalopathy with Brain stem and Spinal cord involvement and Lactate elevation (LBSL), Mitochondrial disease resulting from SDH mutations, Spastic paraplegia, Paragangliomas, Pheochromocytoma, Optic atrophy type 1, Ethylmalonic encephalopathy, Carnitine-acylcarnitine translocase deficiency, Primary systemic carnitine deficiency, Cerebral creatine deficiency syndrome- 1, Cerebral creatine deficiency syndrome-2 or Cerebral creatine deficiency syndrome-3, Carnitine palmitoyltransferase 1 (CPT 1) deficiency, Carnitine palmitoyltransferase 2 (CPT II) deficiency, Short-chain acyl-CoA dehydrogenase deficiency, Very long chain acyl-CoA dehydrogenase deficiency, Long-chain 3-hydroxyl-CoA dehydrogenase (LCHAD) deficiency, Multiple acyl-CoA dehydrogenase deficiency, Glutaric acidemia ΠΑ, Glutaric acidemia IIB, Glutaric acidemia IIC, Pyruvate carboxylase deficiency, Pymvate dehydrogenase deficiency, Pyruvate dehydrogenase El -alpha deficiency, Pyruvate dehydrogenase phosphatase deficiency, Pyruvate dehydrogenase E3-binding protein deficiency, Pyruvate dehydrogenase E2 deficiency, Pyruvate dehydrogenase El -beta deficiency, 3- hydroxyacyl-CoA dehydrogenase (SCHAD) deficiency/(HADH) deficiency, and Perrault syndrome,
29. The method of claim 21, wherein the mitochondrial disease is selected from the group consisting of Metabolic syndrome, Obesity, Diabetes, Blindness, Deafness, Cardiovascular diseases, Movement disorders, Neurometabolic and neuromuscular pathologies, Epilepsy, Autoimmune diseases, Dementia, Schizophrenia, Bipolar disorder, Parkinson's disease, Sarcopenia, Cancer, Stroke, Cachexia, Cataracts, Infertility and Skin aging.
30. The method of claim 21, wherein the mitochondrial product is selected from the group consisting of Lon peptidase 1, mitochondrial (LONP1), NADH:ubiquinone oxidoreductase complex assembly factor 1 (NDUFAF1), NADH: ubiquinone oxidoreductase complex assembly factor 2 (NDUFAF2), NADH:ubiquinone oxidoreductase complex assembly factor 3 (NDUFAF3), NADH:ubiquinone oxidoreductase complex assembly factor 4 (NDUFAF4), NADH:ubiquinone oxidoreductase complex assembly factor 5 ( DUFAF5), Nucleotide-binding protein-like (NUBPL), FAD-dependent oxidoreductase domain containing 1 (FOXRED1), Acyl- CoA dehydrogenase family member 9 (ACAD9), Succinate dehydrogenase complex assembly factor 1 (SDHAF1), Succinate dehydrogenase complex assembly factor 2 (SDHAF2), BCS1 homolog, ubiquinol-cytochrome c reductase complex chaperone (BCS1L), Surfeit 1 (SURF1), SCOl cytochrome c oxidase assembly protein (SCOl), COX10 heme A:farnesyltransfeiase cytochrome c oxidase assembly factor (COX10), Cytochrome c oxidase assembly homolog 15 (COX 15), Leucine rich pentatricopeptide repeat containing (L PPRC), FAST kinase domains 2 (FASTKD2), Translational activator of mitochondrially encoded cytochrome c oxidase I (TACOl), ATP synthase mitochondrial Fl complex assembly factor 2 (ATPAF2), Transmembrane protein 70 (TMEM70), Polymerase (DNA directed), gamma (POLG), Polymerase (DNA directed), gamma 2, accessory subunit (POLG2), MpV17 mitochondrial inner membrane protein (MPV17), Chromosome 10 open reading frame 2 (C10orf2)} Thymidine phosphorylase (TYMP), Deoxyguanosine kinase (DGUOK), Ribonucleotide reductase M2 B (RRM2B), Succinate-CoA ligase, ADP-forming, beta subunit (SUCLA2), Succinate-CoA ligase, alpha subunit (SUCLG1), Thymidine kinase 2, mitochondrial (TK2), Translocase of inner mitochondrial membrane 8 homolog A (TIMM8A), DnaJ heat shock protein family (Hsp40) member CI 9 (DNAJC19), G elongation factor, mitochondrial 1 (GFMl), Leucyl-tRNA synthetase 2 (LARS2), TyrosyL-tRNA synthetase 2 (YARS2), Seryl-tRNA synthetase 2, mitochondrial (SARS2), Aspartyl-tRNA synthetase 2, mitochondrial (DARS2), Arginyi-tRNA synthetase 2, mitochondrial (RARS2), Mitochondrial ribosomal protein S16 (MRPS16), Mitochondrial ribosomal protein S22 (MRPS22), Ts translation elongation factor, mitochondrial (TSFM), Tu translation elongation factor, mitochondrial (TUFM), Frataxin (FXN), ATP-binding cassette subfamily B member 7 (ABCB7), Solute carrier family 25 member 38 (SLC25A38), Iron-sulfur cluster assembly enzyme (ISCU), BolA family member 3 (BOLA3), NFU1 iron- sulfur cluster scaffold (NFU3), Coenzyme Q2 4-hydroxybenzoate polyprenyltransferase (COQ2), Coenzyme Q4 (COQ4), Coenzyme Q9 (COQ9), Aprataxin (APTX), Prenyl (decaprenyl) diphosphate synthase, subunit 1 (PDSS1), Prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2), AarF domain containing kinase 3 (ADCK3), Paraplegin (SPG7), Heat shock protein family D (Hsp60) member 1 (HSPD1), Optic atrophy 1 (autosomal dominant) (OPA1), Mitofusin 2 (MFN2), Dynamin 1-like (DNM1L), Tafazzin (TAZ), Pyruvate dehydrogenase (lipoamide) alpha 1 (PDHA1), Ethylmalonic encephalopathy 1 (ETHE1), Pseudouridylate synthase 1 (PUS1), NADH:ubiquinone oxidoreductase core subunit SI (NDUFS1), NADH: ubiquinone oxidoreductase core subunit S2 (NDUFS2), NADH: ubiquinone oxidoreductase core subunit S3 (NDUFS3), NADH:ubiquinone oxidoreductase subunit S4 (NDUFS4), NADH:ubiqumone oxidoreductase subunit S6 (NDUFS6), NADH:ubiquinone oxidoreductase core subunit S7 (NDUFS7), NADH: ubiquinone oxidoreductase core subunit S8 (NDUFS8), NADH:ubiquinone oxidoreductase subunit B3 (NDUFB3), NADH:ubiquinone oxidoreductase core subunit VI (NDUFV1), NADH:ubiquinone oxidoreductase core subunit V2 (NDUFV2), NADH:ubiquinone oxidoreductase subunit Al (NDUFA1), NADH: ubiquinone oxidoreductase subunit A2 (NDUFA2), NADH ubiquinone oxidoreductase subunit A10 (NDUFA10), NADH:ubiquinone oxidoreductase subunit Al l (NDUFA11), Succinate dehydrogenase complex subunit A, flavoprotein (Fp) (SDHA), Succinate dehydrogenase complex subunit B, iron sulfur (Ip) (SDHB), Succinate dehydrogenase complex, subunit C, integral membrane protein, 15kDa (SDHC), Succinate dehydrogenase complex subunit D, integral membrane protein (SDHD), Ubiquinol- cytochrome c reductase binding protein (UQCRB), Ubiquinol-cytochrome c reductase complex III subunit VII (UQCRQ), Cytochrome c oxidase subunit VIb polypeptide 1 (ubiquitous) (COX6B1), ATP synthase, H+ transporting, mitochondrial Fl complex, epsilon subunit (ATP5E), Solute carrier family 22 (organic cation/carnitine transporter), member 5 (SLC22A5), Carnitine palmitoyltransferase 2 (CPT2), Carnitine palmitoyltransferase 1A (liver) (CPT1A), Solute carrier family 6 (neurotransmitter transporter), member 8 (SLC6A8), Guanidinoacetate N-methyltransferase (GAMT), Glycine amidinotransferase (GATM), Pyruvate carboxylase (PC), hydroxyacyl-CoA dehydrogenase/3- ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit (H ADHA)/ (LCH AD), Acyl-CoA dehydrogenase, very long chain (ACADVL), Acyl-CoA dehydrogenase, C-2 to C-3 short chain (ACADS), Hydroxyacyl-CoA dehydrogenase (HADH)/(SCHAD), Electron transfer flavoprotein alpha subunit (ETFA), Electron transfer flavoprotein beta subunit (ETFB), Electron transfer flavoprotein dehydrogenase (ETFDH), Solute carrier family 25 (SLC25A20), Pyruvate dehyrogenase phosphatase catalytic subunit 1 (PDP1), Pyruvate dehydrogenase complex component X (PDHXD), Dihydrolipoamide S- acetyltransferase (DLAT), Pyruvate dehydrogenase (l poamide) beta (PDHB), Solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member (4SLC25A4)/(ANT)} Mitochondrially encoded NADH dehydrogenase 1 (MT-NDl), Mitochondrially encoded NADH dehydrogenase 2 (MT-ND2), Mitochondrially encoded NADH dehydrogenase 3 (MT-ND3), Mitochondrially encoded NADH dehydrogenase 4 (MT-ND4), Mitochondrially encoded NADH dehydrogenase 4L (MT-ND4L), Mitochondrially encoded NADH dehydrogenase 5 (MT-ND5), Mitochondrially encoded NADH dehydrogenase 6 (MT-ND6), Mitochondrially encoded cytochrome b (MT-CYB), Mitochondrially encoded cytochrome c oxidase I (MT-COI), Mitochondrially encoded cytochrome c oxidase II (MT-COII), Mitochondrially encoded cytochrome c oxidase III (MT-COIII), ATPase6 (MT-ATP6), ATPaseS (MT-ATP8), 12S rRNA (MT-RNR1), 16S rRNA (MT-RNR2), Mitochondrially encoded tRNA threonine (MT-TT), Mitochondrially encoded tRNA proline (MT-TP), Mitochondrially encoded tRNA phenylalanine (MT-TF), Mitochondrially encoded tRNA valine (MT-TV), Mitochondrially encoded tRNA leucine 1 (UUA/G) (MT-TL1), Mitochondrially encoded tRNA isoleucine (MT-TI), Mitochondrially encoded tRNA glutamine (MT-TQ), Mitochondrially encoded tRNA Methionine (MT-TM), Mitochondrially encoded tRNA tryptophan (MT-TW), Mitochondrially encoded tRNA alanine (MT-TA), Mitochondrially encoded tRNA asparagine (MT-TN), Mitochondrially encoded tRNA cysteine (MT-TC), Mitochondrially encoded tRNA tyrosine (MT-TY), Mitochondrially encoded tRNA serine 1 (UCN) ( T-TSl), Mitochondrially encoded tRNA aspartic acid (MT-TD), Mitochondrially encoded tRNA lysine (MT-TK), Mitochondrially encoded tRNA glycine (MT-TG), Mitochondrially encoded tRNA arginine (MT-TR), Mitochondrially encoded tRNA histidine (MT-TH), Mitochondrially encoded tRNA Serine 2 (AGY) (MT-TS2), Mitochondrially encoded tRNA Leucine 2 (CUN) (MT-TL2), and Mitochondrially encoded tRNA glutamic acid (MT-TE).
31. The method of claim 21, wherein the mitochondrial disease is AIpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS) (including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO) and the mitochondrial product is DNA polymerase gamma (POLG).
32. The method of claim 21, wherein the mitochondrial disease is Alpers-Huttenlocher syndrome (AHS), Childhood Myocerebrohepatopathy Spectrum (MCHS), Myoclonic Epilepsy Myopathy Sensory Ataxia (MEMSA), Ataxia Neuropathy Spectrum (ANS) (including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia neuropathy dysarthria and ophthalmoplegia (SANDO)), Autosomal Recessive Progressive External Ophthalmoplegia (arPEO), Autosomal Dominant Progressive External Ophthalmoplegia (adPEO) and the mitochondrial product is mitochondrial total RNA.
33. The method of claim 21, wherein the mitochondrial disease is mitochondrial complex I deficiency and/or Leber hereditary optic neuropathy (LHON) and the mitochondrial product is mitochondrially encoded NADH dehydrogenase 4 ( D4).
34, The method of claim 21, wherein the mitochondrial disease is mitochondrial complex IV deficiency and/or mitochondrial myopathy, encephalomyopathy with lactic acidosis and strokelike episodes (MELAS) and the mitochondrial product is tRNALeu(UUR).
35. The method of claim 21, wherein the mitochondrial disease is mitochondrial complex IV deficiency or cerebral, ocular, dental, auricular, and skeletal anomalies syndrome (CODAS) and the mitochondrial product is LONP1.
36. The method of claim 21, wherein the mitochondrial disease is Leigh syndrome, or mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like episodes (MELAS) mitochondrial complex I deficiency and/or Leber hereditary optic neuropathy (LHON) and the mitochondrial product is mitochondrially encoded NADH dehydrogenase 6 ( D6).
37. The method of claim 21, wherein a dosage of exosomes sufficient to deliver about 0,1 mg/kg to about 100 mg/kg of a mitochondrial product that is a protein, or a dosage of exosomes sufficient to deliver about 1 ng/kg to about 100 ug kg of a mitochondrial product that is nucleic acid, is administered to the mammal,
38. The exosomes of claim 1, wherein the exosomes are isolated from a biological sample using a method comprising the following steps: i) exposing the biological sample to a first centnfugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centnfugation; ii) subjecting the supernatant from step i) to centnfugation to remove microvesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the microfiltered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) resuspending. the exosome pellet from step iv) in a physiological solution and conducting a second ultracentrifugation in a density gradient and removing the exosome pellet fraction therefrom.
39. The method of claim 18, wherein the exosomes are isolated from a biological sample using a method comprising the following steps: i) exposing the biological sample to a first centrifugation to remove cellular debris greater than about 7-10 microns in size from the sample and obtaining the supernatant following centrifugation; ii) subjecting the supernatant from step i) to centrifugation to remove miciovesicles therefrom; iii) microfiltering the supernatant from step ii) and collecting the microfiltered supernatant; iv) subjecting the micro filtered supernatant from step iii) to at least one round of ultracentrifugation to obtain an exosome pellet; and v) resuspending. the exosome pellet from step iv) in a physiological solution and conducting a second ultracentrifugation in a density gradient and removing the exosome pellet fraction therefrom.
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