US20180030413A1 - Mammalian cells enriched with functional mitochondria - Google Patents

Mammalian cells enriched with functional mitochondria Download PDF

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US20180030413A1
US20180030413A1 US15/551,600 US201615551600A US2018030413A1 US 20180030413 A1 US20180030413 A1 US 20180030413A1 US 201615551600 A US201615551600 A US 201615551600A US 2018030413 A1 US2018030413 A1 US 2018030413A1
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marrow cells
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Natalie Yivgi-Ohana
Uriel Halavee
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Minovia Therapeutics Ltd
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Definitions

  • the present invention relates to cells derived from human bone-marrow and enriched with functional mitochondria, methods for their production, and therapeutic methods utilizing such cells.
  • the mitochondrion is a membrane bound organelle found in most eukaryotic cells, ranging from 0.5 to 1.0 ⁇ m in diameter. Mitochondria are found in nearly all eukaryotic cells and vary in number and location depending on the cell type. Mitochondria contain their own DNA (mtDNA) and their own machinery for synthesizing RNA and proteins. The mtDNA contains only 37 genes, thus most of the gene products in the mammalian body are encoded by nuclear DNA.
  • Mitochondria perform numerous essential tasks in the eukaryotic cell such as pyruvate oxidation, the Krebs cycle and metabolism of amino acids, fatty acids and steroids.
  • the primary function of mitochondria is the generation of energy as adenosine triphosphate (ATP) by means of the electron-transport chain and the oxidative-phosphorylation system (the “respiratory chain”).
  • ATP adenosine triphosphate
  • Additional processes in which mitochondria are involved include heat production, storage of calcium ions, calcium signaling, programmed cell death (apoptosis) and cellular proliferation. Therefore, there are many diseases and disorders known in the art associated with malfunction or dysfunction of mitochondria which require treatment.
  • ATP concentration inside the cell is typically 1-10 mM.
  • ATP can be produced by redox reactions using simple and complex sugars (carbohydrates) or lipids as an energy source.
  • simple and complex sugars carbohydrates
  • lipids as an energy source.
  • complex fuels to be synthesized into ATP they first need to be broken down into smaller, simpler molecules.
  • Complex carbohydrates are hydrolyzed into simple sugars, such as glucose and fructose. Fats (triglycerides) are metabolized to give fatty acids and glycerol.
  • the overall process of oxidizing glucose to carbon dioxide is known as cellular respiration and can produce about 30 molecules of ATP from a single molecule of glucose.
  • ATP can be produced by a number of distinct cellular processes.
  • the three main pathways used to generate energy in eukaryotic organisms are glycolysis and the citric acid cycle/oxidative phosphorylation, both components of cellular respiration, and beta-oxidation.
  • the majority of this ATP production by a non-photosynthetic aerobic eukaryote takes place in the mitochondria, which can make up nearly 25% of the total volume of a typical cell.
  • Mitochondrial diseases are a group of disorders caused by dysfunctional mitochondria. Mitochondrial diseases may be caused by mutations in the mitochondrial DNA that affect mitochondrial function. Other causes of mitochondrial disease are mutations in genes of the nuclear DNA, whose gene products are imported into the Mitochondria (Mitochondrial proteins) as well as acquired mitochondrial conditions. Mitochondrial diseases take on unique characteristics both because of the way the diseases are often inherited and because mitochondria are so critical to cell function. The subclass of these diseases that have neuromuscular disease symptoms are often called a mitochondrial myopathy.
  • Mitochondrial disorders may be caused by mutations, acquired or inherited, in mitochondrial DNA (mtDNA) or in nuclear genes that code for mitochondrial components. They may also be the result of acquired mitochondrial dysfunction due to adverse effects of drugs, infections, or other environmental causes. Mitochondrial disease may become clinically apparent once the number of affected mitochondria reaches a certain level; this phenomenon is called “threshold expression”. Mitochondrial DNA mutations occur frequently, due to the lack of error checking capability. This means that mitochondrial DNA disorders may occur spontaneously and relatively often. Defects in enzymes that control mitochondrial DNA replication (all of which are encoded for by genes in the nuclear DNA) may also cause mitochondrial DNA mutations. Most mitochondrial function and biogenesis is controlled by nuclear DNA.
  • Human mitochondrial DNA encodes only 13 proteins of the respiratory chain, while most of the estimated 1,500 proteins and components targeted to mitochondria are nuclear-encoded. Defects in nuclear-encoded mitochondrial genes are associated with hundreds of clinical disease phenotypes including anemia, dementia, hypertension, lymphoma, retinopathy, seizures, and neurodevelopmental disorders.
  • LHON Leber's hereditary optic neuropathy
  • RRCs retinal ganglion cells
  • LHON is only transmitted through the mother as it is primarily due to mutations in the mitochondrial (not nuclear) genome and only the egg contributes mitochondria to the embryo.
  • LHON is usually due to one of three pathogenic mitochondrial DNA (mtDNA) point mutations.
  • MELAS Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
  • MELAS mitochondrial cytopathies, which also include MERRF, and Leber's hereditary optic neuropathy. The disease can manifest in both sexes.
  • MELAS is caused by mutations in the genes in mitochondrial DNA. Some of the genes (MT-ND1, MT-ND5) affected in MELAS encode proteins that are part of NADH dehydrogenase (also called complex I) in mitochondria, that helps convert oxygen and simple sugars to energy. Other genes (MT-TH, MT-TL1, and MT-TV) encode mitochondrial specific transfer RNAs (tRNAs). Mutations in MT-TL1 cause more than 80% of all cases of MELAS. They impair the ability of mitochondria to make proteins, use oxygen, and produce energy.
  • tRNAs mitochondrial specific transfer RNAs
  • WO 2013/002880 describes compositions and methods comprising bio-energetic agents for restoring the quality of aged oocytes, enhancing oogonial stem cells or improving derivatives thereof (e.g., cytoplasm or isolated mitochondria) for use in fertility-enhancing procedures.
  • WO 2013/035101 to the present inventors relates to mitochondrial compositions and therapeutic methods of using same, and discloses compositions of partially purified functional mitochondria and methods of using the compositions to treat conditions which benefit from increased mitochondrial function by administering the compositions to a subject in need thereof.
  • the present invention provides cells and methods for treating a variety of mitochondrial diseases.
  • the present invention provides compositions comprising bone-marrow cells which have been enriched with functional mitochondria obtained from healthy donors.
  • the present invention further provides methods for the use of heterologous or autologous “mitochondrially-enriched” bone-marrow cells for treatment of patients.
  • the provision of bone-marrow cells of a patient afflicted with a mitochondrial disease, treated ex-vivo and returned to the same patient provides great benefits over other methods involving allogeneic cell therapy.
  • the provided methods eliminate the need to screen the population and find a donor which is human leukocyte antigen (HLA)-matched with the patient, which is a lengthy and costly process, and not always successful.
  • HLA human leukocyte antigen
  • the methods further advantageously eliminate the need for life-long immunosuppression therapy of the patient, so that his body does not reject allogeneic cell populations.
  • the present invention advantageously provides a unique methodology of ex-vivo corrective therapy, in which defective human cells are removed from the patient's body, treated ex-vivo, and returned to the same patient.
  • the present invention relates to the administration of bone-marrow cells which, without being bound to any theory or mechanism, are distributed throughout the body in different tissues, and transfer the healthy mitochondria with which they were enriched to the patient's damaged cells.
  • the present invention is based, in part, on the surprising findings that isolated functional mitochondria can enter intact fibroblasts and bone marrow cells, and that treatment of fibroblasts and bone marrow cells having defective mitochondria with functional mitochondria increases mitochondrial content, cell survival and ATP production.
  • bone-marrow cells are more receptive to be enriched with mitochondria than fibroblast cells, and that human bone-marrow cells are more receptive to be enriched with mitochondria than murine bone-marrow cells.
  • the extent of enrichment of bone-marrow cells with functional mitochondria is dependent in their concentrations or relative proximity, and thus may be manipulated.
  • the present invention is based, in part, on the surprising finding that functional mitochondria, initially comprised in bone-marrow cells, may spontaneously be distributed in-vivo throughout organs, such as the eye, upon injection of the bone-marrow cells directly into the organs.
  • Such findings lay the base for a variety of cellular platforms for delivery of functional mitochondria, and their use in therapy of mitochondrial diseases.
  • the present invention provides, for the first time, bone-marrow cells of patients of mitochondrial diseases having satisfactory, non-pathologic mitochondrial activity.
  • compositions and methods provided by the present invention may be regarded as a form of “replacement therapy”.
  • replacement therapy either the mutated genes and/or their missing or un-functional protein products are replaced (or made irrelevant) by wild-type, functional mitochondrial genes and/or functional proteins. Fusion or entry of intact functional mitochondria into bone-marrow cells of mitochondrial disease' patients provides both wild-type mitochondrial genes and functional mitochondrial proteins.
  • the present invention provides, in one aspect, an ex-vivo method for enriching human bone-marrow cells with functional mitochondria, the method comprising the steps of (i) providing a first composition, comprising a plurality of human bone-marrow cells obtained or derived from a patient afflicted with a mitochondrial disease or from a subject not afflicted with a mitochondrial disease; (ii) providing a second composition, comprising a plurality of isolated human functional mitochondria obtained from a subject not afflicted with a mitochondrial disease; (iii) contacting the human bone-marrow cells of the first composition with the human functional mitochondria of the second composition, thus forming a third composition; and (iv) incubating the third composition under conditions allowing the human functional mitochondria to enter the human bone-marrow cells thereby enriching said human bone-marrow cells with said human functional mitochondria, thus forming a fourth composition; wherein the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 50% higher than the mitochondrial content of the human bone-marrow
  • the mitochondrial content of the bone-marrow cells in the first composition or in the fourth composition is determined by determining the content or activity level of citrate synthase.
  • the bone-marrow cells comprise myelopoietic cells. In certain embodiments, the bone-marrow cells comprise erythropoietic cells. In certain embodiments, the bone-marrow cells comprise multi-potential hematopoietic stem cells (HSCs). In certain embodiments, the bone-marrow cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof.
  • the bone-marrow cells comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof.
  • the bone-marrow cells comprise mesenchymal stem cells. Each possibility represents a separate embodiment of the invention.
  • the bone-marrow cells express the bone-marrow progenitor cell antigen CD34 (CD34 + ).
  • the bone-marrow cells in the first composition are obtained from the bone marrow of the patient afflicted with a mitochondrial disease, or are obtained from the bone marrow of the subject not afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are mobilized from the bone marrow of the patient afflicted with a mitochondrial disease, or are mobilized from the bone marrow of the subject not afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are directly obtained from the bone marrow of the patient afflicted with a mitochondrial disease, or are directly obtained from the bone marrow of the subject not afflicted with a mitochondrial disease.
  • the bone-marrow cells in the first composition are indirectly obtained from the bone marrow of the patient afflicted with a mitochondrial disease, or are indirectly obtained from the bone marrow of the subject not afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are obtained from the peripheral blood of the patient afflicted with a mitochondrial disease, or are obtained from the peripheral blood of the subject not afflicted with a mitochondrial disease.
  • a mitochondrial disease obtained from the peripheral blood of the subject not afflicted with a mitochondrial disease.
  • the method described above further comprises a preceding step, the step comprising administering to the patient afflicted with a mitochondrial disease or to the subject not afflicted with a mitochondrial disease an agent which induces mobilization of bone-marrow cells to peripheral blood.
  • the agent is selected from the group consisting of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), 1,1′-[1,4-Phenylenebis(methylene)]-bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor), a salt thereof, and any combination thereof.
  • G-CSF granulocyte-colony stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the method described above further comprises a step of isolating the bone-marrow cells from the peripheral blood of the patient afflicted with a mitochondrial disease or from the peripheral blood of the subject not afflicted with a mitochondrial disease.
  • the isolation is performed by apheresis.
  • the method described above further comprises concentrating the bone-marrow cells and the functional mitochondria in the third composition before or during incubation. In certain embodiments, the method described above further comprises centrifugation of the third composition before, during or after incubation.
  • the bone-marrow cells in the first composition are obtained from a patient afflicted with a mitochondrial disease, and have (i) a sub-normal rate of oxygen (O 2 ) consumption; (ii) a sub-normal content or activity level of citrate synthase; (iii) a sub-normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and (iii).
  • O 2 oxygen
  • ATP a sub-normal rate of adenosine triphosphate
  • the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 50% lower than the heteroplasmy level of the bone-marrow cells in the first composition.
  • the bone-marrow cells in the first composition are obtained from a subject not afflicted with a mitochondrial disease, and have (i) a normal rate of oxygen (O 2 ) consumption; (ii) a normal content or activity level of citrate synthase; (iii) a normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and (iii).
  • O 2 oxygen
  • ATP adenosine triphosphate
  • the bone-marrow cells in the fourth composition have (i) an above-normal rate of oxygen (O 2 ) consumption; (ii) an above-normal content or activity level of citrate synthase; (iii) an above-normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and (iii).
  • O 2 above-normal rate of oxygen
  • ATP adenosine triphosphate
  • the total amount of mitochondrial proteins in the second composition is between 20%-80% of the total amount of cellular proteins within the sample.
  • the fourth composition is not enriched with cytochrome C reductase or cytochrome C reductase activity compared to the first composition.
  • the mitochondrial disease is a mitochondrial respiratory chain disease (MRCD).
  • the mitochondrial disease is selected from the group consisting of LHON (Leber's hereditary optic neuropathy); MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms); Pearson syndrome; Leigh syndrome; NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis); MERRF (myoclonic epilepsy with ragged red fibers); KSS (Kearns-Sayre Syndrome); MNGIE (myoneurogenic gastrointestinal encephalopathy); Friedreich Ataxia; and Alpers' disease.
  • LHON Leber's hereditary optic neuropathy
  • MELAS mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms
  • Pearson syndrome Leigh syndrome
  • NARP neuroopathy, ataxia, retinitis pigmentosa, and ptosis
  • MERRF my
  • the mitochondrial disease is selected from the group consisting of LHON, MELAS, Pearson syndrome, Leigh syndrome, NARP, MERRF, and KSS. Each possibility represents a separate embodiment of the invention.
  • the mitochondrial disease is LHON. In certain embodiments, the mitochondrial disease is MELAS.
  • the present invention further provides, in another aspect, a plurality of human bone-marrow cells enriched with functional mitochondria, obtained by any one of the embodiments of the method described above.
  • the present invention further provides, in another aspect, a plurality of human bone-marrow cells, wherein the bone-marrow cells (a) have an above-normal mitochondrial content (b) have an above-normal rate of oxygen (O 2 ) consumption; (c) have an above-normal content or activity level of citrate synthase; (d) are CD34 + ; or (e) any combination thereof.
  • the bone-marrow cells (a) have an above-normal mitochondrial content (b) have an above-normal rate of oxygen (O 2 ) consumption; (c) have an above-normal content or activity level of citrate synthase; (d) are CD34 + ; or (e) any combination thereof.
  • the plurality of human bone-marrow cells described above is having an above-normal mitochondrial content; having an above-normal rate of oxygen (O 2 ) consumption; having an above-normal content or activity level of citrate synthase; and are CD34 + .
  • the present invention further provides, in another aspect, a pharmaceutical composition comprising a plurality of the human bone-marrow cells described above.
  • the present invention further provides, in another aspect, the pharmaceutical composition described above for use in a method of treating a mitochondrial disease in a human patient.
  • the present invention further provides, in another aspect, a method of treating a mitochondrial disease in a human patient in need thereof, comprising the step of administering to the patient the pharmaceutical composition described above.
  • the bone-marrow cells are autologous to the patient afflicted with the mitochondrial disease. In certain embodiments, the bone-marrow cells are exogenous to the patient afflicted with the mitochondrial disease. In certain embodiments, the method described above further comprises a step of administering to the patient an agent which promotes mitochondrial biogenesis. In certain embodiments, the agent which promotes mitochondrial biogenesis is erythropoietin (EPO) or a salt thereof. In certain embodiments, the method described above further comprises a step of administering to the patient an agent which prevents, delays, minimizes or abolishes an adverse immunogenic reaction between the patient and the bone-marrow cells. Each possibility represents a separate embodiment of the invention. In certain embodiments, the adverse immunogenic reaction is a graft-versus-host disease (GvHD). In certain embodiments, the method described above further comprises a step of administering to the patient a pre-transplant conditioning agent prior to the administration of the pharmaceutical composition.
  • GvHD graft-versus-host disease
  • the mitochondrial disease is associated with a mutation in the mitochondrial DNA. In certain embodiments, the mitochondrial disease is associated with a mutation in the nuclear DNA. In certain embodiments, the mitochondrial disease is a MRCD. In certain embodiments, the mitochondrial disease is selected from the group consisting of LHON; MELAS; Pearson syndrome; Leigh syndrome; NARP; MERRF; KSS; MNGIE; Friedreich Ataxia; and Alpers' disease. In certain embodiments, the mitochondrial disease is selected from the group consisting of LHON, MELAS, Pearson syndrome, Leigh syndrome, NARP, MERRF, and KSS. Each possibility represents a separate embodiment of the invention. In certain embodiments, the mitochondrial disease is LHON. In certain embodiments, the mitochondrial disease is MELAS.
  • FIG. 1 is a bar graph showing a comparison of citrate synthase (CS) activity in human hepatocyte cells incubated with (Mito) or without (Control) mitochondria isolated from human placental cells.
  • CS citrate synthase
  • FIG. 2 is three micrographs showing mouse fibroblast cell expressing mitochondrial GFP (left panel), incubation with isolated RFP-labeled mitochondria (middle panel), and an overlay (right panel), obtained by fluorescence confocal microscopy.
  • FIG. 3 is a bar graph showing a comparison of ATP levels in mouse fibroblast cells which were either untreated (Control), treated with a mitochondrial complex I irreversible inhibitor (Rotenone), or treated with Rotenone and mouse placental mitochondria (Rotenone+Mitochondria). Data is presented as mean values ⁇ SEM, (*) p value ⁇ 0.05. RLU ⁇ relative luminescence units.
  • FIG. 4 is four micrographs obtained by fluorescence confocal microscopy showing mouse bone marrow cells incubated with GFP-labeled mitochondria isolated from mouse melanoma cells.
  • FIG. 5 is a bar graph showing a comparison of citrate synthase (CS) activity in mouse bone marrow (BM) cells incubated with varying amounts of GFP-labeled mitochondria isolated from mouse melanoma cells, with or without centrifugation.
  • CS citrate synthase
  • FIG. 6A is a bar graph showing a comparison of CS activity in murine BM cells after enrichment with increasing amounts of GFP-labeled mitochondria.
  • FIG. 6B is a bar graph showing a comparison of cytochrome c reductase activity in these cells (black bars), compared to the activity in GFP-labeled mitochondria (gray bar).
  • FIG. 7A depict the results of GFP staining in control, untreated murine eyeballs.
  • FIG. 7B depict the results of GFP staining in murine eyeballs injected with murine BM cells incubated with GFP-labeled mitochondria after centrifugation.
  • FIG. 8A depicts the increase in viability of mitochondrial DNA-(mtDNA-) deficient human 143B osteosarcoma cells (Control, upper three panels) by mitochondria isolated from mtDNA-containing cells (Mitochondria, lower three panels).
  • FIG. 8B is a bar graph showing a comparison of ATP levels in the cells presented in FIG. 8A .
  • FIG. 9A is a bar graph showing a comparison of CS activity in control, untreated human BM cells and human BM cells incubated with GFP-labeled mitochondria isolated from human placental cells, with or without centrifugation.
  • FIG. 9B is a bar graph showing a comparison of ATP levels in control, untreated human BM cells and human BM cells incubated with GFP-labeled mitochondria isolated from human placental cells, with centrifugation.
  • FIG. 10A depict the result of a FACS analysis in human BM cells not incubated with GFP-labeled mitochondria.
  • FIG. 10B depict the result of a FACS analysis in human BM cells incubated with GFP-labeled mitochondria after centrifugation.
  • the present invention provides cellular platforms, more specifically bone-marrow-derived cellular platforms, for targeted and systemic delivery of therapeutically-significant amounts of fully functional, healthy mitochondria.
  • the present invention further provides methods for producing such cellular platforms, and methods for their utilization in treating mitochondrial diseases.
  • mitochondrial diseases associated with mutations (including deletions/insertions) in mitochondrial DNA such as LHON (Leber's hereditary optic neuropathy) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) can now be treated by transplanting functional mitochondria into diseased cells, leading to long-term, potentially life-long annulment of the disease.
  • LHON Leber's hereditary optic neuropathy
  • MELAS mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes
  • the affected cells are bone-marrow cells or are derived from bone-marrow cells
  • the administered bone-marrow cells may also replace the affected cells, again leading to long-term, life-long annulment of the disease.
  • the mitochondrial disease is associated with a mutation (including deletions/insertions) in nuclear DNA and the affected cells are bone-marrow cells or are derived from bone-marrow cells, such as MNGIE and Friedreich's ataxia
  • the administered bone-marrow cells can replace the affected cells, again leading to long-term, potentially life-long annulment of the disease. It should be emphasized that the present invention provides, for the first time, means and methods for the sustained correction of pathological states of mitochondrial diseases, and therefore annulment of these diseases.
  • the present invention is based on several surprising findings, amongst which are that (i) the extent of enrichment of bone-marrow cells with intact, functional mitochondria is dependent in their concentrations, and thus may be manipulated, (ii) bone-marrow cells are more receptive to be enriched with mitochondria than fibroblast cells, (iii) human bone-marrow cells are more receptive to be enriched with mitochondria than murine bone-marrow cells, reaching over 8 fold of their natural mitochondrial content, and that (iv) bone marrow cells enriched with normal, functional mitochondria can transfer such mitochondria to the patient's cells.
  • the present invention provides, in one aspect, an ex-vivo method for enriching human bone-marrow cells with functional mitochondria, the method comprising the steps of (i) providing a first composition, comprising a plurality of human bone-marrow cells obtained or derived from a patient afflicted with a mitochondrial disease or from a subject not afflicted with a mitochondrial disease; (ii) providing a second composition, comprising a plurality of isolated human functional mitochondria obtained from a subject not afflicted with a mitochondrial disease; (iii) contacting the human bone-marrow cells of the first composition with the human functional mitochondria of the second composition, thus forming a third composition; and (iv) incubating the third composition under conditions allowing the human functional mitochondria to enter the human bone-marrow cells thereby enriching said human bone-marrow cells with said human functional mitochondria, thus forming a fourth composition; wherein the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 50% higher than the mitochondrial content of the human bone-marrow
  • ex-vivo method refers to any method comprising steps performed exclusively outside the human body.
  • enriching refers to any action designed to increase the mitochondrial content, e.g. the number of intact mitochondria, of a human cell.
  • human bone-marrow cells generally refers to all human cells naturally found in the bone-marrow of humans, and to all cell populations naturally found in the bone-marrow of humans.
  • the term “functional mitochondria” as used herein refers to mitochondria displaying normal, non-pathologic levels of activity.
  • the activity of mitochondria can be measured by a variety of methods well known in the art, such as O 2 consumption, ATP production, and CS activity level.
  • bone-marrow cells obtained from a patient afflicted with a mitochondrial disease or from a subject not afflicted with a mitochondrial disease refers to cells that were bone-marrow cells in the patient or subject at the time of their isolation from the patient or subject.
  • bone-marrow cells derived from a patient afflicted with a mitochondrial disease or from a subject not afflicted with a mitochondrial disease refers to cells that were not bone-marrow cells in the patient or subject, and have been manipulated to become bone-marrow cells.
  • the term “manipulated” as used herein refers to the use of any one of the methods known in the field (Yu J. et al., Science, 2007, Vol. 318(5858), pages 1917-1920) for reprograming somatic cells to an undifferentiated state and becoming induced pluripotent stem cells (iPSc), and, optionally, further reprograming the iPSc to become cells of a desired lineage or population (Chen M. et al., IOVS, 2010, Vol. 51(11), pages 5970-5978), such as bone-marrow cells (Xu Y. et al., 2012, PLoS ONE, Vol. 7(4), page e34321).
  • a patient afflicted with a mitochondrial disease refers to a human subject diagnosed with a mitochondrial disease, suspected to have a mitochondrial disease, or in a risk group of developing a mitochondrial disease. As certain mitochondrial diseases are inherited, the progeny of subjects diagnosed with a mitochondrial disease are considered a risk group of developing a mitochondrial disease.
  • a subject not afflicted with a mitochondrial disease refers to human subject not diagnosed with a mitochondrial disease, not suspected to have a mitochondrial disease, and/or not in a risk group of developing a mitochondrial disease.
  • iPSc induced pluripotent stem cells
  • isolated human functional mitochondria refers to intact mitochondria isolated from cells obtained from a subject not afflicted with a mitochondrial disease.
  • condition allowing the human functional mitochondria to enter the human bone-marrow cells generally refers to parameters such as time, temperature, and proximity between the mitochondria and the bone-marrow cells. While identifying those conditions are within the capabilities of any man of ordinary skill in the field, such conditions are provided by the present invention.
  • human cells and human cell lines are routinely incubated in liquid medium, and kept in sterile environments, such as in tissue culture incubators, in 37° C. and 5% CO 2 atmosphere.
  • mitochondria content refers to the amount of functional mitochondria within a cell.
  • the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%, 800%, 850%, 900%, 950%, or 1000% higher than the mitochondrial content of the human bone-marrow cells in the first composition.
  • the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 100% higher than the mitochondrial content of the human bone-marrow cells in the first composition.
  • the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 200% higher than the mitochondrial content of the human bone-marrow cells in the first composition.
  • the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 300% higher than the mitochondrial content of the human bone-marrow cells in the first composition. In certain embodiments, the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 400% higher than the mitochondrial content of the human bone-marrow cells in the first composition. In certain embodiments, the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 500% higher than the mitochondrial content of the human bone-marrow cells in the first composition. In certain embodiments, the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 600% higher than the mitochondrial content of the human bone-marrow cells in the first composition.
  • the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 700% higher than the mitochondrial content of the human bone-marrow cells in the first composition. In certain embodiments, the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 750% higher than the mitochondrial content of the human bone-marrow cells in the first composition. In certain embodiments, the mitochondrial content of the human bone-marrow cells in the fourth composition is at least 800% higher than the mitochondrial content of the human bone-marrow cells in the first composition.
  • the mitochondrial content of the human bone-marrow cells in the fourth composition is 100%-7900%, 200%-6900%, 300%-5900%, 400%-4900%, 500%-3900%, 600%-2900%, 700%-1900%, or 800%-1400%, higher than the mitochondrial content of the human bone-marrow cells in the first composition.
  • Each possibility represents a separate embodiment of the invention.
  • the first composition is fresh. In certain embodiments, the first composition was frozen and then defrosted. In certain embodiments, the second composition is fresh. In certain embodiments, the second composition was frozen and then defrosted.
  • Citrate synthase is localized in the mitochondrial matrix, but is encoded by nuclear DNA. Citrate synthase is involved in the first step of the Krebs cycle, and is commonly used as a quantitative enzyme marker for the presence of intact mitochondria (Larsen S. et al., 2012, J. Physiol., Vol. 590(14), pages 3349-3360; Cook G. A. et al., Biochim. Biophys. Acta., 1983, Vol. 763(4), pages 356-367).
  • the mitochondrial content of the bone-marrow cells in the first composition or in the fourth composition is determined by determining the content of citrate synthase.
  • the mitochondrial content of the bone-marrow cells in the first composition or in the fourth composition is determined by determining the activity level of citrate synthase. In certain embodiments, the mitochondrial content of the bone-marrow cells in the first composition or in the fourth composition correlates with the content of citrate synthase. In certain embodiments, the mitochondrial content of the bone-marrow cells in the first composition or in the fourth composition correlates with the activity level of citrate synthase.
  • the bone-marrow cells comprise myelopoietic cells.
  • myelopoietic cells refers to cells involved in myelopoiesis, e.g. in the production of bone marrow and of all cells that arise from it, namely, all blood cells.
  • the bone-marrow cells comprise erythropoietic cells.
  • erythropoietic cells refers to cells involved in erythropoiesis, e.g. in the production of red blood cells (erythrocytes).
  • the bone-marrow cells comprise multi-potential hematopoietic stem cells (HSCs).
  • HSCs multi-potential hematopoietic stem cells
  • hemocytoblasts refers to the stem cells that give rise to all the other blood cells through the process of haematopoiesis.
  • the bone-marrow cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof.
  • common myeloid progenitor refers to the cells that generate myeloid cells.
  • common lymphoid progenitor refers to the cells that generate lymphocytes.
  • the bone-marrow cells comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof.
  • NK natural killer
  • the bone-marrow cells comprise mesenchymal stem cells.
  • mesenchymal stem cells refers to multipotent stromal cells that can differentiate into a variety of cell types, including osteoblasts (bone cells), chondrocytes (cartilage cells), myocytes (muscle cells) and adipocytes (fat cells).
  • the bone-marrow cells consist of myelopoietic cells. In certain embodiments, the bone-marrow cells consist of erythropoietic cells. In certain embodiments, the bone-marrow cells consist of multi-potential hematopoietic stem cells (HSCs). In certain embodiments, the bone-marrow cells consist of common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof.
  • HSCs multi-potential hematopoietic stem cells
  • the bone-marrow cells consist of megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof.
  • the bone-marrow cells consist of mesenchymal stem cells. Each possibility represents a separate embodiment of the invention.
  • Hematopoietic progenitor cell antigen CD34 also known as CD34 antigen is a protein that in humans is encoded by the CD34 gene.
  • CD34 is a cluster of differentiation in a cell surface glycoprotein and functions as a cell-cell adhesion factor.
  • the bone-marrow cells express the bone-marrow progenitor cell antigen CD34 (are CD34 + ).
  • the bone-marrow cells present the bone-marrow progenitor cell antigen CD34 on their external membrane.
  • the bone-marrow cells in the first composition are directly derived from the patient afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are directly derived from the subject not afflicted with a mitochondrial disease.
  • the term “directly derived” as used herein refers to bone-marrow cells which were derived directly from other cells. In certain embodiments, the bone-marrow cells were derived from hematopoietic stem cells (HSc).
  • the bone-marrow cells in the first composition are indirectly derived from the patient afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are indirectly derived from the subject not afflicted with a mitochondrial disease.
  • the term “indirectly derived” as used herein refers to bone-marrow cells which were derived from non-bone-marrow cells. In certain embodiments, the bone-marrow cells were derived from somatic cells which were manipulated to become induced pluripotent stem cells (iPSc).
  • the bone-marrow cells in the first composition are directly obtained from the bone marrow of the patient afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are directly obtained from the bone marrow of the subject not afflicted with a mitochondrial disease.
  • the term “directly obtained” as used herein refers to bone-marrow cells which were obtained from the bone-marrow itself, e.g. by means such as surgery or suction through a needle by a syringe.
  • the bone-marrow cells in the first composition are indirectly obtained from the bone marrow of the patient afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are indirectly obtained from the bone marrow of the subject not afflicted with a mitochondrial disease.
  • the term “indirectly obtained” as used herein refers to bone-marrow cells which were obtained from a location other than the bone-marrow itself.
  • the bone-marrow cells in the first composition are obtained from the peripheral blood of the patient afflicted with a mitochondrial disease. In certain embodiments, the bone-marrow cells in the first composition are obtained from the peripheral blood of the subject not afflicted with a mitochondrial disease.
  • peripheral blood refers to blood circulating in the blood system.
  • the method described above further comprises a preceding step, the step comprising administering to the patient afflicted with a mitochondrial disease an agent which induces mobilization of bone-marrow cells to peripheral blood. In certain embodiments, the method described above further comprises a preceding step, the step comprising administering to the subject not afflicted with a mitochondrial disease an agent which induces mobilization of bone-marrow cells to peripheral blood.
  • the agent which induces mobilization of bone-marrow cells to peripheral blood is selected from the group consisting of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), Phenylenebis(methylene)bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor, CAS number 155148-31-5), a salt thereof, and any combination thereof.
  • G-CSF granulocyte-colony stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Phenylenebis(methylene)bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor, CAS number 155148-31-5), a salt thereof, and any combination thereof.
  • the method described above further comprises a step of isolating the bone-marrow cells from the peripheral blood of the patient afflicted with a mitochondrial disease. In certain embodiments, the method described above further comprises a step of isolating the bone-marrow cells from the peripheral blood of the subject not afflicted with a mitochondrial disease.
  • isolated from the peripheral blood refers to the isolation of bone-marrow cells from other constituents of the blood.
  • the blood of a donor or patient is passed through an apparatus that separates out one particular constituent and returns the remainder to the circulation. It is thus a medical procedure which is performed outside the body.
  • the isolation is performed by apheresis.
  • the method described above further comprises concentrating the bone-marrow cells and the functional mitochondria in the third composition before incubation. In certain embodiments, the method described above further comprises concentrating the bone-marrow cells and the functional mitochondria in the third composition during incubation.
  • the method described above further comprises centrifugation of the third composition before incubation. In certain embodiments, the method described above further comprises centrifugation of the third composition during incubation. In certain embodiments, the method described above further comprises centrifugation of the third composition after incubation.
  • the bone-marrow cells in the first composition are obtained from a patient afflicted with a mitochondrial disease, and the bone-marrow cells have (i) a sub-normal rate of oxygen (O 2 ) consumption; (ii) a sub-normal content or activity level of citrate synthase; (iii) a sub-normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and (iii).
  • O 2 oxygen
  • ATP a sub-normal rate of adenosine triphosphate
  • sub-normal rate of oxygen (O 2 ) consumption refers to a rate of oxygen (O 2 ) consumption which is substantially lower than a control rate of oxygen (O 2 ) consumption which is derived from or corresponds to the rate of oxygen (O 2 ) consumption found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • sub-normal content or activity level of citrate synthase refers to a content or activity level of citrate synthase which is substantially lower than a control content value or activity level of citrate synthase which is derived from or corresponds to the content or activity level of citrate synthase of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • control rate of adenosine triphosphate (ATP) production which is derived from or corresponds to the rate of adenosine triphosphate (ATP) production found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the term “substantially lower” as used herein refers to a statistically-significant decrease between the normal and sub-normal values. In certain embodiments, the term “substantially lower” as used herein refers to a pathological decrease, i.e. to a level in which at least one pathological symptom associated with the substantially lower value becomes apparent. In certain embodiments, the term “sub-normal” as used herein refers to a value which is 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, or 10 fold lower than the corresponding value found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the term “sub-normal” as used herein refers to a value which is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 9 fold, or at least 10 fold lower than the corresponding value found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • Each possibility represents a separate embodiment of the invention.
  • the bone-marrow cells in the first composition are obtained from a patient afflicted with a mitochondrial disease, and the bone-marrow cells have (i) a sub-normal rate of oxygen (O 2 ) consumption compared to the rate of oxygen (O 2 ) consumption in bone-marrow cells obtained from a subject or of a plurality of subjects not afflicted with a mitochondrial disease; (ii) a sub-normal content or activity level of citrate synthase compared to the content or activity level of citrate synthase in bone-marrow cells obtained from a subject or of a plurality of subjects not afflicted with a mitochondrial disease; (iii) a sub-normal rate of adenosine triphosphate (ATP) production compared to the rate of adenosine triphosphate (ATP) production in bone-marrow cells obtained from a subject or of a plurality of subjects not afflicted with a mitochondrial disease; or (iv) any combination of (i), (i), (i
  • any reference to any measurable feature or characteristic or aspect directed to a plurality of cells or mitochondria is directed to the measurable average feature or characteristic or aspect of the plurality of cells or mitochondria.
  • Heteroplasmy is the presence of more than one type (wild-type/functional vs. mutated/non-functional) of mitochondrial DNA within a cell or individual, and is an important factor in considering the severity of mitochondrial diseases. While low levels of heteroplasmy (sufficient amount of mitochondria are functional) are associated with a healthy phenotype, high levels of heteroplasmy (insufficient amount of mitochondria are functional) are associated with pathologies.
  • the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 50% lower than the heteroplasmy level of the bone-marrow cells in the first composition.
  • the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 66% lower than the heteroplasmy level of the bone-marrow cells in the first composition. In certain embodiments, the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 75% lower than the heteroplasmy level of the bone-marrow cells in the first composition. In certain embodiments, the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 80% lower than the heteroplasmy level of the bone-marrow cells in the first composition. In certain embodiments, the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 87% lower than the heteroplasmy level of the bone-marrow cells in the first composition. In certain embodiments, the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 90% lower than the heteroplasmy level of the bone-marrow cells in the first composition.
  • the bone-marrow cells in the first composition are obtained from a subject not afflicted with a mitochondrial disease, and have (i) a normal rate of oxygen (O 2 ) consumption; (ii) a normal content or activity level of citrate synthase; (iii) a normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and (iii).
  • O 2 oxygen
  • ATP adenosine triphosphate
  • normal rate of oxygen (O 2 ) consumption and “control rate of oxygen (O 2 ) consumption” as used herein refer to the rate of oxygen (O 2 ) consumption found in cells or mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • normal content or activity level of citrate synthase and “control content or activity level of citrate synthase” as used herein refer to the content or activity level of citrate synthase of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • normal rate of adenosine triphosphate (ATP) production and “control rate of adenosine triphosphate (ATP) production” as used herein refer to the rate of adenosine triphosphate (ATP) production found in cells or mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the isolated human functional mitochondria in the second composition are obtained from a subject not afflicted with a mitochondrial disease, and have (i) a normal rate of oxygen (O 2 ) consumption; (ii) a normal content or activity level of citrate synthase; (iii) a normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and Each possibility represents a separate embodiment of the invention.
  • the bone-marrow cells in the fourth composition have (i) an above-normal rate of oxygen (O 2 ) consumption; (ii) an above-normal content or activity level of citrate synthase; (iii) an above-normal rate of adenosine triphosphate (ATP) production; or (iv) any combination of (i), (ii) and (iii).
  • O 2 above-normal rate of oxygen
  • ATP adenosine triphosphate
  • above-normal rate of oxygen (O 2 ) consumption refers to a rate of oxygen (O 2 ) consumption which is substantially higher than a control rate of oxygen (O 2 ) consumption which is derived from or corresponds to the rate of oxygen (O 2 ) consumption found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • above-normal content or activity level of citrate synthase refers to a content or activity level of citrate synthase which is substantially higher than a control content value or activity level of citrate synthase which is derived from or corresponds to the content or activity level of citrate synthase of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • ATP adenosine triphosphate
  • ATP adenosine triphosphate
  • control rate of adenosine triphosphate (ATP) production which is derived from or corresponds to the rate of adenosine triphosphate (ATP) production found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the term “substantially higher” as used herein refers to a statistically-significant increase between the normal and above-normal values. In certain embodiments, the term “substantially higher” as used herein refers to a non-pathological increase, i.e. to a level in which no pathological symptom associated with the substantially higher value becomes apparent. In certain embodiments, the term “above-normal” as used herein refers to a value which is 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 8.5 fold, 9 fold, or 10 fold higher than the corresponding value found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the term “above-normal” as used herein refers to a value which is at least 2 fold, at least 3 fold, at least 4 fold, at least 5 fold, at least 6 fold, at least 7 fold, at least 8 fold, at least 8.5 fold, at least 9 fold, or at least 10 fold higher than the corresponding value found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the term “above-normal” as used herein refers to a value which is 2-80 fold, 3-70 fold, 4-60 fold, 5-50 fold, 6-40 fold, 7-30 fold, 8-20 fold, 8.5-20 fold, or 9-15 fold higher than the corresponding value found in corresponding cells or corresponding mitochondria of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • Each possibility represents a separate embodiment of the invention.
  • the bone-marrow cells in the fourth composition have (i) an above-normal rate of oxygen (O 2 ) consumption compared to the rate of oxygen (O 2 ) consumption in bone-marrow cells obtained from a subject or of a plurality of subjects not afflicted with a mitochondrial disease; (ii) an above-normal content or activity level of citrate synthase compared to the content or activity level of citrate synthase in bone-marrow cells obtained from a subject or of a plurality of subjects not afflicted with a mitochondrial disease; (iii) an above-normal rate of adenosine triphosphate (ATP) production compared to the rate of adenosine triphosphate (ATP) production in bone-marrow cells obtained from a subject or of a plurality of subjects not afflicted with a mitochondrial disease; or (iv) any combination of (i), (ii) and (iii). Each possibility represents a separate embodiment of the invention.
  • O 2 above-normal rate of oxygen
  • the total amount of mitochondrial proteins in the second composition is between 20%-80% of the total amount of cellular proteins within the sample.
  • Eukaroytic NADPH-cytochrome C reductase (cytochrome C reductase) is a flavoprotein localized to the endoplasmic reticulum. It transfers electrons from NADPH to several oxygenases, the most important of which are the cytochrome P450 family of enzymes, responsible for xenobiotic detoxification. Cytochrome C reductase is widely used as an endoplasmic reticulum marker.
  • the second composition is substantially free from cytochrome C reductase or cytochrome C reductase activity.
  • the fourth composition is not enriched with cytochrome C reductase or cytochrome C reductase activity compared to the first composition.
  • Mitochondrial Respiratory Chain Disorders are a heterogeneous group of disorders that share the involvement of the cellular bio-energetic machinery due to molecular defects affecting the mitochondrial oxidative phosphorylation system (OXPHOS). Clinically, they usually involve multiple tissues although they tend to mainly affect nervous system and skeletal muscle. Cardiologic manifestations are frequent and include hypertrophic or dilated cardiomyopathies and heart conduction defects, being part of adult or infantile multi-systemic mitochondrial disorders or, less frequently, presenting as isolated clinical condition. In certain embodiments, the mitochondrial disease is a mitochondrial respiratory chain disease (MRCD).
  • MRCD mitochondrial respiratory chain disease
  • the mitochondrial disease is selected from the group consisting of LHON (Leber's hereditary optic neuropathy); MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms); Pearson syndrome; Leigh syndrome; NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis); MERRF (myoclonic epilepsy with ragged red fibers); KSS (Kearns-Sayre Syndrome); MNGIE (myoneurogenic gastrointestinal encephalopathy); Friedreich Ataxia; and Alpers' disease.
  • LHON Leber's hereditary optic neuropathy
  • MELAS mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms
  • Pearson syndrome Leigh syndrome
  • NARP neuroopathy, ataxia, retinitis pigmentosa, and ptosis
  • MERRF myoclonic epilepsy with ragged red fibers
  • KSS Kern
  • the mitochondrial disease is selected from the group consisting of LHON, MELAS, Pearson syndrome, Leigh syndrome, NARP, MERRF, and KSS. Each possibility represents a separate embodiment of the invention.
  • the mitochondrial disease is LHON. In certain embodiments, the mitochondrial disease is MELAS.
  • the method further comprises freezing the fourth composition. In certain embodiments, the method further comprises freezing and then defrosting the fourth composition.
  • the present invention further provides, in another aspect, a plurality of human bone-marrow cells enriched with functional mitochondria, obtained by the method described above.
  • the plurality is frozen. In certain embodiments, the plurality is frozen and then defrosted.
  • the present invention further provides, in another aspect, a plurality of human bone-marrow cells, wherein the bone-marrow cells (a) have an above-normal mitochondrial content (b) have an above-normal rate of oxygen (O 2 ) consumption; (c) have an above-normal content or activity level of citrate synthase; (d) are CD34 + ; or (v) any combination of (a), (b), (c) and (d).
  • the bone-marrow cells (a) have an above-normal mitochondrial content (b) have an above-normal rate of oxygen (O 2 ) consumption; (c) have an above-normal content or activity level of citrate synthase; (d) are CD34 + ; or (v) any combination of (a), (b), (c) and (d).
  • above-normal mitochondrial content refers to a mitochondrial content which is substantially higher than a control mitochondrial content which is derived from or corresponds to the mitochondrial content found in corresponding cells of a subject or of a plurality of subjects not afflicted with a mitochondrial disease.
  • the plurality is frozen. In certain embodiments, the plurality is frozen and then defrosted.
  • the plurality of human bone-marrow cells described above is having an above-normal mitochondrial content; having an above-normal rate of oxygen (O 2 ) consumption; having an above-normal content or activity level of citrate synthase; and are CD34 + .
  • the present invention further provides, in another aspect, a pharmaceutical composition comprising a plurality of human bone-marrow cells as described above.
  • composition refers to any composition comprising at least one biologically active agent.
  • biologically active agent refers to any molecule capable of eliciting a response in a biological system such as, for example, living cell(s), tissue(s), organ(s), and being(s).
  • biologically active agents include cells, intact mitochondria, mitochondrial DNA, and a mitochondrial protein.
  • the pharmaceutical composition is frozen. In certain embodiments, the pharmaceutical composition is frozen and then defrosted.
  • the pharmaceutical composition described above is for use in a method of treating a mitochondrial disease in a human patient.
  • treating includes the diminishment, alleviation, or amelioration of at least one symptom associated or induced by a disease or condition.
  • treating as used herein also includes preventative (e.g., prophylactic), palliative and curative treatment.
  • the present invention further provides, in another aspect, a method of treating a mitochondrial disease in a human patient in need thereof, comprising the step of administering to the patient the pharmaceutical composition described above.
  • method generally refers to manners, means, techniques and procedures for accomplishing a given task, including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the pharmaceutical composition is frozen, and the method described above further comprises defrosting the frozen pharmaceutical composition.
  • the bone-marrow cells are autologous to the patient afflicted with the mitochondrial disease.
  • autologous to the patient refers to the bone-marrow cells and other cells of the patient being HLA-matched.
  • the bone-marrow cells are exogenous to the patient afflicted with the mitochondrial disease.
  • exogenous to the patient refers to the bone-marrow cells and other cells of the patient not being HLA-matched.
  • the method described above further comprises a step of administering to the patient an agent which promotes mitochondrial biogenesis.
  • mitochondrial biogenesis refers to the growth and division of mitochondria.
  • the agent which promotes mitochondrial biogenesis is erythropoietin (EPO) or a salt thereof.
  • the agent is selected from the group consisting of recombinant human erythropoietin and isolated human erythropoietin.
  • the method described above further comprises a step of administering to the patient an agent which prevents, delays, minimizes or abolishes an adverse immunogenic reaction between the patient and the bone-marrow cells.
  • the adverse immunogenic reaction is a graft-versus-host disease (GvHD).
  • the GvHD is an acute form of the disease (aGvHD).
  • the GvHD is a chronic form of the disease (cGvHD).
  • the method described above further comprises a preceding step of administering to the patient a pre-transplant conditioning agent prior to the administration of the pharmaceutical composition.
  • pre-transplant conditioning agent refers to any agent capable of killing bone-marrow cells within the bone-marrow of a human subject.
  • the pre-transplant conditioning agent is Busulfan.
  • the mitochondrial disease is associated with a mutation in the mitochondrial DNA. In certain embodiments, the mitochondrial disease is associated with a mutation in the nuclear DNA.
  • mutation refers to an insertion, deletion or replacement of at least one nucleotide in mitochondrial or nuclear DNA.
  • the mitochondrial disease is a MRCD.
  • the mitochondrial disease is selected from the group consisting of LHON; MELAS; Pearson syndrome; Leigh syndrome; NARP; MERRF; KSS; MNGIE; Friedreich Ataxia; and Alpers' disease.
  • the mitochondrial disease is selected from the group consisting of LHON, MELAS, Pearson syndrome, Leigh syndrome, NARP, MERRF, and KSS.
  • the mitochondrial disease is LHON.
  • the mitochondrial disease is MELAS.
  • the pharmaceutical composition is administered locally. In certain embodiments, the administration of the pharmaceutical composition to a subject is by direct administration to the bone-marrow of the subject. In certain embodiments, the administration of the pharmaceutical composition to a subject is to a tissue or an organ. In certain embodiments, the administration of the pharmaceutical composition to a subject is to the eye.
  • the vitreous humour is a transparent, colorless, gelatinous mass that fills the space in the eye between the lens and the retina. In certain embodiments, the administration of the pharmaceutical composition to a subject is to the vitreous humour of the eye. In certain embodiments, the administration of the pharmaceutical composition to a subject is by direct intramuscular injection. In certain embodiments, the pharmaceutical composition is administered systemically.
  • the administration of the pharmaceutical composition to a subject is by a route selected from the group consisting of intravenous, intraarterial, intramuscular, subcutaneous, and direct injection into a tissue or an organ.
  • the functional mitochondria are obtained from a human cell or a human tissue selected from the group consisting of placenta, placental cells grown in culture, and blood cells.
  • a human cell or a human tissue selected from the group consisting of placenta, placental cells grown in culture, and blood cells.
  • the functional mitochondria have undergone a freeze-thaw cycle.
  • mitochondria that have undergone a freeze-thaw cycle demonstrate a comparable oxygen consumption rate following thawing, as compared to control mitochondria that have not undergone a freeze-thaw cycle.
  • the freeze-thaw cycle comprises freezing said functional mitochondria for at least 24 hours prior to thawing. According to some embodiments, the freeze-thaw cycle comprises freezing said functional mitochondria for at least 1 month prior to thawing, several months prior to thawing or longer. According to another embodiment, the oxygen consumption of the functional mitochondria after the freeze-thaw cycle is equal or higher than the oxygen consumption of the functional mitochondria prior to the freeze-thaw cycle.
  • freeze-thaw cycle refers to freezing of the functional mitochondria to a temperature below 0° C., maintaining the mitochondria in a temperature below 0° C. for a defined period of time and thawing the mitochondria to room temperature or body temperature or any temperature above 0° C. which enables treatment of the hematopoietic cells with the mitochondria.
  • room temperature refers to a temperature of between 18° C. and 25° C.
  • body temperature refers to a temperature of between 35.5° C. and 37.5° C., preferably 37° C.
  • mitochondria that have undergone a freeze-thaw cycle are functional mitochondria.
  • the mitochondria that have undergone a freeze-thaw cycle were frozen at a temperature of ⁇ 70° C. or lower. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle were frozen at a temperature of ⁇ 20° C. or lower. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle were frozen at a temperature of ⁇ 4° C. or lower. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle were frozen at a temperature of 0° C. or lower. According to another embodiment, freezing of the mitochondria is gradual. According to some embodiment, freezing of mitochondria is through flash-freezing. As used herein, the term “flash-freezing” refers to rapidly freezing the mitochondria by subjecting them to cryogenic temperatures.
  • the mitochondria that underwent a freeze-thaw cycle were frozen for at least 30 minutes prior to thawing.
  • the freeze-thaw cycle comprises freezing the functional mitochondria for at least 30, 60, 90, 120, 180, 210 minutes prior to thawing.
  • Each possibility represents a separate embodiment of the present invention.
  • the mitochondria that have undergone a freeze-thaw cycle were frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48, 72, 96, or 120 hours prior to thawing. Each freezing time presents a separate embodiment of the present invention.
  • the mitochondria that have undergone a freeze-thaw cycle were frozen for at least 4, 5, 6, 7, 30, 60, 120, 365 days prior to thawing.
  • the freeze-thaw cycle comprises freezing the functional mitochondria for at least 1, 2, 3 weeks prior to thawing. Each possibility represents a separate embodiment of the present invention. According to another embodiment, the freeze-thaw cycle comprises freezing the functional mitochondria for at least 1, 2, 3, 4, 5, 6 months prior to thawing. Each possibility represents a separate embodiment of the present invention.
  • the mitochondria that have undergone a freeze-thaw cycle were frozen at ⁇ 70° C. for at least 30 minutes prior to thawing.
  • the possibility to freeze mitochondria and thaw them after a long period enables easy storage and use of the mitochondria with reproducible results even after a long period of storage.
  • thawing is at room temperature. In another embodiment, thawing is at body temperature. According to another embodiment, thawing is at a temperature which enables administering the mitochondria according to the methods of the invention. According to another embodiment, thawing is performed gradually.
  • the mitochondria that underwent a freeze-thaw cycle were frozen within a freezing buffer.
  • the mitochondria that underwent a freeze-thaw cycle were frozen within the isolation buffer.
  • the term “isolation buffer” refers to a buffer in which the mitochondria of the invention have been isolated.
  • the isolation buffer is a sucrose buffer.
  • the freezing buffer comprises a cryoprotectant.
  • the cryoprotectant is a saccharide, an oligosaccharide or a polysaccharide.
  • the saccharide concentration in the freezing buffer is a sufficient saccharide concentration which acts to preserve mitochondrial function.
  • the isolation buffer comprises a saccharide.
  • the saccharide concentration in the isolation buffer is a sufficient saccharide concentration which acts to preserve mitochondrial function.
  • the saccharide is sucrose.
  • the intactness of a mitochondrial membrane may be determined by any method known in the art.
  • intactness of a mitochondrial membrane is measured using the tetramethylrhodamine methyl ester (TMRM) or the tetramethylrhodamine ethyl ester (TMRE) fluorescent probes.
  • TMRM tetramethylrhodamine methyl ester
  • TMRE tetramethylrhodamine ethyl ester
  • Human HepG2 cells (10 5 , ATCC HB-8065) were untreated (control) or incubated for 24 hours with mitochondria isolated from human placental cells. Before plating the cells, mitochondria were mixed with the cells, centrifuged at 8000 g and re-suspended. After incubation, the cells were washed twice with PBS, and CS activity was measured using the CS0720 Sigma kit ( FIG. 1 ).
  • Mouse fibroblast cells (3T3) expressing green fluorescent protein (GFP) in their mitochondria were incubated for 24 hours with red fluorescent protein (RFP)-labeled mitochondria isolated from mouse fibroblasts (3T3) expressing RFP in their mitochondria (middle panel). Fluorescent confocal microscopy was used to identify fibroblasts labeled with both GFP and RFP, which appear yellow (right panel) ( FIG. 2 ).
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • Mouse fibroblast cells (10 4 , 3T3) were either not treated (control) or treated with 0.5 ⁇ M Rotenone (Rotenone, mitochondrial complex I irreversible inhibitor, CAS number 83-79-4) for 4 hours, washed, and further treated with 0.02 mg/ml mouse placental mitochondria (Rotenone+Mitochondria) for 3 hours.
  • the cells were washed and ATP level was determined using the Perkin Elmer ATPlite kit ( FIG. 3 ). As seen in FIG. 3 , the production of ATP was completely rescued in cells incubated with mitochondria compared to control.
  • Mouse bone-marrow cells (10 5 ) were incubated for 24 hours with GFP-labeled mitochondria, isolated from mouse melanoma cells. Fluorescence confocal microscopy was used to identify GFP-labeled mitochondria inside the bone marrow cells ( FIG. 4 ).
  • Mouse bone marrow cells (10 6 ) were untreated or incubated for 15 hours with different amounts of GFP-labeled mitochondria isolated from mouse melanoma cells. Before plating the cells, mitochondria were mixed with the cells and either left to stand for 5 minutes at room temperature (( ⁇ ) Cent) or centrifuged for 5 minutes at 8,000 g at 4° C. ((+) Cent). The cells were then plated in 24 wells (10 6 cells/well). After 15 hours of incubation, the cells were washed twice to remove any mitochondria that did not enter the cells. Citrate synthase activity was determined using the CS0720 Sigma kit ( FIG. 5 ). The CS activity levels measured under the conditions specified above are summarized in Table 1.
  • Mouse bone-marrow cells (10 6 ) were untreated or incubated for 24 hours with GFP-labeled mitochondria isolated from mouse melanoma cells (17 U or 34 U, indicating the level of citrate synthase activity as a marker for mitochondria content). The cells were mixed with mitochondria, centrifuged at 8000 g and re-suspended. After 24 hour incubation, the cells were washed twice with PBS and the level of citrate synthase (CS) activity ( FIG. 6A ) and cytochrome c reductase activity ( FIG. 6B ) were measured using the CS0720 and CY0100 kits (Sigma), respectively.
  • CS citrate synthase
  • FIG. 6B cytochrome c reductase activity
  • FIG. 7A depicts the staining of slices of Control 1 (no cells or mitochondria injected, left panel) and Control 2 (cells loaded with mitochondria injected, but no anti-GFP used in staining, right panel).
  • FIG. 7B depicts the staining of slices of an injected eyeball. GFP-positive cells were observed in the Retinal Ganglion Cell (RGC) layer (two upper panels), in blood vessels (*) and in cells lining the wall of blood vessels (arrow).
  • RRC Retinal Ganglion Cell
  • 143B Rho0 cells human bone osteosarcoma, mitochondrial-DNA deficient (mtDNA ⁇ ) were seeded in 96 wells (3*10 4 cells/well) and cultured in medium containing pyruvate and uridine. Mitochondria were isolated from 143B TK cells (human bone osteosarcoma, thymidine kinase positive (TK + ), mtDNA + , 2*10 6 cells per well) and incubated with the 143B Rho0 cells. Twenty four hours later, the medium was replaced and the cells were treated with Ganciclovir to eliminate any 143B TK + cells.
  • the remaining cells were cultured for additional 3 days, and then the medium was replaced with pyruvate and uridine-free medium. After 24 hours, the cells were trypsinized and transferred to 10 cm dish. The medium was replaced every 3 days for the next 9 days.
  • One set of dishes (control and mitochondria-treated) was fixed in methanol and stained with giemsa ( FIG. 8A ).
  • the second set was trypsinized and assayed for ATP level as an indicator for cell number and mitochondrial activity ( FIG. 8B ). As seen in FIG. 8B , ATP levels in cells incubated with mitochondria were increased by about 5.3 compared to control.
  • Human CD34 + cells (1.4*10 5 , ATCC PCS-800-012) were untreated or incubated for 20 hours with GFP-labeled mitochondria isolated from human placental cells. Before plating the cells, mitochondria were mixed with the cells, centrifuged at 8000 g and re-suspended. After incubation, the cells were washed twice with PBS and CS activity was measured using the CS0720 Sigma kit ( FIG. 9A ). ATP content was measured using ATPlite (Perkin Elmer) ( FIG. 9B ). The CS activity levels ( FIG. 9A ) measured under the conditions specified above are summarized in Table 2.
  • FIG. 9B The cell populations depict in FIG. 9B were further evaluated by FACS analysis. While in the CD34 + cells not incubated with GFP-labeled mitochondria only a minor portion (0.9%) of the cells were fluorescent ( FIG. 10A ), the CD34 + cells incubated with GFP-labeled mitochondria after centrifugation were substantially fluorescent (28.4%) ( FIG. 10B ).
  • Mouse mitochondria from WT murine placenta are harvested and used to treat bone marrow of the mouse model of mitochondrial disease (such as with a mutation in mtDNA tRNA-Ala). Briefly, diseased bone marrow cells are treated ex-vivo with murine placental mitochondria at different concentrations and incubation times, and injected to another diseased mouse. For comparison, healthy bone marrow transplantation from a healthy mouse to a diseased mouse is also conducted. Assays of efficacy are performed at various time points (before transplantation: ATP level, O 2 consumption, citrate synthase (CS) activity; after transplantation: ATP synthesis, O 2 consumption, heteroplasmy of mtDNA, CS activity and presence of lactic acid in blood. Biodistribution of mitochondria is tested at 1 day, 1 week, and 1 month after injection by PCR analysis of the mutated vs. wild type mitochondrial tRNA gene in different tissues (mainly bone marrow, heart, skeletal muscle, liver and brain).
  • High-purity active human placental mitochondria grown in 3D cultures are isolated and characterized in terms of CS activity and O 2 consumption.
  • Patients with mtDNA mutations or deletions presenting with mitochondrial disease (LHON, MELAS, Leigh syndrome, Pearson's disease, Alper's syndrome) provide fibroblasts isolated from their skin biopsies. Mitochondria augmentation and rescue capabilities are tested both in patient's fibroblasts and in fibroblasts converted in-vitro to induced pluripotent stem (iPS) cells reprogrammed to hematopoietic lineage (to avoid the need in bone marrow aspiration from the patients).
  • the cells are treated with human mitochondria and mtDNA heteroplasmy, CS activity, ATP content and O 2 consumption are tested at different time points after incubation with mitochondria (3 hours, 24 hours, and 48 hours).
  • Mitochondria are introduced into bone marrow cells of control healthy mice from two different backgrounds: the source of mitochondria will be from mice with different mtDNA sequences (Jenuth J P et al., Nature Genetics, 1996, Vol. 14, pages 146-151).
  • the steps of the method are (1) isolation mitochondria from placenta/livers of Balb/C mice, frozen at ⁇ 80 and defrosted, or used fresh; (2) bone marrow isolation from e.g. NZB strain; (3) mixing the mitochondria and bone marrow and centrifugation at 8000 g, 5 minutes, resuspending and incubating for 24 hours; (4) washing the bone marrow cells twice with PBS and injecting into a tail vain of a mouse. After 24 hours, a week, a month and 3 months post transplantation tissues (blood, bone marrow, brain, heart, kidney, liver, lung, spleen, skeletal muscle, eye) are collected and DNA is extracted for further sequence analysis.
  • tissue blood, bone marrow, brain, heart, kidney, liver, lung, spleen, skeletal muscle, eye
  • Mitochondria are introduced into bone marrow cells of ND6, a mitochondrial disease mouse model, similar to LHON (Chun Shi Lin et al., PNAS, 2012, Vol. 109(49), pages 20065-20070) to prove that healthy mitochondria may partly rescue the phenotype of ND6 mouse model.
  • the steps of the method are (1) isolation of mitochondria from placenta/livers of B6ME mice (WT strain of the ND6 mouse), frozen at ⁇ 80 and defrosted, or used fresh; (2) bone marrow isolation from 1A1B6ME mice (ND6 mutant mouse on B6ME background); (3) mixing the mitochondria and bone marrow and centrifugation at 8000 g, 5 minutes, resuspending and incubating for 24 hours; (4) washing the bone marrow cells twice with PBS and injecting into the tail vain of a 1A1B6ME mouse. After 24 hours, a week, a month and 3 months post transplantation tissues (blood, bone marrow, brain, heart, kidney, liver, lung, spleen, skeletal muscle, eye) are collected and DNA is extracted for further sequence analysis.
  • Human blood mitochondria are introduced into mouse bone marrow cells for safety studies and bio-distribution.
  • the steps of the method are (1) isolation of mitochondria from human white blood cells and platelets, frozen at ⁇ 80 and defrosted, or used fresh; (2) bone marrow isolation from e.g. C57/b1 mice; (3) mixing the mitochondria and bone marrow and centrifugation at 8000 g, 5 minutes, resuspending and incubating for 24 hours; (4) washing the bone marrow cells twice with PBS and injecting into the tail vain of e.g. C57/b1 mouse. After 24 hours, a week, a month and 3 months post transplantation tissues (blood, bone marrow, brain, heart, kidney, liver, lung, spleen, skeletal muscle, eye) are collected and DNA is extracted for further sequence analysis. During the entire period, evaluating changes in food consumption, body weight, lactic acidosis, blood counts and biochemical blood markers.
  • Example 15 Therapy of Human Patients Afflicted by a Mitochondrial Disease
  • the steps of the method for therapy of human patients afflicted by a mitochondrial disease are (1) administering to a patient afflicted by a mitochondrial disease, for example LHON, MELAS or Pearson Syndrome, G-CSF in a dosage of 10-16 ⁇ g/kg for 5 days; (2) on day 6, performing apheresis on the blood of the patient to obtain bone marrow cells; (3) in parallel, isolating functional mitochondria from a blood sample of a healthy donor; (4) incubation of bone marrow cells with functional mitochondria for 24 hours; (5) washing the bone marrow cells; and (6) infusion of bone marrow cells loaded with mitochondria to the patient.
  • a mitochondrial disease for example LHON, MELAS or Pearson Syndrome, G-CSF in a dosage of 10-16 ⁇ g/kg for 5 days
  • a mitochondrial disease for example LHON, MELAS or Pearson Syndrome, G-CSF in a dosage of 10-16 ⁇ g/kg for 5 days
  • on day 6 performing apheresis on

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JP7015169B2 (ja) 2022-02-02
IL299482A (en) 2023-02-01
WO2016135723A1 (en) 2016-09-01
US11441124B2 (en) 2022-09-13
IL299482B1 (en) 2024-10-01
EP3261649A1 (en) 2018-01-03

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