阅读说明：本技术 使用富集有功能性线粒体的干细胞的线粒体增强疗法 (Mitochondrion-enhanced therapy using stem cells enriched for functional mitochondria ) 是由 娜塔莉·伊夫吉奥哈纳 乌列·哈拉维 史密尔·布克施班 诺亚·谢尔 于 2019-07-22 设计创作，主要内容包括：本发明提供了富集有健康的功能性线粒体的干细胞和利用此类细胞在需要的对象中缓解包括衰老和年龄相关疾病以及抗癌疗法的衰弱效应在内的令人衰弱的病症的治疗方法。(The present invention provides stem cells enriched in healthy functional mitochondria and methods of using such cells to alleviate debilitating conditions, including aging and age-related diseases, as well as the debilitating effects of anti-cancer therapies, in a subject in need thereof.)

1. A pharmaceutical composition for treating or alleviating a debilitating condition in a subject, said pharmaceutical composition comprising at least 10⁵To 2x10⁷Human stem cells suspended in a pharmaceutically acceptable liquid medium capable of supporting the survival of said cells per kilogram body weight of a subject, wherein said human stem cells are enriched in freeze-thawed, healthy, functional, exogenous mitochondria, and wherein said debilitating conditionSelected from the group consisting of aging, age-related diseases and sequelae of anti-cancer therapy.

2. The pharmaceutical composition of claim 1, wherein the enriching comprises introducing mitochondria at a dose of at least 0.088 up to 176 milliunits CS activity per million cells in the stem cells.

3. The pharmaceutical composition of claim 2, wherein the enriching comprises contacting the stem cells with mitochondria at a dose of 0.88 up to 17.6 milliunits CS activity per million cells.

4. The pharmaceutical composition of claim 1, wherein the anti-cancer treatment is selected from the group consisting of radiation, chemotherapy, and immunotherapy using monoclonal antibodies.

5. The pharmaceutical composition of claim 1, wherein the stem cells are autologous, syngeneic, or derived from a donor.

6. The pharmaceutical composition of claim 1, wherein the stem cell is a Pluripotent Stem Cell (PSC) or an Induced Pluripotent Stem Cell (iPSC).

7. The pharmaceutical composition of claim 1, wherein the stem cell is a mesenchymal stem cell.

8. The pharmaceutical composition of claim 1, wherein the stem cells are derived from adipose tissue, oral mucosa, blood or umbilical cord blood.

9. The pharmaceutical composition of claim 1, wherein the stem cells are derived from bone marrow cells.

10. The pharmaceutical composition of claims 1-9, wherein the human stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof.

11. The pharmaceutical composition of any one of the preceding claims, wherein the stem cell is CD34⁺A cell.

12. The pharmaceutical composition of any one of the preceding claims, wherein the stem cells are at least partially purified.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the healthy functional mitochondria are derived from a cell or tissue selected from the group consisting of: placenta, placental cells and blood cells grown in culture.

14. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is administered to a subject suffering from a debilitating condition selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy.

15. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is administered to a specific tissue or organ.

16. The pharmaceutical composition of claim 14, wherein the pharmaceutical composition is administered by systemic parenteral administration.

17. The pharmaceutical composition of claim 16, comprising at least about 10⁶Individual mitochondrially enriched human stem cells per kilogram patient body weight.

18. The pharmaceutical composition of any one of claims 1-15, comprising a total of about 5x10⁵To 5x10⁹Human stem cells enriched in human mitochondria.

19. The pharmaceutical composition of any one of claims 1 to 18, wherein administration of the pharmaceutical composition to a subject is by a parenteral route selected from intravenous, intra-arterial, intramuscular, subcutaneous, intraperitoneal, and direct injection into a tissue or organ.

20. The pharmaceutical composition of any one of claims 1 to 19, wherein the mitochondrially enriched human stem cell has:

corresponding levels in the stem cells prior to enrichment relative to mitochondria

(i) Increased mitochondrial DNA content;

(ii) increased CS activity levels;

(iii) increased levels of at least one mitochondrial protein selected from succinate dehydrogenase complex subunit a (sdha) and cytochrome C oxidase (COX 1);

(iv) increased O₂A consumption rate;

(v) increased ATP production rate; or

(vi) Any combination thereof.

21. An ex vivo method of enriching human stem cells for functional exogenous mitochondria, said method comprising the steps of:

(i) providing a first composition comprising a plurality of isolated or partially purified human stem cells from an individual having a debilitating condition or from a donor;

(ii) providing a second composition comprising a plurality of isolated or partially purified freeze-thawed human functional mitochondria obtained from a healthy donor;

(iii) (ii) freeze-thawed human functional mitochondria of said first composition of human stem cells and said second composition at 0.088-176mU CS activity/10⁶Proportional contact of individual stem cells; and

(iv) (iv) incubating the composition of (iii) under conditions that allow the freeze-thawed human functional mitochondria to enter the human stem cells, thereby enriching the human stem cells with the human functional mitochondria;

wherein the functional mitochondrial content of the enriched human stem cells is detectably higher than the functional mitochondrial content of the human stem cells in the first composition.

22. The method of claim 21, wherein the stem cells in the first composition are obtained from an aging subject or donor.

23. An ex vivo method of enriching human stem cells for functional exogenous mitochondria, said method comprising the steps of:

(i) providing a first composition comprising a plurality of isolated or partially purified human stem cells from an individual having a malignant disease or from a healthy donor;

(ii) providing a second composition comprising a plurality of isolated or partially purified freeze-thawed human functional mitochondria obtained from the same individual or from a healthy donor;

(iii) (ii) freeze-thawed human functional mitochondria of said first composition of human stem cells and said second composition at 0.088-176mU CS activity/10⁶Proportional contact of individual stem cells; and

(iv) (iv) incubating the composition of (iii) under conditions that allow the human functional mitochondria to enter the human stem cell, thereby enriching the human stem cell with the human functional mitochondria;

wherein the functional mitochondrial content of the enriched human stem cells is detectably higher than the functional mitochondrial content of the human stem cells in the first composition.

24. The method of claim 23, wherein the stem cells in the first composition are obtained from a subject having a non-hematopoietic malignant disease or a healthy donor not having a malignant disease.

25. The method of any one of claims 21 to 24, wherein the conditions that allow healthy functional exogenous mitochondria to enter human stem cells comprise incubating the human stem cells with the healthy functional exogenous mitochondria at a temperature in the range of 16 to 37 ℃ for a time in the range of 0.5 to 30 hours.

26. The method of claim 25, wherein prior to incubating, the method further comprises single centrifugation of the human stem cells and healthy functional exogenous mitochondria above 2500 xg.

27. The method of any one of claims 21 and 26, wherein the stem cell is a bone marrow cell.

28. The method of claim 23, wherein the functional mitochondria in the second composition are obtained from a subject having a malignant disease prior to an anti-cancer treatment.

29. The method of any one of claims 21 to 28, further comprising expanding the stem cells prior to or after enrichment with the healthy, functional, exogenous mitochondria.

30. The method of any one of claims 21 to 28, wherein autologous human stem cells are frozen and stored prior to suffering from the debilitating condition.

31. The method of any one of claims 21 to 28, wherein enriching the human stem cells for mitochondria is performed prior to freezing the cells.

32. The method of any one of claims 21 to 28, wherein enriching the human stem cells for mitochondria is performed after freezing and thawing the cells.

33. The method of any one of claims 21 and 23, wherein the detectable enrichment of functional mitochondrial content of the stem cell prior to mitochondrial enrichment or after mitochondrial enrichment is determined by an assay selected from the group consisting of: (i) the level of at least one mitochondrial protein selected from SDHA and COX 1; (ii) the level of citrate synthase activity; (iii) oxygen (O)₂) A consumption rate; (iv) adenosine Triphosphate (ATP) production rate; (v) mitochondrial DNA content; and any of themAnd (4) combining.

34. The method of any one of claims 21 and 23, wherein the stem cell is a Pluripotent Stem Cell (PSC) or an Induced Pluripotent Stem Cell (iPSC).

35. The method of any one of claims 21 and 23, wherein the stem cell is a mesenchymal stem cell.

36. The method of any one of claims 21 and 23, wherein the stem cells are derived from adipose tissue, skin fibroblasts, oral mucosa, blood, or umbilical cord blood.

37. The method of any one of claims 21 and 23, wherein the stem cell is CD34⁺A cell.

38. The method of any one of claims 21 and 23, wherein the human stem cells are derived from adipose tissue, oral mucosa, blood, cord blood, or bone marrow.

39. The method of any one of claims 21 and 23, wherein the stem cells are derived from bone marrow cells.

40. The method of any one of claims 21-39, wherein the mitochondrially-enriched stem cells have:

(i) increased levels of at least one mitochondrial protein selected from SDHA and COX 1;

(ii) enhanced oxygen (O)₂) A consumption rate;

(iii) increased level of citrate synthase activity;

(iv) increased rate of Adenosine Triphosphate (ATP) production;

(v) increased mitochondrial DNA content; or

(vi) Any combination thereof.

41. The method of any one of claims 21 and 23, wherein the total amount of mitochondrial proteins in the partially purified mitochondria is between 20% and 80% of the total amount of cellular proteins in the sample.

42. A plurality of human stem cells enriched for functional mitochondria obtained by the method of any one of claims 21 and 23.

43. A pharmaceutical composition comprising a plurality of human stem cells according to claim 42.

44. The pharmaceutical composition of claim 43, for treating or ameliorating a debilitating condition in a human subject, wherein said debilitating condition is selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy.

45. A method of treating a debilitating condition in a human subject in need thereof, comprising the step of administering to said subject a pharmaceutical composition of claim 43.

46. The method of claim 45, wherein the stem cells are autologous or syngeneic to the subject with the debilitating condition.

47. The method of claim 45, wherein the stem cells are allogeneic to the subject with the debilitating condition.

48. The method of claim 45, further comprising the step of administering to a subject having a debilitating condition selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy an agent that prevents, delays, minimizes or eliminates an adverse immunogenic reaction between the subject and stem cells of an allogeneic donor.

49. Is used forA method of treating or ameliorating a debilitating condition in a subject, said method comprising parenterally administering to said subject a composition comprising at least 5x10⁵To 5X10⁹A pharmaceutical composition enriched for freeze-thawed, healthy, functional, exogenous mitochondrial human stem cells, wherein the debilitating condition is selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy.

Technical Field

The present invention relates to functional mitochondrially enriched stem cells and methods of treatment using such cells to alleviate the debilitating effects of a variety of conditions including aging and age-related diseases, as well as the debilitating effects of anti-cancer therapy treatments.

Background

Mitochondria are membrane-bound organelles present in most eukaryotic cells, ranging in diameter from 0.5 to 1.0 μm. Mitochondria are present in almost all eukaryotic cells, and their number and location vary with cell type. Mitochondria contain their own dna (mtdna) and their own mechanisms for synthesizing RNA and proteins. mtDNA contains only 37 genes, and therefore most gene products in mammals are encoded by nuclear DNA.

Mitochondria perform a number of essential tasks in eukaryotic cells, such as pyruvate oxidation, the krebs cycle, and the metabolism of amino acids, fatty acids, and steroids. However, the main function of mitochondria is to utilize the electron transport chain and the oxidative phosphorylation system ("respiratory chain") to generate energy as Adenosine Triphosphate (ATP). Other processes in which mitochondria are involved include thermogenesis, calcium ion storage, calcium signaling, programmed cell death (apoptosis) and cell proliferation.

Intracellular ATP concentrations are typically 1-10 mM. ATP can be produced by redox reactions using monosaccharides and complex sugars (carbohydrates) or lipids as energy sources. For complex fuels to be used for the synthesis of ATP, it is first necessary to break them down into smaller, simpler molecules. Complex carbohydrates are hydrolyzed into monosaccharides such as glucose and fructose. Fat (triglycerides) is metabolized to give fatty acids and glycerol.

The overall process of oxidation of glucose to carbon dioxide is known as cellular respiration, and about 30 ATP molecules can be produced from a single glucose molecule. ATP may be produced by a number of different cellular processes. The three major pathways for energy production in eukaryotic organisms are glycolysis and citric acid cycle/oxidative phosphorylation (both components of cellular respiration) and β -oxidation. This ATP production by non-photosynthetic eukaryotes occurs mostly in mitochondria, which can account for nearly 25% of the total volume of a typical cell. Various mitochondrial disorders are known to be caused by defective genes in mitochondrial DNA.

WO2016/135723 to the present inventors discloses mitochondria-enriched mammalian bone marrow cells for use in the treatment of mitochondrial diseases.

US 2012/0058091 discloses diagnostic and therapeutic treatments related to mitochondrial disorders. The method comprises microinjecting a heterologous mitochondrion into an oocyte or embryonic cell, wherein the heterologous mitochondrion is capable of achieving at least a normal level of mitochondrial membrane potential in the oocyte or embryonic cell.

WO 2001/046401 discloses embryonic or stem-like cells produced by cross-species nuclear transfer. Nuclear transfer efficiency is enhanced by the introduction of compatible cytoplasmic or mitochondrial DNA (of the same or similar species as the donor cell or nucleus).

WO 2013/002880 describes compositions and methods comprising bioenergy agents for restoring the quality of aged oocytes, enhancing oocytes or improving their derivatives (e.g. cytoplasm or isolated mitochondria) for use in procedures for enhancing fertility.

US 20130022666 provides compositions comprising a lipid carrier and mitochondria and methods of delivering exogenous mitochondria to cells and methods of treating or reversing the progression of disorders associated with mitochondrial dysfunction in a mammalian subject in need thereof.

WO 2017/124037 relates to compositions comprising isolated mitochondrial or combined mitochondrial agents and methods of treating disorders using such compositions.

US 20080275005 relates to mitochondrially targeted antioxidant compounds. The compounds of the invention comprise a lipophilic cation covalently coupled to an antioxidant moiety.

US 9855296 describes a method of enhancing cardiac or cardiovascular function in a human subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising isolated and substantially pure mitochondria, wherein the mitochondria are syngeneic or allogeneic mitochondria, in an amount sufficient to enhance the cardiac or cardiovascular function.

US 9603872 provides methods, kits and compositions for mitochondrial replacement in the treatment of disorders caused by mitochondrial dysfunction. The invention also describes methods for diagnosing neuropsychiatric (e.g., bipolar disorder) and neurodegenerative disorders based on abnormalities in mitochondrial structure.

US 20180071337 discloses a therapeutic composition comprising human mitochondria isolated from a cell, wherein said mitochondria can be present with or without at least one polypeptide or glycoprotein in a carrier, vesicle or liposome comprising a lipid bilayer, and a pharmaceutically acceptable excipient.

US 20010021526 provides cellular and animal models of diseases associated with mitochondrial defects. A cybrid cell line is described as a model system for studying disorders associated with mitochondrial defects.

WO 2013/035101 to the present inventors relates to mitochondrial compositions and methods of treatment using them and discloses partially purified functional mitochondrial compositions and methods of using the compositions for treating conditions that benefit from increased mitochondrial function comprising administering the compositions to a subject in need thereof.

Attempts to induce mitochondrial transfer into host cells or tissues have been reported. Most methods require active transfer of mitochondria by injection (e.g., McCully et al, Am J Physiol Heart physiol.2009,296(1): H94-H105). Transfer of mitochondria engulfed within a vehicle such as a liposome is also known (e.g., Shi et al, Ethicity and Disease, 2008; 18(S1): 43).

It has been shown that mitochondrial transfer can occur spontaneously between cells in vitro, although only mtDNA can be determined to be transferred, rather than intact functional mitochondria (e.g., Plotnikov et al, Exp Cell Res.2010,316(15): 2447-55; Spees et al, Proc Natl Acad Sci, 2006; 103(5): 1283-8). In vitro mitochondrial transfer by endocytosis or internalization has also been demonstrated (Clark et al, Nature,1982:295: 605-.

US 20110105359 provides compositions for cryopreservation of cells and cellular and subcellular fractions in the form of self-maintenance bodies. On the other hand, attempts to inject isolated mitochondria during early reperfusion to provide cardioprotection have shown that freshly isolated mitochondria are required for cardioprotection, as frozen mitochondria do not provide cardioprotection and exhibit significantly reduced oxygen consumption compared to freshly isolated mitochondria (McCully et al, supra).

WO 2016/008937 relates to a method of transferring mitochondrial cells isolated from a donor cell population into a recipient cell population. The method shows improved transport efficiency for a certain amount of mitochondria.

US 2012/0107285 relates to mitochondrial enhancement of cells. Certain embodiments include, but are not limited to, methods of modifying stem cells or methods of administering modified stem cells to at least one biological tissue.

Aging is one of the largest known risk factors for many human diseases. Age-related diseases are the most common diseases, and their frequency increases with aging. In essence, age-related diseases are complications caused by aging. Age-related diseases should be distinguished from the aging process itself, as all adult animals will age, but not all will experience age-related diseases.

The decline in mitochondrial quality and activity has been associated with normal aging and with the development of a wide range of age-related diseases. Mitochondria contribute to specific aspects of the aging process including cellular aging, chronic inflammation, and age-dependent decline in stem cell activity. There is a large body of supportive evidence that mitochondrial dysfunction occurs with aging due to the accumulation of mutations in mitochondrial DNA. It has been shown that in human brain, heart, skeletal muscle and liver tissues, a variety of different mitochondrial DNA point mutations increase significantly with aging. The frequency of mitochondrial DNA deletions/insertions has also been reported to increase with age in both animal models and humans. It is speculated that the replication cycle and accumulation of mitochondrial DNA mutations may be conserved mechanisms behind stem Cell senescence, allowing mitochondria to influence or regulate many key aspects of senescence (Sun et al, Cell,2016,61: 654-66; Srivastava, Genes,2017,8: 398; Ren et al, Genes,2017,8: 397).

Cancer is caused by the uncontrolled proliferation of abnormal cells in organs or tissues of the body. Various different types of cancer treatments are available, including: surgery, chemotherapy, radiation therapy, immunotherapy, targeted therapy, hormonal therapy or stem cell transplantation. Such cancer treatments often cause serious adverse effects, including: fatigue, nausea and vomiting, anemia, diarrhea, loss of appetite, thrombocytopenia, delirium, hair loss, fertility problems, peripheral neuropathy, pain, lymphedema. These debilitating effects significantly reduce the quality of life of cancer patients. It is well known to use bone marrow cells to replenish bone marrow of cancer patients who have hematopoietic malignancies and have undergone bone marrow ablation. Bone marrow transplantation most commonly uses matched healthy donors. However, in certain cases, such as multiple myeloma, autologous bone marrow transplantation may be performed. Bone marrow cells are not routinely used in the treatment of non-hematopoietic cancer patients.

There is an unmet need to improve the quality of life of subjects suffering from debilitating effects due to a variety of different conditions, such as aging and age-related diseases, as well as cancer patients undergoing chemical or radiation therapy. Reversing the decline in mitochondrial function can slow the effects of aging and alleviate the debilitating effects of age-related diseases and anticancer treatments.

Disclosure of Invention

The present invention provides mammalian stem cells enriched for healthy functional mitochondria and methods of alleviating the debilitating effects and adverse events of anti-cancer therapy for a number of conditions including aging and age-related diseases. Surprisingly, it has now been shown for the first time that transplantation of robust cells enriched with healthy mitochondria can significantly delay the symptoms of aging and the progression of age-related diseases. In addition, mitochondrion-enhanced therapy using stem cells enriched in healthy mitochondria can mitigate the debilitating effects of chemotherapy, radiation therapy and/or immunotherapy using monoclonal antibodies in cancer patients undergoing anti-cancer therapy. In particular, the invention provides compositions comprising stem cells enriched for functional mitochondria, including autologous or donor stem cells. These cells, when introduced into a subject to be treated, can be used to alleviate or reduce the effects of debilitating conditions.

In particular embodiments, the subject is treated with stem cells enriched for functional mitochondria obtained from healthy donors. Convenient sources of healthy donor mitochondria include, but are not limited to, placental mitochondria or mitochondria derived from blood cells. Accordingly, the present invention provides methods of using allogeneic, autologous, or syngeneic "mitochondrially enriched" stem cells to treat or ameliorate the debilitating effects of anti-cancer therapy in aging and age-related diseases, as well as in cancer patients.

The present invention is based, in part, on the discovery that aged C57BL mice receiving bone marrow cells enriched in healthy mitochondria from a murine placenta, exhibit improvements in functional, cognitive and physiological blood tests as compared to age-matched mice receiving bone marrow not enriched for mitochondria.

According to various embodiments, the source of the stem cells may be autologous, syngeneic, or derived from a donor. Providing stem cells from a subject with a debilitating condition, enriching ex vivo with healthy mitochondria and returning them to the same subject provides benefits compared to other methods involving allogeneic cell therapy. For example, the provided methods eliminate the need for a long, costly and not always successful process of screening a population and finding donors that match the subject Human Leukocyte Antigens (HLA). The method also advantageously eliminates the need for life-long immunosuppressive therapy of the subject in order that the subject's body does not reject the population of allogeneic cells. Thus, the present invention advantageously provides a unique method of ex vivo therapy, wherein human stem cells are removed from the body of the subject, enriched ex vivo with healthy functional mitochondria and returned to the same subject. Furthermore, the present invention relates to the administration of stem cells that, without being bound by any theory or mechanism, circulate in different tissues throughout the body and thereby improve the quality of life of subjects with debilitating conditions.

The present invention is based, in part, on the surprising discovery that functional mitochondria can enter intact fibroblasts, hematopoietic stem cells, and bone marrow cells, and that treatment of fibroblasts, hematopoietic stem cells, and bone marrow cells with functional mitochondria increases mitochondrial content, cell viability, and ATP production.

The present invention provides for the first time stem cells of an aging subject or a cancer patient having enhanced or increased mitochondrial activity. These stem cells can be enriched with healthy functional mitochondria from a suitable source. Typically, the mitochondria can be obtained from blood cells, placental cell cultures, or other suitable cell lines. Each possibility is a separate embodiment of the invention.

In one aspect, the invention provides a method of treating or alleviating the debilitating effects of a variety of different conditions, said method comprising introducing isolated or partially purified freeze-thawed functional human mitochondria into stem cells obtained or derived from a subject or donor suffering from a debilitating condition, and introducing at least 10% of said cells in a pharmaceutically acceptable liquid medium capable of supporting the survival of said cells⁵To 2x10⁷(ii) transplanting individual "mitochondrially enriched" human stem cells per kilogram patient body weight into the subject suffering from the debilitating condition.

According to another aspect, the present invention provides a method of treating or alleviating a debilitating condition in a subject, said method comprising parenterally administering to said subject a composition comprising at least 5x10⁵To 5X10⁹A pharmaceutical composition enriched for freeze-thawed, healthy, functional, exogenous mitochondrial human stem cells, wherein the debilitating condition is selected fromFrom aging, age-related diseases and sequelae of anti-cancer treatments.

According to yet another aspect, the present invention provides a pharmaceutical composition for treating or alleviating a debilitating condition in a subject, said pharmaceutical composition comprising at least 10⁵To 2x10⁷Human stem cells suspended in a pharmaceutically acceptable liquid medium capable of supporting the survival of said cells per kilogram of body weight of a subject, wherein said human stem cells are enriched in freeze-thawed, healthy, functional, exogenous mitochondria, and wherein said debilitating condition is selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy. According to certain embodiments, the mitochondrial enrichment of the stem cell comprises introducing a dose of mitochondria of at least 0.088 up to 176 milliunits CS activity per million cells into the stem cell. According to other embodiments, the mitochondrial enrichment of the stem cell comprises introducing a dose of 0.88 up to 17.6 milliunits CS activity per million cells of mitochondria in the stem cell.

In certain embodiments, the amount of isolated mitochondria is added to the recipient cell at a desired concentration. The ratio of the number of mitochondrial donor cells to the number of mitochondrial acceptor cells is higher than 2:1 (donor cells compared to acceptor cells). In typical embodiments, the ratio is at least 5 or at least 10 or higher. In particular embodiments, the ratio of donor cells to recipient cells from which mitochondria are collected is at least 20, 50, 100, or higher. Each possibility is a separate embodiment.

In certain embodiments, the subject with a debilitating condition is a subject with aging. In certain embodiments, the subject with a debilitating condition has one or more age-related diseases. In other embodiments, the subject with a debilitating condition is a cancer patient undergoing chemotherapy, radiation therapy, immunotherapy with monoclonal antibodies, or a combination thereof. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the healthy functional human allogenic mitochondria are allogeneic mitochondria. In other embodiments, the healthy functional human extramitochondrial is autologous or syngeneic, i.e. of the same maternal lineage.

In another aspect, the present invention provides an in vitro method of enriching human stem cells for healthy mitochondria, the method comprising the steps of: (i) providing a first composition comprising a plurality of human stem cells obtained or derived from an individual having a debilitating condition or a healthy donor not having a debilitating condition; (ii) providing a second composition comprising a plurality of isolated or partially purified freeze-thawed, human functional, exogenous mitochondria obtained from a healthy donor who has not suffered a debilitating condition; (iii) (ii) freeze-thawed human functional mitochondria of said first composition of human stem cells and said second composition at 0.088-176mU CS activity/10⁶Proportional contact of individual stem cells; and (iv) incubating the composition of (iii) under conditions that allow the freeze-thawed human functional mitochondria to enter the human stem cells, thereby enriching the freeze-thawed human stem cells with the human functional mitochondria; wherein the functional mitochondrial content of the enriched human stem cells is detectably higher than the healthy functional mitochondrial content of the human stem cells in the first composition.

In particular embodiments, the subject having a debilitating condition is a cancer patient after treatment with a debilitating anti-cancer treatment. Accordingly, the present invention provides an in vitro method for enriching human stem cells with healthy functional exogenous mitochondria, said method comprising the steps of: (i) providing a first composition comprising a plurality of human stem cells from an individual having a malignant disease or a healthy subject not having a malignant disease; (ii) providing a second composition comprising a plurality of isolated or partially purified freeze-thawed human functional mitochondria obtained from the same individual having said malignant disease or a healthy subject not having a malignant disease prior to anti-cancer treatment; (iii) (ii) freeze-thawed human functional mitochondria of said first composition of human stem cells and said second composition at 0.088-176mU CS activity/10⁶Proportional contact of individual stem cells; and (iv) subjecting said compound (iii) to(ii) incubating the composition under conditions that allow the human functional mitochondria to enter the freeze-thawed human stem cells, thereby enriching the human stem cells with the human functional mitochondria; wherein the functional mitochondrial content of the enriched human stem cells is detectably higher than the functional mitochondrial content of the human stem cells in the first composition.

In certain embodiments, the conditions that allow healthy functional human exogenous mitochondria to enter human stem cells comprise incubating the human stem cells with the healthy functional human exogenous mitochondria for a time in the range of 0.5 to 30 hours at a temperature in the range of 16 to 37 ℃. In certain embodiments, the conditions that allow healthy functional human exogenous mitochondria to enter human stem cells comprise incubating the human stem cells with the healthy functional human exogenous mitochondria in a culture medium at a temperature in the range of 16 to 37 ℃ for a time in the range of 0.5 to 30 hours in an environment that supports cell survival. According to certain embodiments, the medium is saline containing human serum albumin. In certain embodiments, the conditions for incubation include a 5% CO₂Of the atmosphere (c). In certain embodiments, the conditions for incubating do not include adding CO above the level present in air₂. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the method further comprises centrifuging the human stem cells and healthy functional exogenous mitochondria before, during, or after the incubating. In certain embodiments, the method further comprises a single centrifugation of the human stem cells and healthy functional human exo-source mitochondria at a centrifugal force above 2500xg prior to incubation. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the mitochondria that have undergone a freeze-thaw cycle exhibit a comparable rate of oxygen consumption after thawing as compared to a control mitochondria that has not undergone a freeze-thaw cycle.

In certain embodiments, the methods further comprise freezing the mitochondria-enriched human stem cells, and optionally further comprise thawing the mitochondria-enriched human stem cells.

In further embodiments, the human stem cells are expanded before or after mitochondrial enhancement.

The detectable enrichment of functional mitochondria by the stem cells can be determined by functional and/or enzymatic assays, including but not limited to oxygen (O)₂) Consumption rate, citrate synthase activity level, Adenosine Triphosphate (ATP) production rate, mitochondrial protein content (e.g. succinate dehydrogenase complex subunit a-SDHA and cytochrome C oxidase-COX 1), mitochondrial DNA content. In an alternative, enrichment of healthy donor mitochondria by the stem cells can be confirmed by detection of donor mitochondrial dna (mtdna). According to certain embodiments, the degree of enrichment of functional mitochondria by stem cells can be determined by the level of heterogeneity variation and/or by the mtDNA copy number per cell. According to certain exemplary embodiments, the enrichment of healthy, functional mitochondria by the stem cells can be determined by routine assays recognized in the art. For example, the presence of donor mitochondria can be determined by a method selected from: (i) the level of citrate synthase activity; or (ii) mtDNA sequencing indicates the source of more than one mtDNA. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, mitochondria may be matched according to mtDNA haplotypes between the donor and the subject being treated. According to other embodiments, mitochondria are selected according to a particular distinct mtDNA haplotype group prior to stem cell enrichment.

In certain embodiments, the mitochondrial content of the stem cells in the first or fourth composition is determined by determining the level of citrate synthase activity. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the process of enriching human stem cells for mitochondria is performed prior to freezing the cells. In other embodiments, the process of enriching human stem cells for mitochondria is performed after freezing and thawing the cells.

In certain embodiments, the autologous human stem cells are frozen and stored prior to suffering from the debilitating condition. In other embodiments, the process of enriching human stem cells for mitochondria is performed after freezing and thawing the cells.

In certain embodiments, the stem cell is a Pluripotent Stem Cell (PSC). In other embodiments, the PSC is a non-embryonic stem cell. In certain embodiments, the stem cell is an induced psc (ipsc). In certain embodiments, the stem cells are derived from bone marrow cells. In certain embodiments, the stem cell expresses the myeloid hematopoietic progenitor antigen CD34(CD 34)⁺). In certain embodiments, the stem cell is a mesenchymal stem cell. In other embodiments, the stem cells are derived from adipose tissue. In yet other embodiments, the stem cells are derived from blood. In other embodiments, the stem cells are derived from cord blood. In other embodiments, the stem cells are derived from the oral mucosa. In other embodiments, the stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cell is a bone marrow cell.

In certain embodiments, the stem cell is a bone marrow-derived stem cell comprising a myeloblast. In certain embodiments, the bone marrow-derived stem cells comprise erythropoietic cells. In certain embodiments, the bone marrow-derived stem cells comprise pluripotent Hematopoietic Stem Cells (HSCs). In certain embodiments, the bone marrow-derived stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow-derived stem cells comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, Natural Killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticulocytes, or any combination thereof. In certain embodiments, the bone marrow-derived stem cells comprise mesenchymal stem cells. Each possibility represents a separate embodiment of the invention.

In a particular embodiment, the stem cell is CD34⁺A cell. In certain embodiments, CD34 is expressed⁺Is obtained from cord blood (i.e., non-bone marrow hematopoietic stem cells). In certain embodiments, the cells used are autologous stem cells, and they can be frozen and stored prior to debilitating conditions associated with aging or cancer therapy. In certain embodiments, the process of enriching the cells for mitochondria is performed prior to freezing. In an alternative embodiment, the process of enriching cells for mitochondria is performed after freezing and thawing the stem cells.

In certain embodiments, the stem cells in the first composition are obtained from an aging subject or donor. In certain embodiments, the stem cells in the first composition are bone marrow cells obtained from bone marrow of an aging subject or donor. In certain embodiments, the stem cells in the first composition are obtained directly or indirectly from bone marrow of the aging subject or bone marrow of a donor. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of the aging subject or from the bone marrow of a donor. In certain embodiments, the stem cells in the first composition are obtained from peripheral blood of the aging subject or from peripheral blood of a donor. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells in the first composition are obtained from a subject having a malignant disease. In certain embodiments, the stem cells in the first composition are obtained from a subject having a non-hematopoietic malignant disease or a healthy subject not having a malignant disease. In certain embodiments, the stem cells in the first composition are obtained from bone marrow of a subject having a non-hematopoietic malignant disease or a healthy subject not having a malignant disease. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of the subject having a non-hematopoietic malignant disease, or from the bone marrow of a healthy subject not having a malignant disease. In certain embodiments, the stem cells in the first composition are obtained directly from bone marrow of the subject having a non-hematopoietic malignant disease, or are obtained directly from bone marrow of a healthy subject not having a malignant disease. In certain embodiments, the stem cells in the first composition are obtained indirectly from bone marrow of the subject having the non-hematopoietic malignant disease, or indirectly from bone marrow of a healthy subject not having the malignant disease. In certain embodiments, the bone marrow cells in the first composition are obtained from peripheral blood of the subject having a non-hematopoietic malignant disease, or from peripheral blood of a healthy subject not having a malignant disease. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells are at least partially purified.

In certain embodiments, the healthy functional mitochondria are derived from a cell or tissue selected from the group consisting of a placenta, placental cells grown in culture, and blood cells.

In certain embodiments, the pharmaceutical composition is administered to a subject suffering from a debilitating condition selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy. In other embodiments, the pharmaceutical composition is administered to a specific tissue or organ. In still other embodiments, the pharmaceutical composition is administered by systemic parenteral administration. In other embodiments, the pharmaceutical composition comprises at least about 10⁶Individual mitochondrially enriched human stem cells per kilogram patient body weight. In further embodiments, the pharmaceutical composition comprises a total of about 5x10⁵To 5x10⁹Human stem cells enriched in human mitochondria. In certain embodiments, administration of the pharmaceutical composition to a subject is by a parenteral route selected from intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, and direct injection into a tissue or organ. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the above methods further comprise a preliminary step comprising administering to a subject having a debilitating condition selected from the group consisting of an aging or non-hematopoietic malignancy or to a healthy donor an agent that induces mobilization of stem cells from bone marrow to peripheral blood. In certain embodiments, the agent is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), 1' - [1, 4-phenylenebis (methylene) ] bis [1,4,8, 11-tetraazacyclotetradecane ] (plexadifen), salts thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the above methods further comprise the step of isolating stem cells from peripheral blood of a subject having a debilitating condition selected from the group consisting of an aging or non-hematopoietic malignant disease, or from peripheral blood of a healthy subject. In certain embodiments, the separation is performed by apheresis.

In certain embodiments, the above methods further comprise the step of administering to a subject having a debilitating condition selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy an agent that prevents, delays, minimizes or eliminates an adverse immunogenic reaction between the subject and stem cells of the allogeneic donor. In additional embodiments, the functional mitochondria in the second composition are obtained from a subject having a malignant disease prior to an anti-cancer treatment.

In certain embodiments, the above methods further comprise concentrating the stem cells and functional mitochondria in the third composition prior to or during the incubating. In certain embodiments, the above methods further comprise centrifuging the third composition before, during, or after the incubating. Each possibility represents a separate embodiment of the invention.

In an alternative embodiment, the aging subject or a subject with one or more age-related diseases is transplanted with mitochondria-enriched stem cells. In certain embodiments, the stem cells are from a donor that does not have an age-related disease. In a particular embodiment, the stem cells are autologous bone marrow stem cells. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of an aging subject or a subject with one or more age-related diseases, or from a healthy donor who has never suffered an age-related disease. In certain embodiments, the stem cells in the first composition are obtained from the peripheral blood of the aging subject or a subject with one or more age-related diseases, or from the peripheral blood of a healthy donor who has not suffered from an age-related disease. Each possibility represents a separate embodiment of the invention.

In an alternative embodiment, the subject has a hematopoietic malignancy and the stem cells transplanted into the subject are enriched for mitochondria. In certain embodiments, the stem cells are from a healthy donor who does not have a malignant disease. In certain embodiments, the stem cells are autologous bone marrow stem cells, such as used in a variety of hematopoietic malignancies including multiple myeloma and certain types of lymphoma. According to these embodiments, the stem cells in the first composition are obtained from bone marrow of a subject having a hematopoietic malignancy or from bone marrow of a healthy subject not having a malignancy. In certain embodiments, the stem cells in the first composition are mobilized from the bone marrow of a subject having a hematopoietic malignancy, or from the bone marrow of a healthy subject not having a malignancy. In certain embodiments, the stem cells in the first composition are obtained from peripheral blood of a subject having a hematopoietic malignancy, or from peripheral blood of a healthy subject not having a malignancy. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the above methods further comprise a preliminary step comprising administering to the subject an agent that induces mobilization of bone marrow stem cells from bone marrow to peripheral blood. In certain embodiments, the agent is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), 1' - [1, 4-phenylenebis (methylene) ] bis [1,4,8, 11-tetraazacyclotetradecane ] (plexadifen), salts thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention. In certain embodiments, the above methods further comprise the step of isolating the stem cells from peripheral blood of a subject having a hematopoietic malignancy or from peripheral blood of a healthy subject not having a malignancy. In certain embodiments, the separation is performed by apheresis.

In certain embodiments, the above method further comprises concentrating the stem cells and functional mitochondria in composition (iii) prior to or during the incubation. In certain embodiments, the above method further comprises centrifuging composition (iii) before, during, or after the incubation. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells in the first composition are obtained from a subject having a debilitating condition selected from the group consisting of aging, an age-related disease, and a malignant disease undergoing a debilitating therapy, and have (i) reduced oxygen (O) compared to a subject not having the debilitating condition₂) A consumption rate; (ii) reduced levels of citrate synthase activity; (iii) a reduced rate of Adenosine Triphosphate (ATP) production; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells in the first composition are obtained from a healthy donor who has not suffered from a debilitating condition, have (i) normal oxygen (O)₂) A consumption rate; (ii) normal citrate synthase activity level; (iii) normal Adenosine Triphosphate (ATP) production rate; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention. In certain embodiments, the isolated or partially purified human functional mitochondria in the second composition are obtained from a donor who has no debilitating condition and has normal mitochondrial DNA. As used herein, the term "normal mitochondrial DNA" refers to mitochondrial DNA without any deletions or mutations known to be associated with a primary mitochondrial disease.

In certain embodiments, the stem cell enriched in healthy functional mitochondria has (i) increased oxygen (O) compared to the stem cell prior to mitochondrial enrichment₂) A consumption rate; (ii) increased level of citrate synthase activity; (iii) increased rate of Adenosine Triphosphate (ATP) production; (iv) increased normal mitochondrial DNA content; or (v) any combination of (i), (ii), (iii), and (iv). Each possibility represents a separate embodiment of the invention.

According to certain exemplary embodiments, the stem cell enriched for healthy functional mitochondria has (i) an increased level of citrate synthase activity as compared to the stem cell prior to mitochondrial enrichment; and (ii) increased normal mitochondrial DNA content.

In certain embodiments, the total amount of mitochondrial proteins in the partially purified mitochondria is between 20% and 80% of the total amount of cellular proteins in the sample. An exemplary method for obtaining such a composition of isolated or partially purified mitochondria is disclosed in WO 2013/035101.

In another aspect, the present invention also provides a plurality of healthy mitochondrially enriched human stem cells obtained by any of the above-described methods. It should be clearly understood that the functional mitochondria-enriched human stem cells according to the invention do not originate from a subject suffering from a primary mitochondrial disease. According to certain specific embodiments, the stem cells enriched for healthy mitochondria are not bone marrow stem cells.

In another aspect, the invention also provides a plurality of human stem cells enriched ex vivo with mitochondria, wherein said stem cells have at least one property selected from the group consisting of: (ii) an increased mitochondrial DNA content relative to the corresponding level in the stem cell prior to mitochondrial enrichment; (b) increased level of citrate synthase activity; (c) increased levels of at least one mitochondrial protein selected from SDHA and COX 1; (d) enhanced oxygen (O)₂) A consumption rate; (e) increased ATP production rate; or (f) any combination thereof. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the stem cell is CD34⁺A stem cell. The ex vivo enriched functional mitochondria human stem cells according to the invention do not originate from a subject with a primary mitochondrial disease.

In certain embodiments, the total amount of mitochondrial proteins in the partially purified mitochondria is between 20% and 80% of the total amount of cellular proteins in the sample.

In certain embodiments, the plurality of human stem cells is CD34⁺And has increased mitochondrial content, increased mitochondrial DNA content, as compared to stem cells prior to mitochondrial enrichmentOxygen (O)₂) Consumption rate, increased level of citrate synthase activity. In certain embodiments, the increased level or activity is greater than the level or activity in the cell when isolated.

In another aspect, the present invention also provides a pharmaceutical composition comprising a plurality of ex vivo human bone marrow stem cells enriched for healthy functional mitochondria as described above.

In another aspect, the invention also provides the above pharmaceutical composition for use in treating a human subject suffering from a debilitating condition. According to some embodiments, the subject having a debilitating condition is a subject of aging. In certain embodiments, the subject having a debilitating condition has an age-related disease or disorder. In certain embodiments, the subject having a debilitating condition has a malignant disease that undergoes debilitating therapy. In other embodiments, the above pharmaceutical composition is used in a human subject in remission or after recovery from a malignant disease.

In another aspect, the invention also provides a method of treating a human subject suffering from a debilitating condition, said method comprising the step of administering to said subject a pharmaceutical composition as described above. According to some embodiments, the subject having a debilitating condition is a subject of aging. In certain embodiments, the subject having a debilitating condition has one or more age-related diseases. In certain embodiments, the subject having a debilitating condition has a malignant disease that undergoes debilitating therapy. In other embodiments, the above pharmaceutical composition is used to treat a human subject in remission or after recovery from a malignant disease. In certain embodiments, the pharmaceutical composition comprises stem cells that are autologous or syngeneic to the subject with the debilitating condition. In certain embodiments, the pharmaceutical composition comprises stem cells that are allogeneic to the subject having the debilitating condition. Each possibility represents a separate embodiment of the invention.

Further embodiments and a full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

Figure 1 is three micrographs obtained by fluorescence confocal microscopy showing mouse fibroblasts expressing mitochondrial GFP (left panel), mouse fibroblasts incubated with isolated RFP-labeled mitochondria (middle panel), and overlay (right panel).

Figure 2 is a bar graph showing a comparison of ATP levels in mouse fibroblasts untreated (control), treated with mitochondrial complex I irreversible inhibitor (rotenone), or treated with rotenone and mouse placental mitochondria (rotenone + mitochondria). Data are presented as mean ± SEM. (x) p value < 0.05. RLU-relative luminescence units.

FIG. 3 is four photomicrographs obtained by fluorescence confocal microscopy showing mouse bone marrow cells incubated with GFP-labeled mitochondria isolated from mouse myeloma cells.

Figure 4 is a bar graph showing the level of C57BL mtDNA in the bone marrow of FVB/N mice at various time points post IV injection of bone marrow cells enriched for exogenous mitochondria from C57BL mice.

FIG. 5 is a bar graph showing a comparison of Citrate Synthase (CS) activity in centrifuged or non-centrifuged mouse Bone Marrow (BM) cells incubated with different amounts of GFP-labeled mitochondria isolated from mouse myeloma cells.

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 cytochrome C reductase activity in these cells (black bars) compared to activity in GFP-labeled mitochondria (grey bars).

Fig. 7A is a bar graph showing copy number of C57BL mtDNA in FVB/N bone marrow cells after incubation with various concentrations (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 milliunits CS activity) of exogenous mitochondria from C57BL mice compared to untreated cells (NTs). Figure 7B is a bar graph showing the level of mtDNA-encoded (COX1) protein in FVB/N bone marrow cells after incubation with various concentrations (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 milliunits of CS activity) of exogenous mitochondria from C57BL mice, normalized to Janus levels, compared to untreated cells (NTs). Figure 7C is a bar graph showing the amount of nuclear-encoded (SDHA) protein in FVB/N bone marrow cells normalized to Janus levels after incubation of the cells with various concentrations (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 milliunits of CS activity) of exogenous mitochondria from C57BL mice compared to untreated cells (NTs).

Figure 8A is a bar graph showing a comparison of CS activity in control untreated human BM cells and centrifuged or non-centrifuged human BM cells incubated with GFP-labeled mitochondria isolated from human placental cells. Figure 8B is a bar graph showing ATP levels in control untreated human BM cells and centrifuged human BM cells incubated with GFP-labeled mitochondria isolated from human placental cells.

Figure 9A depicts the results of FACS analysis in human BM cells that were not incubated with GFP-labeled mitochondria. Figure 9B depicts the results of FACS analysis in human BM cells incubated with GFP-labeled mitochondria after centrifugation.

FIG. 10A is a bar graph showing human CD34 from healthy donors untreated (NT) or treated with blood-derived mitochondria (MNV-BLD)⁺ATP content of the cells. FIG. 10B is a bar graph showing human CD34 from a healthy donor treated with or without blood-derived mitochondria⁺CS activity of the cell.

FIG. 11 is three micrographs obtained by fluorescence confocal microscopy showing CD34+ cells incubated with GFP-labeled mitochondria isolated from HeLa-TurboGFP-mitochondrial cells.

FIG. 12A is a graphical illustration of mtDNA deletion in cord blood cells of a patient with Pearson's syndrome and a southern blot analysis showing the deletion. Figure 12B is a bar graph showing human mtDNA copy number in bone marrow of NSGS mice 2 months after mitochondrial-enhanced therapy with human mitochondrial enriched cord blood cells of pearson syndrome (UCB + Mito) compared to mice injected with non-enhanced cord blood cells (UCB).

Figure 13A is a bar graph showing the level of FVB/N ATP8 mutant mtDNA in the bone marrow of FVB/N mice 1 month after administration of stem cells enriched with healthy functional mitochondria obtained from the C57BL placenta. Figure 13B is a bar graph showing the levels of FVB/N ATP8 mutant mtDNA in the liver of FVB/N mice at 3 months after administration of stem cells enriched with healthy functional mitochondria obtained from the C57BL placenta.

Fig. 14A-14C are bar graphs illustrating the biodistribution of mitochondria-enriched bone marrow cells by the amount of C57BL mtDNA in the bone marrow (fig. 14A), brain (fig. 14B), and heart (fig. 14C) of mice up to 3 months after MAT. White bars and accompanying dots indicate enhanced bone marrow samples, gray bars are controls.

FIG. 15 is a bar graph showing the results in untreated FVB/N mice (naive)) Comparison of FVB/N ATP8 mutated mtDNA levels in the brain of FVB/N mice 1 month after administration of stem cells enriched with healthy wild-type mitochondria (isolated from the liver of C57BL mice), in FVB/N mice (TBI C57BL Mito) administered stem cells enriched with healthy C57BL mitochondria and systemically irradiated (TBI) prior to administration of stem cells, and FVB/N mice (busulfan C57BL Mito) administered stem cells enriched with healthy, functional, wild-type mitochondria (isolated from the liver of C57BL mice) administered stem cells enriched with healthy, functional, healthy, liver mitochondria, and administered stem cells that received busulfan chemotherapeutic agents prior to administration of stem cells.

Figures 16A-16C show line graphs illustrating the open field behavior test performance of 12-month old C57BL/6J mice treated with the following substances before and 9 months after treatment: mitochondria-enriched BM cells (MNV-BM-PLC, 1X 10)⁶Individual cell) Bone marrow cells (BM control, 1X 10)⁶Individual cells) or control medium solution (control, 4.5% albumin in 0.9% w/v NaCl). Fig. 16A shows the quantification of the distance moved during the open field test. FIG. 16B shows the center duration (% of time(s) or change from baseline); fig. 16C shows the peripheral wall duration (% time(s) or change from baseline).

Fig. 16D is a line graph illustrating Blood Urea Nitrogen (BUN) levels in 12-month old C57BL/6J mice treated with the following substances before and 9 months after treatment: mitochondria-enriched BM cells (MNV-BM-PLC, 1X 10)⁶Individual cells), bone marrow cells (BM control, 1x 10)⁶Individual cells) or control medium solution (control, 4.5% albumin in 0.9% w/v NaCl).

FIGS. 16E-16F show bar graphs illustrating Bone Marrow (BM) cells enriched with mitochondria (MNV-BM-PLC, 1x 10)⁶Individual cell), bone marrow cell (BM, 1x 10)⁶Individual cells) or control vehicle solution (vehicle, 4.5% albumin in 0.9% w/v NaCl) 12-month old C57BL/6J mice. Results are presented before and 1 and 3 months after treatment. Figure 16E shows the rotarod scores (in seconds (s)) for each of the different treatment trial groups at the indicated time points. Figure 16F shows the rotarod scores (presented as a percentage of each different treatment trial group compared to baseline) for each different treatment trial group at the indicated time points.

FIGS. 16G-16J show bar graphs illustrating Bone Marrow (BM) cells enriched with mitochondria (MNV-BM-PLC, 1x 10)⁶Individual cell), bone marrow cell (BM, 1x 10)⁶Individual cells) or control vehicle solution (vehicle, 4.5% albumin in 0.9% w/v NaCl) 12-month old C57BL/6J mice. Results are presented before and 1 and 3 months after treatment. FIGS. 16G-16H-grip strength (% change in G or compared to baseline); FIGS. 16I-16J-grip time (% change in time(s) or compared to baseline) (%).

Fig. 17A is a planned graph depicting the course of treatment and assessment in a clinical trial on patient 1, a young patient with Pearson Syndrome (PS) and PS-associated Fanconi Syndrome (FS), with a deletion mutation in their mtDNA encompassing ATP 8. Figure 17B is a bar graph showing aerobic task Metabolic Equivalent (MET) scores before, 2.5 months, and 8 months after administration of stem cells enriched in functional mitochondria. Fig. 17C is a bar graph illustrating lactate levels in the blood of PS patients treated by the methods provided in the present invention before and after therapy as a function of time. Figure 17D is a line graph illustrating the standard deviation scores for weight and height of PS patients treated by the methods provided in the present invention before and after therapy as a function of time. Fig. 17E is a line graph illustrating the change over time in alkaline phosphatase (ALP) levels in PS patients treated by the methods provided in the invention before and after therapy. Fig. 17F is a line graph illustrating the long-term elevation of blood Red Blood Cell (RBC) levels in PS patients before and after the therapy provided by the present invention. Fig. 17G is a line graph illustrating the long-term elevation of blood Hemoglobin (HGB) levels in PS patients before and after the therapy provided by the present invention. Fig. 17H is a line graph illustrating the long-term increase in blood Hematocrit (HCT) levels in PS patients before and after the therapy provided by the present invention. Fig. 17I is a line graph illustrating the change over time of creatinine levels in PS patients treated by the methods provided in the present invention before and after therapy. Fig. 17J is a line graph illustrating the bicarbonate levels over time for PS patients treated by the methods provided in the present invention before and after therapy. Fig. 17K is a line graph illustrating the change over time of base excess levels in PS patients treated by the methods provided in the present invention before and after therapy. Fig. 17L is a bar graph illustrating the change over time in blood magnesium levels in PS patients treated by the methods provided in the present invention before and after therapy, before and after magnesium supplementation. Figure 17M is a bar graph illustrating the change over time in the glucose to creatinine ratio in urine of PS patients treated by the methods provided in the present invention before and after therapy. Figure 17N is a bar graph illustrating the change over time in the potassium to creatinine ratio in urine of PS patients treated by the methods provided in the present invention before and after therapy. Figure 17O is a bar graph illustrating the change over time in the chloride to creatinine ratio in urine of PS patients treated by the methods provided in the present invention before and after therapy. Figure 17P is a bar graph illustrating the sodium to creatinine ratio in urine over time for PS patients treated by the methods provided in the present invention before and after therapy.

Fig. 18A is a line graph showing the change in the amount of normal mtDNA before and after therapy in 3 PS patients (pt.1, pt.2 and pt.3) treated by the method provided in the present invention, measured for the deletion region (in each patient) by digital PCR, compared to 18S genomic DNA representing the normal mtDNA amount per cell, and normalized according to baseline.

Fig. 18B is a line graph showing the level of heterogeneity (presence of deleted mtDNA compared to total mtDNA) in 3 PS patients (pt.1, pt.2, and pt.3) at baseline after MAT. The dashed line represents the baseline for each patient.

FIG. 19A is another plan view of different stages of treatment for a patient with Pearson Syndrome (PS) further provided by the present invention. Fig. 19B is a bar graph showing the variation of lactate levels in the blood over time before (B) and after therapy in PS patients treated by the methods provided in the present invention. Fig. 19C is a bar graph showing the change in sitting trial score over time before and after therapy for PS patients treated by the methods provided in the present invention. Fig. 19D is a bar graph showing the 6 minute walk test score of PS patients treated by the methods provided in the present invention as a function of time before and after therapy. Figure 19E is a bar graph showing the ergometer score of three consecutive replicates (R1, R2, R3) of PS patients treated by the methods provided in the present invention as a function of time before and after therapy. Figure 19F is a bar graph showing the change in urinary magnesium to creatinine ratio over time before and after therapy in PS patients treated by the methods provided in the present invention. Fig. 19G is a bar graph showing the change in urinary potassium to creatinine ratio over time before and after therapy in PS patients treated by the methods provided in the present invention. Fig. 19H is a bar graph showing the change in urinary calcium to creatinine ratio over time before and after therapy in PS patients treated by the methods provided in the present invention. Fig. 19I is a bar graph showing the ratio of ATP8 to 18S copy number in urine of PS patients treated by the methods provided in the present invention as a function of time before and after therapy. Fig. 19J is a bar graph showing ATP levels in lymphocytes of PS patients treated by the methods provided in the invention as a function of time before and after therapy.

FIG. 20A is yet another plan view of different stages of treatment for a patient with Pearson Syndrome (PS) and a patient with Kearns-Sayre syndrome (KSS) further provided by the present invention. Fig. 20B is a bar graph illustrating the variation of lactate levels in the blood over time before (B) and after therapy in PS patients treated by the methods provided in the present invention. Fig. 20C is a bar graph showing the AST and ALT levels over time before and after therapy for PS patients treated by the methods provided in the present invention. Fig. 20D is a bar graph showing the change in triglyceride, total cholesterol, and VLDL cholesterol levels over time before and after therapy in PS patients treated by the methods provided in the present invention. Fig. 20E is a bar graph showing the change in hemoglobin A1C (HbA1C) score over time before and after therapy for PS patients treated by the methods provided in the present invention. Fig. 20F is a line graph showing the change in sitting trial score over time before and after therapy for PS patients (pt.3) treated by the methods provided herein. Fig. 20G is a line graph showing the change in 6-minute walk test score before and after therapy for PS patients (pt.3) treated by the methods provided herein over time.

FIG. 21 is a bar graph showing ATP levels in peripheral blood of KSS patients treated by methods provided herein before and after therapy.

Detailed Description

The present invention provides cell platforms, more specifically stem cell-derived cell platforms, for the targeted and systemic delivery of significant amounts of fully functional healthy mitochondria and methods of using them in subjects with debilitating conditions, including aging subjects and subjects with one or more age-related diseases and cancer patients with the sequelae of anti-cancer therapy including chemotherapy, radiation therapy or immunotherapy using monoclonal antibodies. The present invention is based on several surprising findings, including the clinical results exemplified herein, which show that intravenous injection of bone marrow-derived hematopoietic stem cells enriched in normal, functional, healthy mitochondria can beneficially affect various tissues of a subject. In other words, functional improvement can be achieved in a variety of different organs and tissues following administration of stem cells enriched in healthy mitochondria.

The present invention is based in part on the finding that bone marrow cells are susceptible to enrichment with intact functional mitochondria, and that human bone marrow cells are particularly susceptible to enrichment with mitochondria, as disclosed in, for example, WO 2016/135723. Without being bound by any theory or mechanism, it is speculated that co-incubation of stem cells with healthy mitochondria promotes the transfer of intact functional mitochondria into stem cells.

It has also been found that the extent to which stem cells, including but not limited to bone marrow derived hematopoietic stem cells, are enriched for mitochondria and the improvement in mitochondrial function of the cells depends on the conditions used for mitochondrial enrichment, including but not limited to the concentration of isolated or partially purified mitochondria and the incubation conditions, and thus can be manipulated in order to produce the desired enrichment.

In one aspect, the invention provides a method of treating and/or alleviating the debilitating effects of a variety of different disorders, said method comprising ex vivo introducing partially purified healthy human mitochondria into stem cells obtained from or derived from a subject or healthy donor suffering from a debilitating disorder, and transplanting the "mitochondria-enriched" stem cells into said subject suffering from a debilitating disorder.

In certain embodiments, the subject having a debilitating condition has aging or one or more age-related diseases. In other embodiments, the subject with debilitating effects is a cancer patient undergoing chemotherapy, radiation therapy, or immunotherapy with monoclonal antibodies. In certain embodiments, the cancer patient is a subject having a non-hematopoietic malignant disease. In other embodiments, the cancer patient is a subject having a hematopoietic malignancy.

In other embodiments, the human stem cells administered to a subject are autologous to the subject. In other embodiments, the human stem cells administered to a subject are from a donor, i.e., are allogeneic to the subject.

In certain embodiments, the autologous or allogeneic human stem cells are Pluripotent Stem Cells (PSCs) or induced pluripotent stem cells (ipscs). In other embodiments, the autologous or allogeneic human stem cells are mesenchymal stem cells.

According to several embodiments, the human stem cells are derived from adipose tissue, oral mucosa, blood, umbilical cord blood or bone marrow. Each possibility represents a separate embodiment of the invention. In certain embodiments, the human stem cells are derived from bone marrow.

In another aspect, the invention provides a pharmaceutical composition for treating or alleviating a debilitating condition in a subject, said pharmaceutical composition comprising at least 10⁵To 2x10⁷Human stem cells suspended in a pharmaceutically acceptable liquid medium capable of supporting the survival of said cells per kilogram of body weight of a subject, wherein said human stem cells are enriched in freeze-thawed, healthy, functional, exogenous mitochondria, and wherein said debilitating condition is selected from the group consisting of aging, age-related diseases, and sequelae of anti-cancer therapy.

In certain embodiments, the pharmaceutical composition comprises at least 10⁵To 2x10⁷Individual mitochondrially enriched human stem cells per kilogram patient body weight. In certain embodiments, the pharmaceutical composition comprises at least 5x10⁵To 1.5x10⁷Individual mitochondrially enriched human stem cells per kilogram patient body weight. In certain embodiments, the pharmaceutical composition comprises at least 5x10⁵To 4x10⁷Individual mitochondrially enriched human stem cells per kilogram patient body weight. In certain embodiments, the pharmaceutical composition comprises at least 10⁶To 10⁷Mitochondrially enriched human stem cells/thousandThe weight of the patient is gram. In other embodiments, the pharmaceutical composition comprises at least 10⁵Or at least 10⁶Individual mitochondrially enriched human stem cells per kilogram patient body weight. Each possibility represents a separate embodiment of the invention. In certain embodiments, the pharmaceutical composition comprises at least 5x10 in total⁵Up to 5x10⁹Individual mitochondrially enriched human stem cells. In certain embodiments, the pharmaceutical composition comprises a total of at least 10⁶Up to 10⁹Individual mitochondrially enriched human stem cells. In other embodiments, the pharmaceutical composition comprises at least 2x10 in total⁶Up to 5x10⁸Individual mitochondrially enriched human stem cells.

In another aspect, the present invention provides an ex vivo method of enriching human stem cells for functional mitochondria, the method comprising the steps of: (i) providing a first composition comprising a plurality of human stem cells obtained or derived from a subject having a debilitating condition or a healthy donor not having a debilitating condition; (ii) providing a second composition comprising a plurality of isolated or partially purified human functional mitochondria obtained from a healthy donor who has not suffered a debilitating condition; (iii) contacting the human stem cells of the first composition with the human functional mitochondria of the second composition, thereby forming a third composition; and (iv) incubating the third composition under conditions that allow the human functional mitochondria to enter the human stem cells, thereby enriching the human stem cells for the human functional mitochondria, thereby forming a fourth composition; wherein the mitochondrial content of the enriched human stem cells in the fourth composition is detectably higher than the mitochondrial content of the human stem cells in the first composition.

In one aspect, the present invention provides an ex vivo method of enriching human bone marrow cells for functional mitochondria, said method comprising the steps of: (i) providing a first composition comprising a plurality of human bone marrow cells obtained or derived from a patient having a malignant disease or a healthy subject not having a malignant disease; (ii) providing a second composition comprising a plurality of isolated human functional mitochondria obtained from the same patient having a malignant disease prior to an anti-cancer treatment or from a healthy subject not having a malignant disease; (iii) mixing the human bone marrow cells of the first composition with the human functional mitochondria of the second composition, thereby forming a third composition; and (iv) incubating the third composition under conditions that allow the human functional mitochondria to enter the human bone marrow cells, thereby enriching the human bone marrow cells for the human functional mitochondria, thereby forming a fourth composition; wherein the mitochondrial content of human bone marrow cells in the fourth composition is detectably higher than the mitochondrial content of human bone marrow cells in the first composition.

As used herein, the term "ex vivo method" refers to any method that includes steps that are performed exclusively outside the human body. In particular, ex vivo methods involve manipulation of cells in vitro, which are subsequently reintroduced or transplanted into the subject to be treated.

As used herein, the term "enrichment" refers to any action designed to increase the mitochondrial content of mammalian cells, such as the number of intact mitochondria or the function of mitochondria. In particular, a stem cell enriched for functional mitochondria will exhibit enhanced function compared to the same stem cell prior to enrichment.

As used herein, the term "stem cell" generally refers to any mammalian stem cell. Stem cells are undifferentiated cells that can differentiate into other types of cells and can divide to produce more stem cells of the same type. The stem cells may be totipotent or pluripotent.

As used herein, the term "human stem cell" generally refers to all stem cells naturally occurring in humans, as well as all stem cells produced or derived ex vivo and compatible with humans. Like stem cells, "progenitor cells" have a tendency to differentiate into a particular type of cell, but are already more specific than stem cells and are pushed to differentiate into its "target" cells. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, while progenitor cells can divide only a limited number of times. As used herein, the term "human stem cell" also includes "progenitor cells" and "stem cells that are not fully differentiated.

In certain embodiments, the enriching of the stem cells with healthy functional human exogenous mitochondria comprises washing the mitochondria-enriched stem cells after incubating the human stem cells with the healthy functional human exogenous mitochondria. This step provides a composition of mitochondrially enriched stem cells that is substantially free of cell debris or mitochondrial membrane residues and mitochondria that do not enter the stem cells. In certain embodiments, washing comprises centrifuging the mitochondrially enriched stem cells after incubating the human stem cells with the healthy functional human exo-source mitochondria. According to some embodiments, the pharmaceutical composition comprising the mitochondrially enriched human stem cell is separated from free mitochondria, i.e. mitochondria or other cell debris that has not entered the stem cell. According to certain embodiments, the pharmaceutical composition comprising mitochondrially enriched human stem cells does not comprise a detectable amount of free mitochondrion.

As used herein, the term "Pluripotent Stem Cell (PSC)" refers to a cell that is capable of immortalizing and producing multiple cell types in the body. Totipotent stem cells are cells that can give rise to every other cell type in the body. Embryonic Stem Cells (ESC) are totipotent stem cells, and Induced Pluripotent Stem Cells (iPSC) are pluripotent stem cells.

As used herein, the term "Induced Pluripotent Stem Cell (iPSC)" refers to a class of pluripotent stem cells that can be generated from human adult somatic cells.

As used herein, the term "Embryonic Stem Cell (ESC)" refers to a type of totipotent stem cell derived from the internal cell mass of the blastocyst.

As used herein, the term "bone marrow cells" generally refers to all human cells that naturally occur in human bone marrow, and refers to all cell populations that naturally occur in human bone marrow. The terms "bone marrow stem cells" and "bone marrow-derived stem cells" refer to a population of stem cells derived from bone marrow.

The terms "functional mitochondria" and "are useful in the treatment of diseases associated with the disease"healthy mitochondria" is used interchangeably herein and refers to mitochondria that exhibit parameters indicative of normal mtDNA and normal, non-pathological levels of activity. Mitochondrial activity can be measured by various methods known in the art, e.g., membrane potential, O₂Consumption, ATP production and Citrate Synthase (CS) activity level.

As used herein, the phrase "stem cells obtained from a subject having a debilitating condition or a donor not having a debilitating condition" refers to cells that, when isolated from a subject, are stem cells in the subject/donor.

As used herein, the phrase "stem cells derived from a subject having a debilitating condition or a donor not having a debilitating condition" refers to cells that are not stem cells in the subject/donor and have been manipulated to become stem cells. As used herein, the term "manipulation" refers to reprogramming somatic cells to an undifferentiated state and into induced pluripotent stem cells (iPSc) using any of the methods known in the art (Yu j. et al, Science,2007, vol.318(5858), pp 1917-1920), and optionally further reprogramming the ipscs to become cells of a desired lineage or population (Chen m. et al, IOVS,2010, vol.51(11), pp 5970-5978), such as bone marrow cells (Xu y. et al, 2012, PLoS ONE, vol.7(4), pp e 34321).

As used herein, the term "CD 34⁺By cells is meant hematopoietic stem cells characterized as being positive for CD34, obtained from stem cells or mobilized from bone marrow or obtained from cord blood.

As used herein, the term "subject having a debilitating condition" refers to a human subject that experiences the debilitating effects caused by certain conditions. The debilitating conditions may refer to aging, age-related diseases, or cancer patients undergoing anti-cancer therapy, among other debilitating conditions.

The term "aging" refers to the inevitable progressive deterioration of physiological function with age, which is characterised demographically by an age-dependent increase in mortality and a decline in various physical and mental capacities.

As used herein, the term "age-related disease" refers to "geriatric disease," a disease that occurs with increasing frequency with aging. Age-related diseases include, but are not limited to, atherosclerosis and cardiovascular disease, cancer, arthritis, cataracts, osteoporosis, type 2 diabetes, hypertension, and dementia disorders such as alzheimer's disease. The incidence of all these diseases increases gradually with age.

As used herein, the term "subject having a malignant disease" refers to a human subject diagnosed as having a malignant disease, suspected of having a malignant disease, or in a risk group for developing a malignant disease. Since certain types of malignancies are inherited, the offspring of subjects diagnosed with a malignant disease are considered to be a risk group for developing a malignant disease.

As used herein, the term "subject/donor not suffering from a malignant disease" refers to a human subject that has not been diagnosed as suffering from a malignant disease and/or is not suspected of suffering from a malignant disease.

As used herein, the term "subject having a non-hematopoietic malignant disease" refers to a human subject diagnosed as having a non-hematopoietic malignant disease and/or suspected of having a non-hematopoietic malignant disease.

As used herein, the term "subject having a hematopoietic malignant disease" refers to a human subject diagnosed as having a hematopoietic malignant disease and/or suspected of having a hematopoietic malignant disease.

The terms "healthy donor" and "healthy subject" are used interchangeably and refer to a subject that does not have the disease or disorder to be treated.

The term "contacting" refers to bringing the composition of mitochondria and cells into sufficient proximity to facilitate entry of the mitochondria into the cells. The term "introducing mitochondria into a target cell" can be used interchangeably with the term "contacting".

As used herein, the term "isolated or partially purified human functional mitochondria" refers to intact mitochondria isolated from cells obtained from a healthy subject not suffering from a mitochondrial disease. The total amount of mitochondrial proteins in the partially purified mitochondria is between 20% and 80% of the total amount of cellular proteins in the sample.

The term "isolated" when used herein and in the claims in the context of mitochondria includes mitochondria that are at least partially purified from other components present in the source. In certain embodiments, the total amount of mitochondrial proteins in the second composition comprising a plurality of isolated healthy, functional, exogenous mitochondria is between 20% -80%, 20-70%, 40-70%, 20-40%, or 20-30% of the total amount of cellular proteins in the sample. Each possibility represents a separate embodiment of the invention. In certain embodiments, the total amount of mitochondrial proteins in the second composition comprising a plurality of isolated healthy, functional, exogenous mitochondria is between 20% and 80% of the total amount of cellular proteins in the sample. In certain embodiments, the total amount of mitochondrial proteins in the second composition comprising a plurality of isolated healthy, functional, exogenous mitochondria is between 20% and 80% of the combined weight of the mitochondria and other subcellular fractions. In other embodiments, the total amount of mitochondrial proteins in the second composition comprising a plurality of isolated healthy functional exogenous mitochondria is greater than 80% of the combined weight of the mitochondria and other subcellular fractions.

According to some embodiments, the method for enriching a human stem cell for healthy, functional exogenous mitochondria does not comprise measuring the membrane potential of the cell.

In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.044 up to 176 milliunits CS activity per million cells into the stem cell. In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.088 up to 176 milliunits CS activity per million cells into the stem cell. In other embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.2 up to 150 milliunits CS activity per million cells into the stem cell. In other embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.4 up to 100 milliunits CS activity per million cells into the stem cell. In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.6 up to 80 milliunits CS activity per million cells into the stem cell. In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.7 up to 50 milliunits CS activity per million cells into the stem cell. In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.8 up to 20 milliunits CS activity per million cells into the stem cell. In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.88 up to 17.6 milliunits CS activity per million cells into the stem cell. In certain embodiments, enriching the stem cell for healthy functional exogenous mitochondria comprises introducing a dose of mitochondria of at least 0.44 up to 17.6 milliunits CS activity per million cells into the stem cell.

Mitochondrial dose can be expressed in terms of units of CS activity or mtDNA copy number or other quantifiable measure of the amount of healthy, functional mitochondria as explained herein. "units of CS activity" is defined as the amount of substrate that is capable of converting 1 micromole in 1 minute in a 1mL reaction volume.

In certain embodiments, the identification/discrimination of the endogenous mitochondria from the exogenous mitochondria after they have been introduced into the target cell can be performed by a variety of different means, including, for example, but not limited to: identifying differences in mtDNA sequence between the endogenous and exogenous mitochondria, e.g., a different haplotype, identifying a particular mitochondrial protein originating from the source tissue of the exogenous mitochondria, e.g., cytochrome P450 cholesterol side chain cleavage (P450SCC) from placenta, UCP1 from brown adipose tissue, etc., or any combination thereof.

The term "exogenous" with respect to mitochondria refers to mitochondria that are introduced into a target cell (e.g., a stem cell) from a source outside the cell. For example, in certain embodiments, the exogenous mitochondria are typically derived or isolated from a donor cell that is different from the target cell. For example, exogenous mitochondria can be produced/manufactured in a donor cell, purified/isolated/obtained from the donor cell, and subsequently introduced into the target cell.

The term "endogenous" with respect to mitochondria refers to mitochondria that are made/expressed/produced by a cell and are not introduced into the cell from an external source. In certain embodiments, the endogenous mitochondria contain proteins and/or other molecules encoded by the genome of the cell. In certain embodiments, the term "endogenous mitochondria" is equivalent to the term "host mitochondria".

As used herein, the term "autologous cells" or "autologous cells" refers to the patient's own cells. The term "autologous mitochondria" refers to mitochondria obtained from the patient's own cells or cells associated with the maternal line. The term "allogeneic cell" or "allogeneic mitochondria" refers to cells from different donor individuals.

The term "syngeneic" as used herein and in the claims refers to genetic identity or genetic near identity sufficient to allow for transplantation between individuals without rejection. In the case of mitochondria, the term "syngeneic" is used interchangeably herein with the term "autologous mitochondria" meaning the same maternal blood system.

The term "exogenous mitochondria" refers to mitochondria or mitochondrial DNA introduced into a target cell (i.e., a stem cell) from a source outside the cell. For example, in certain embodiments, the exogenous mitochondria can be derived or isolated from a cell different from the target cell. For example, exogenous mitochondria can be produced/manufactured in a donor cell, purified/isolated/obtained from the donor cell, and subsequently introduced into the target cell.

As used herein, the phrase "conditions that allow the human functional mitochondria to enter the human stem cells" generally refers to parameters such as time, temperature, culture medium, and proximity between the mitochondria and stem cells, among othersAnd (4) counting. For example, human cells and human cell lines are routinely incubated in liquid media and maintained in a sterile environment, e.g., in a tissue culture incubator, at 37 ℃ and 5% CO₂Under an atmosphere. According to alternative embodiments disclosed and exemplified herein, the cells may be incubated at room temperature in saline supplemented with human serum albumin. According to some embodiments, the human functional mitochondria are centrifuged prior to incubation with human stem cells. According to other embodiments, the incubation occurs prior to centrifugation. In still other embodiments, centrifugation occurs during the incubation. In certain embodiments, the speed of centrifugation is 8,000 g. In certain embodiments, the speed of centrifugation is 7,000 g. According to other embodiments, the centrifugation is performed at a speed between 5,000 and 10,000 g. According to other embodiments, the centrifugation is performed at a speed between 7,000 and 8,000 g.

In certain embodiments, the human stem cells are incubated with the healthy, functional, exogenous mitochondria at a temperature in the range of about 16 to about 37 ℃ for a time in the range of 0.5 to 30 hours. In certain embodiments, the human stem cells are incubated with the healthy, functional, exogenous mitochondria for a time in the range of 1 to 30 or 5 to 25 hours. Each possibility represents a separate embodiment of the invention. In a particular embodiment, the incubation is performed for 20 to 30 hours. In certain embodiments, the incubation is performed for at least 1, 5, 10, 15, or 20 hours. Each possibility represents a separate embodiment of the invention. In other embodiments, the incubation is performed for up to 5, 10, 15, 20, or 30 hours. Each possibility represents a separate embodiment of the invention. In a particular embodiment, the incubation is performed for 24 hours. In certain embodiments, the incubation is performed at room temperature (16 ℃ to 30 ℃). In other embodiments, the incubation is performed at 37 ℃. In certain embodiments, the incubation is at 5% CO₂Is carried out in an atmosphere. In other embodiments, the incubating does not include adding CO above the level present in air₂. In certain embodiments, the incubation is carried out until the mitochondrial content in the stem cells is increased by an average of 1% to 45% compared to their initial mitochondrial content.

In still other embodiments, the incubation is performed in medium supplemented with Human Serum Albumin (HSA). In a further embodiment, the incubation is performed in saline supplemented with HSA. According to certain exemplary embodiments, the conditions that allow functional mitochondria to enter human stem cells, thereby enriching the human stem cells for the functional mitochondria of humans, comprise incubation at room temperature in saline supplemented with 4.5% human serum albumin.

By manipulating the conditions of the incubation, one can manipulate the characteristics of the product. In certain embodiments, the incubation is performed at 37 ℃. In certain embodiments, the incubation is performed for at least 6 hours. In certain embodiments, the incubation is performed for at least 12 hours. In certain embodiments, the incubation is performed for 12 to 24 hours. In certain embodiments, the incubation is 1x10 in an amount of exogenous mitochondria that have or exhibit 4.4 milliunits CS per serving⁵To 1X10⁷The proportion of primary stem cells. In certain embodiments, the incubation is 1x10 in an amount of exogenous mitochondria that have or exhibit 4.4 milliunits CS per serving⁶The proportion of primary stem cells. In certain embodiments, the conditions are sufficient to increase mitochondrial content of the naive stem cell as determined by CS activity by at least about 3%, 5%, or 10%. Each possibility represents a separate embodiment of the invention.

As used herein, the term "mitochondrial content" refers to the amount of functional mitochondria within a cell or the average amount of functional mitochondria within a plurality of cells.

As used herein and in the claims, the term "mitochondrial disease" and the term "primary mitochondrial disease" are used interchangeably. As used herein, the term "primary mitochondrial disease" refers to a mitochondrial disease that is diagnosed by known or undisputed pathogenic mutations in the mitochondrial DNA or by mutations in the genes of the nuclear DNA whose gene products are imported into the mitochondria. According to some embodiments, the primary mitochondrial disease is a congenital disease. According to certain embodiments, the primary mitochondrial disease is not secondary mitochondrial dysfunction. The terms "secondary mitochondrial dysfunction" and "acquired mitochondrial dysfunction" are used interchangeably throughout this application.

In certain embodiments, the methods described above in the various embodiments further comprise centrifuging before, during, or after incubating the stem cells with the exogenous mitochondria. Each possibility represents a separate embodiment of the invention. In certain embodiments, the methods described above in the various embodiments comprise a single centrifugation step before, during, or after incubating the stem cells with the exogenous mitochondria. In certain embodiments, the centrifugal force is in the range of 1000g to 8500 g. In certain embodiments, the centrifugal force is in the range of 2000g to 4000 g. In certain embodiments, the centrifuge force is greater than 2500 g. In certain embodiments, the centrifugal force is in the range of 2500g to 8500 g. In certain embodiments, the centrifugal force is in the range of 2500g to 8000 g. In certain embodiments, the centrifugal force is in the range of 3000g to 8000 g. In other embodiments, the centrifugal force is in the range of 4000g to 8000 g. In a particular embodiment, the centrifugal force is 7000 g. In other embodiments, the centrifugal force is 8000 g. In certain embodiments, centrifugation is performed for a time in the range of 2 minutes to 30 minutes. In certain embodiments, centrifugation is performed for a time in the range of 3 minutes to 25 minutes. In certain embodiments, centrifugation is performed for a time in the range of 5 minutes to 20 minutes. In certain embodiments, centrifugation is performed for a time in the range of 8 minutes to 15 minutes.

In certain embodiments, centrifugation is performed at a temperature in the range of 4 to 37 ℃. In certain embodiments, centrifugation is performed at a temperature in the range of 4 to 10 ℃ or 16-30 ℃. Each possibility represents a separate embodiment of the invention. In a particular embodiment, centrifugation is performed at 2-6 ℃. In a particular embodiment, centrifugation is performed at 4 ℃. In certain embodiments, the methods described above, in various embodiments thereof, comprise a single centrifugation before, during, or after incubating the stem cells with the exogenous mitochondria, followed by resting the cells at a temperature below 30 ℃. In certain embodiments, the conditions that allow the human functional mitochondria to enter the human stem cell comprise a single centrifugation before, during, or after incubating the stem cell with the exogenous mitochondria, followed by resting the cell at a temperature in the range of 16 to 28 ℃.

In certain embodiments, the first composition is fresh. In certain embodiments, the first composition is frozen and then thawed prior to incubation. In certain embodiments, the second composition is fresh. In certain embodiments, the second composition is frozen and then thawed prior to incubation. In certain embodiments, the fourth composition is fresh. In certain embodiments, the fourth composition is frozen and then thawed prior to administration.

In certain embodiments, the stem cells obtained from a patient or healthy subject having a malignant disease are bone marrow cells or bone marrow-derived stem cells.

The term "mammalian stem cells enriched for functional mitochondria" refers to both human and non-human mammals.

According to the principles of the present invention, healthy functional human extramitochondrial is introduced into human stem cells, thereby enriching these cells with healthy functional human mitochondria. It will be appreciated that this enrichment alters the mitochondrial content of the human stem cells: naive human stem cells have essentially one host/autologous mitochondrial population, whereas human stem cells enriched for exogenous mitochondria have essentially two mitochondrial populations, the first population being host/autologous/endogenous mitochondria and the other population being introduced mitochondria (i.e., exogenous mitochondria). Thus, the term "enriched" refers to the state of the cell after receiving/incorporating exogenous mitochondria. Determining the number of and/or ratio between the two mitochondrial populations is straightforward, as the two populations differ in several respects, for example in their mitochondrial DNA. Thus, the phrase "human stem cells enriched in healthy functional human mitochondria" is equivalent to the phrase "human stem cells comprising endogenous mitochondria and healthy functional exogenous mitochondria". For example, a human stem cell comprising at least 1% of healthy functional exogenous mitochondria of total mitochondria is considered to comprise host/autologous/endogenous mitochondria and healthy functional exogenous mitochondria in a ratio of 99: 1. For example, "3% of total mitochondria" means that the original (endogenous) mitochondrial content after enrichment is 97% of total mitochondria and the introduced (exogenous) mitochondria is 3% of total mitochondria, which is equivalent to an enrichment of (3/97 ═ 3.1%. Another example, "33% of total mitochondria" means that after enrichment the original (endogenous) mitochondrial content is 67% of total mitochondria and the introduced (exogenous) mitochondria is 33% of total mitochondria, which is equivalent to an enrichment of (33/67 ═ 49.2%.

Heterogeneity is the presence of more than one type of mitochondrial DNA within a cell or individual. The level of heterogeneity is the ratio of mutant mtDNA molecules relative to wild type/functional mtDNA molecules and is an important factor in considering the severity of mitochondrial disease. A lower level of heterogeneity (sufficient numbers of mitochondria are functional) is associated with a healthy phenotype, while a higher level of heterogeneity (insufficient numbers of mitochondria are functional) is associated with pathology. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 1% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 3% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 5% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 10% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 15% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 20% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 25% less than the level of heterogeneity of stem cells in the first composition. In certain embodiments, the level of heterogeneity of stem cells in the fourth composition is at least 30% less than the level of heterogeneity of stem cells in the first composition.

In certain embodiments, the mitochondrial content of the human stem cell enriched for healthy mitochondria (also referred to herein as a fourth composition of cells) is detectably higher than the mitochondrial content of the human stem cell in the first composition. According to various embodiments, the mitochondrial content of the fourth composition is at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200% or higher than the mitochondrial content of the first composition. In certain embodiments, the first composition is used fresh.

In certain embodiments, the first composition is frozen and then stored and used after thawing. In other embodiments, the second composition comprising a plurality of functional human mitochondria is used fresh. In other embodiments, the second composition is frozen and thawed prior to use. In other embodiments, the fourth composition is used without refrigeration and storage. In still other embodiments, the fourth composition is used after freezing, storing, and thawing. Methods suitable for freezing and thawing cell preparations to preserve viability are well known in the art. Methods suitable for freezing and thawing mitochondria to preserve structure and function are disclosed in WO 2013/035101 and WO2016/135723 belonging to the present inventors and references cited therein.

Citrate Synthase (CS) is located in the mitochondrial matrix but is encoded by nuclear DNA. Citrate synthase participates in the first step of the krebs cycle and is commonly used as a quantitative enzymatic marker for the presence of intact mitochondria (Larsen s. et al, 2012, j. physiol., vol.590(14), p.3349-3360; Cook g. a. et al, biochim. biophysis. acta, 1983, vol.763(4), p.356-367).

In certain embodiments, the mitochondrial content of the stem cells in the first, second, or fourth compositions is determined by determining the level of citrate synthase. In certain embodiments, the mitochondrial content of the stem cells in the first, second, or fourth composition is determined by determining the level of citrate synthase activity. In certain embodiments, the mitochondrial content of the stem cell in the first, second, or fourth composition is related to the level of citrate synthase. In certain embodiments, the mitochondrial content of the stem cell in the first, second, or fourth composition is correlated with the level of citrate synthase activity. CS activity can be measured by commercially available kits, for example using CS activity kit CS0720 (Sigma).

Eukaryotic 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 is the enzyme of the cytochrome P450 family responsible for xenobiotic detoxification. Cytochrome C reductase is widely used as an endoplasmic reticulum marker. In certain embodiments, the second composition is substantially free of cytochrome C reductase or cytochrome C reductase activity. In certain embodiments, the fourth composition is not enriched for cytochrome C reductase or cytochrome C reductase activity as compared to the first composition.

In certain embodiments, the stem cell is a Pluripotent Stem Cell (PSC). In other embodiments, the PSC is a non-embryonic stem cell. According to some embodiments, embryonic stem cells are specifically excluded from the scope of the present invention. In certain embodiments, the stem cell is an inducible psc (ipsc). In certain embodiments, the stem cell is an embryonic stem cell. In certain embodiments, the stem cells are derived from bone marrow cells. In a particular embodiment, the stem cell is CD34⁺A cell. In certain embodiments, the stem cell is a mesenchymal stem cell. In other embodiments, the stem cells are derived from adipose tissue. In yet other embodiments, the stem cells are derived from blood. In other embodiments, the stem cellDerived from umbilical cord blood. In other embodiments, the stem cells are derived from the oral mucosa.

In certain embodiments, the bone marrow-derived stem cells comprise myeloblasts. As used herein, the term "myeloblasts" refers to cells involved in bone marrow formation, e.g., in the production of bone marrow and all cells produced therefrom, i.e., all blood cells.

In certain embodiments, the bone marrow-derived stem cells comprise erythropoietic cells. As used herein, the term "erythropoietic cell" refers to a cell that is involved in erythropoiesis, e.g., in the production of red blood cells (erythrocytes).

In certain embodiments, the bone marrow-derived stem cells comprise pluripotent Hematopoietic Stem Cells (HSCs). As used herein, the term "pluripotent hematopoietic stem cell" or "hematopoietic cell" refers to a stem cell that produces all other blood cells by the hematopoietic process.

In certain embodiments, the bone marrow-derived stem cells comprise common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow-derived stem cells comprise mesenchymal stem cells. As used herein, the term "common myeloid progenitor cell" refers to a cell that produces myeloid cells. As used herein, the term "common lymphoid lineage progenitor" refers to a cell that produces lymphocytes.

In certain embodiments, the bone marrow-derived stem cells of the first composition further comprise megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, Natural Killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticulocytes, or any combination thereof. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the bone marrow-derived stem cells comprise mesenchymal stem cells. As used herein, the term "mesenchymal stem cell" refers to a pluripotent stromal cell that can differentiate into a variety of different cell types including osteoblasts (osteocytes), chondrocytes, myocytes (myocytes), and adipocytes.

In certain embodiments, the bone marrow-derived stem cells are comprised of myeloblasts. In certain embodiments, the bone marrow-derived stem cells are comprised of erythropoietic cells. In certain embodiments, the bone marrow-derived stem cells are comprised of pluripotent Hematopoietic Stem Cells (HSCs). In certain embodiments, the bone marrow-derived stem cells are comprised of common myeloid progenitor cells, common lymphoid progenitor cells, or any combination thereof. In certain embodiments, the bone marrow-derived stem cells are comprised of megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, Natural Killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticulocytes, or any combination thereof. In certain embodiments, the bone marrow-derived stem cells are comprised of mesenchymal stem cells. Each possibility represents a separate embodiment of the invention.

The hematopoietic progenitor antigen, CD34, also known as the CD34 antigen, is a protein encoded by the CD34 gene in humans. CD34 is a cluster of differentiation antigens in cell surface glycoproteins and acts as a cell-cell adhesion factor. In certain embodiments, the bone marrow stem cells express the bone marrow progenitor antigen CD34 (which is CD 34)⁺Of (d). In certain embodiments, the bone marrow stem cells present the bone marrow progenitor antigen CD34 on their outer membrane. In certain embodiments, the CD34⁺The cells are derived from cord blood.

In certain embodiments, the stem cells in the first composition are derived directly from the subject having a debilitating condition. In certain embodiments, the stem cells in the first composition are derived directly from a donor that does not have a debilitating condition. As used herein, the term "directly derived from" refers to stem cells that are directly derived from other cells. In certain embodiments, the Hematopoietic Stem Cells (HSCs) are derived from bone marrow cells. In certain embodiments, the Hematopoietic Stem Cells (HSCs) are derived from peripheral blood.

In certain embodiments, the stem cells in the first composition are derived indirectly from the subject having a debilitating condition. In certain embodiments, the stem cells in the first composition are derived indirectly from a donor that does not have a debilitating condition. As used herein, the term "indirectly derived from" refers to stem cells that are derived from non-stem cells. In certain embodiments, the stem cells are derived from somatic cells manipulated to become induced pluripotent stem cells (ipscs).

In certain embodiments, the stem cells in the first composition are obtained directly from bone marrow of the subject having the debilitating condition. In certain embodiments, the stem cells in the first composition are obtained directly from bone marrow of a donor who has no debilitating condition. As used herein, the term "directly obtained" refers to stem cells obtained from the bone marrow itself, for example, by means such as surgery or sucking through a needle with a syringe.

In certain embodiments, the stem cells in the first composition are obtained indirectly from the bone marrow of the patient having the debilitating condition. In certain embodiments, the stem cells in the first composition are obtained indirectly from bone marrow of a donor who has no debilitating condition. As used herein, the term "indirectly obtained" refers to bone marrow cells obtained from a location other than the bone marrow itself.

In certain embodiments, the stem cells in the first composition are obtained from peripheral blood of the subject having a debilitating condition. In certain embodiments, the stem cells in the first composition are obtained from the peripheral blood of the subject not having a debilitating condition or from the peripheral blood of a subject not having a debilitating condition. As used herein, the term "peripheral blood" refers to blood circulating in the blood system.

In certain embodiments, the first composition comprises a plurality of human bone marrow stem cells obtained from peripheral blood, wherein the first composition further comprises megakaryocytes, erythrocytes, mast cells, myeloblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, Natural Killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticulocytes, or any combination thereof. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the above methods further comprise a preliminary step comprising administering to the subject having the debilitating condition an agent that induces mobilization of bone marrow cells to peripheral blood. In certain embodiments, the above methods further comprise a preliminary step comprising administering to a donor not suffering from a debilitating condition an agent that induces mobilization of bone marrow cells into peripheral blood.

In certain embodiments, the agent that induces mobilization of bone marrow cells/stem cells produced in bone marrow to peripheral blood is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF), 1' - [1, 4-phenylenebis (methylene) ] bis [1,4,8, 11-tetraazacyclotetradecane ] (plexafof, CAS No. 155148-31-5), salts thereof, and any combination thereof. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the above methods further comprise the step of isolating the stem cells from peripheral blood of a subject having a debilitating condition. In certain embodiments, the above method further comprises the step of isolating the stem cells from peripheral blood of a donor not suffering from a debilitating disease. As used herein, the term "isolated from peripheral blood" refers to the separation of stem cells from the other constituents of blood.

During apheresis, the subject's or donor's blood is passed through a device that separates out one particular constituent and returns the remaining components to circulation. It is therefore a medical procedure that is performed outside the body. In certain embodiments, the separation is performed by apheresis.

In certain embodiments, the above method further comprises concentrating the stem cells and functional mitochondria in the third composition prior to incubation. In certain embodiments, the above method further comprises concentrating the stem cells and functional mitochondria in the third composition during the incubating.

In certain embodiments, the above methods further comprise centrifuging the third composition prior to incubating. In certain embodiments, the above methods further comprise centrifuging the third composition during the incubating. In certain embodiments, the above methods further comprise centrifuging the third composition after incubation.

In certain embodiments, the stem cells in the first composition are obtained from a subject having a debilitating condition, and the stem cells have (i) normal oxygen (O)₂) A consumption rate; (ii) normal citrate synthase content or activity level; (iii) normal Adenosine Triphosphate (ATP) production rate; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells in the first composition are obtained from a subject having a debilitating condition, and the stem cells have (i) reduced oxygen (O) compared to a subject not having a debilitating condition₂) A consumption rate; (ii) reduced level or activity of citrate synthase; (iii) a reduced rate of Adenosine Triphosphate (ATP) production; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention.

It should be emphasized that any reference to any measurable characteristic or condition for a plurality of cells or mitochondria is to the measurable average characteristic or condition of the plurality of cells or mitochondria.

In certain embodiments, the stem cells in the first composition are obtained from a donor who has no debilitating condition and have (i) normal oxygen (O)₂) A consumption rate; (ii) normal citrate synthase content or activity level; (iii) normal Adenosine Triphosphate (ATP) production rate; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention.

In certain embodiments, the isolated human functional mitochondria in the second compositionThe body is obtained from a healthy subject with normal mitochondrial DNA and has (i) normal oxygen (O)₂) A consumption rate; (ii) normal citrate synthase content or activity level; (iii) normal Adenosine Triphosphate (ATP) production rate; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells in the fourth composition have (i) increased oxygen (O) as compared to the stem cells in the first composition₂) A consumption rate; (ii) increased level or activity of citrate synthase; (iii) increased rate of Adenosine Triphosphate (ATP) production; (iv) (iv) increased mitochondrial DNA content, or (v) any combination of (i), (ii), (iii), and (iv). Each possibility represents a separate embodiment of the invention.

As used herein, the term "elevated oxygen (O)₂) By consumption rate "is meant a rate detectably higher than the oxygen (O) in the first composition prior to mitochondrial enrichment₂) Consumption rate of oxygen (O)₂) The rate of consumption.

As used herein, the term "increased level or activity level of citrate synthase" refers to a level or activity of citrate synthase that is detectably higher than the level or activity of citrate synthase in the first composition prior to mitochondrial enrichment.

As used herein, the term "increased rate of Adenosine Triphosphate (ATP) production" refers to a rate of Adenosine Triphosphate (ATP) production that is detectably higher than the rate of Adenosine Triphosphate (ATP) production in the first composition prior to mitochondrial enrichment.

As used herein, the term "increased mitochondrial DNA content" refers to a mitochondrial DNA content that is detectably higher than the mitochondrial DNA content in the first composition prior to mitochondrial enrichment. Mitochondrial content can be determined by measuring the SDHA or COX1 content. In the context of the present specification and claims, the term "normal mitochondrial DNA" refers to mitochondrial DNA with/without mutations or deletions known to be associated with mitochondrial disease. As used herein, the term "normal oxygen (O)₂) Rate of consumption "Refers to the average O of cells from healthy individuals₂And (4) consumption. As used herein, the term "normal level of citrate synthase activity" refers to the average level of citrate activity in cells from a healthy individual. As used herein, the term "normal rate of Adenosine Triphosphate (ATP) production" refers to the average ATP production rate in cells from healthy individuals.

According to certain aspects, the present invention provides a method of treating a debilitating condition, or a symptom thereof, in a human patient in need of such treatment, comprising the step of administering to said patient a pharmaceutical composition comprising a plurality of human stem cells enriched in healthy, functional, exogenous mitochondria that have been freeze-thawed, and that are free of pathogenic mutations in mitochondrial DNA.

In certain embodiments, the symptom is selected from the group consisting of impaired walking ability, impaired motor skills, impaired language skills, impaired memory, weight loss, cachexia, low blood alkaline phosphatase level, low blood magnesium level, high blood creatinine level, low blood bicarbonate level, low blood alkaloid excess level, high urine glucose/creatinine ratio, high urine chloride/creatinine ratio, high urine sodium/creatinine ratio, high blood lactate level, high urine magnesium/creatinine ratio, high urine potassium/creatinine ratio, high urine calcium/creatinine ratio, diabetes, magnesium urine, high blood urea level, low C-peptide level, high HbA1C level, hypoparathyroidism, ptosis, hearing loss, cardiac conduction disorders, low ATP content and oxygen consumption in lymphocytes, mood disorders including bipolar disorder, cardiac disorder, and combinations thereof, Obsessive compulsive disorder, depression, and personality disorders. Each possibility represents a separate embodiment of the invention. It is understood that the definition of a symptom as "high" and "low" corresponds to "detectably higher than normal" and "detectably lower than normal", respectively, wherein the normal level is the corresponding level in a plurality of subjects not suffering from a mitochondrial disorder.

In certain embodiments, the pharmaceutical composition is administered to a specific tissue or organ. In certain embodiments, the pharmaceutical composition comprises at least 10⁴Individual mitochondrially enriched human stem cellsAnd (4) cells. In certain embodiments, the pharmaceutical composition comprises about 10⁴To about 10⁸Individual mitochondrially enriched human stem cells.

In certain embodiments, the pharmaceutical composition is administered by parenteral administration. In certain embodiments, the pharmaceutical composition is administered by systemic administration. In certain embodiments, the pharmaceutical composition is administered by intravenous injection. In certain embodiments, the pharmaceutical composition is administered by intravenous infusion. In certain embodiments, the pharmaceutical composition comprises at least 10⁵Individual mitochondrially enriched human stem cells. In certain embodiments, the pharmaceutical composition comprises about 10⁶To about 10⁸Individual mitochondrially enriched human stem cells. In certain embodiments, the pharmaceutical composition comprises at least about 10⁵-2×10⁷Individual mitochondrially enriched human stem cells per kilogram patient body weight. In certain embodiments, the pharmaceutical composition comprises at least about 10⁵Individual mitochondrially enriched human stem cells per kilogram patient body weight. In certain embodiments, the pharmaceutical composition comprises about 10⁵To about 2X10⁷Individual mitochondrially enriched human stem cells per kilogram patient body weight. In certain embodiments, the pharmaceutical composition comprises about 10⁶To about 5X10⁶Individual mitochondrially enriched human stem cells per kilogram patient body weight.

Mitochondrial DNA content can be measured by quantitative PCR of mitochondrial genes and normalization to nuclear genes before and after mitochondrial enrichment.

In particular cases, the same cells prior to mitochondrial enrichment were used as controls to measure CS and ATP activity and determine the level of enrichment.

In certain embodiments, the term "detectably higher" as used herein refers to a statistically significant increase between the stated normal and increased values. In certain embodiments, the term "detectably higher" as used herein refers to a non-pathological increase, i.e., to a level where no pathological symptoms associated with the substantially higher values become apparent. In certain embodiments, the term "increased", as used herein, refers to a value that is 1.05-fold, 1.1-fold, 1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold or higher than the corresponding value present in the corresponding cell or corresponding mitochondrion of a healthy subject or healthy subjects or prior to mitochondrial enrichment in the stem cell of the first composition. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the stem cells in the fourth composition have at least one of: (i) an increased normal mitochondrial DNA content compared to the mitochondrial DNA content of the stem cell prior to mitochondrial enrichment; (ii) with oxygen (O) in the stem cells prior to mitochondrial enrichment₂) Oxygen (O) consumption rate is increased compared to₂) A consumption rate; (iii) an increased level of citrate synthase content or activity compared to the level of citrate synthase content or activity in the stem cells prior to mitochondrial enrichment; (iv) an increased rate of Adenosine Triphosphate (ATP) production compared to the rate of Adenosine Triphosphate (ATP) production in the stem cells prior to mitochondrial enrichment; or (v) any combination of (i), (ii), (iii), and (iv). Each possibility represents a separate embodiment of the invention.

In certain embodiments, the total amount of mitochondrial proteins in the second composition is between 20% and 80% of the total amount of cellular proteins in the sample.

As used herein, the term "about" refers to ± 10% of the indicated numerical value. Generally, numerical values used herein refer to ± 10% of the stated numerical value.

In certain embodiments, the method further comprises freezing the fourth composition. In certain embodiments, the method further comprises freezing and then thawing the fourth composition.

In another aspect, the present invention also provides a plurality of functional mitochondria-enriched human stem cells obtained by the above method.

In certain embodiments, the plurality of stem cells are frozen prior to enrichment with functional mitochondria. In other embodiments, the plurality of stem cells are frozen and then thawed prior to enrichment with functional mitochondria, and in other embodiments, the plurality of functional mitochondria-enriched stem cells are frozen. In other embodiments, the plurality of functional mitochondrially enriched stem cells are frozen and then thawed prior to use.

In another aspect, the present invention also provides a plurality of human stem cells, wherein the stem cells have at least one property selected from the group consisting of: (a) increased mitochondrial content; (b) enhanced oxygen (O)₂) A consumption rate; (c) increased level or activity of citrate synthase; (d) increased mitochondrial DNA content; or (e) any combination of (a), (b), (c), and (d). Each possibility represents a separate embodiment of the invention. According to certain embodiments, the stem cell is CD34⁺A stem cell.

As used herein, the term "increased mitochondrial content" refers to a mitochondrial content that is detectably higher than the mitochondrial content of the first composition prior to mitochondrial enrichment.

In certain embodiments, the plurality of cells is frozen. In certain embodiments, the plurality of cells are frozen and then thawed prior to use.

In certain embodiments, the plurality of human stem cells is CD34⁺And has increased mitochondrial content, increased normal mitochondrial DNA levels, increased oxygen (O)₂) Consumption rate and increased level of citrate synthase activity. Each possibility represents a separate embodiment of the invention.

In certain embodiments, the plurality of human stem cells have increased mitochondrial content, increased normal mitochondrial DNA levels, increased (O)₂) Consumption rate and with increased levels of citrate synthase activity.

In another aspect, the present invention also provides a pharmaceutical composition comprising a plurality of functional mitochondria-enriched human stem cells as described above.

As used herein, the term "pharmaceutical composition" refers to any composition comprising a cell, and further comprising a vehicle or carrier that maintains the cell therein in a viable state.

In certain embodiments, the pharmaceutical composition is frozen. In certain embodiments, the pharmaceutical composition is frozen and then thawed prior to use.

In certain embodiments, the above-described pharmaceutical compositions are used in methods of treating certain conditions in a human subject having a debilitating condition. As used herein, the term "treating" includes the alleviation, alleviation or amelioration of at least one symptom associated with or caused by the debilitating effect of the condition from which the subject is suffering.

In another aspect, the present invention also provides a method of alleviating or reducing the debilitating effects of conditions including, but not limited to, aging, age-related diseases or anti-cancer therapies in a human subject suffering from a malignant disease, said method comprising the step of administering to said subject a pharmaceutical composition as described above.

As used herein, the term "method" refers generally 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, pharmaceutical, biological, biochemical and medical arts.

In certain embodiments, the pharmaceutical composition is frozen and the above method comprises thawing the frozen pharmaceutical composition prior to use.

In certain embodiments, the stem cells are autologous to the subject having the debilitating condition.

Contacting functional mitochondria with stem cells autologous to the subject having the debilitating condition, resulting in rejuvenation/resuscitation of the stem cells.

In certain embodiments, the methods described above, in various embodiments thereof, further comprise expanding the stem cells of the first composition by culturing the stem cells in an expansion medium capable of expanding stem cells. In other embodiments, the method further comprises expanding the cells by culturing the mitochondrially enriched stem cells of the fourth composition in a medium capable of expanding stem cells or a proliferation medium. The term "culture or propagation medium" as used throughout this application is a fluid medium such as cell culture medium, cell growth medium, buffer that provides nutrients to the cells. The term "pharmaceutical composition" as used throughout the present application and claims encompasses fluid carriers such as cell culture media, cell growth media, buffers that provide nutrients to the cells.

In certain embodiments, the administration of the stem cells rejuvenated by functional mitochondria in the subject with debilitating effects may reduce these effects. In certain embodiments, administration of the rejuvenated stem cells may restore the organization and distribution of epithelial cells in the intestinal villi of the subject with the debilitating condition. In other embodiments, the administration of the rejuvenated stem cells may restore the activity of epithelial stem cells in the intestinal crypts of the subject. In other embodiments, the administration of the rejuvenated stem cells may restore dermal thickness in the subject. In still other embodiments, the administration of the rejuvenated stem cells may restore hair follicle activity in the subject. In another embodiment, the administration of the rejuvenated stem cells may restore wound healing activity in dermal tissue of the subject. According to certain embodiments, the functional mitochondrially enriched stem cells may rejuvenate blood precursor cells in an autologous hematopoietic stem cell graft. According to other embodiments, the functional mitochondrially enriched stem cells can rejuvenate blood precursor cells in an allogeneic hematopoietic stem cell transplant. According to still other embodiments, the functional mitochondrially enriched stem cells may rejuvenate dermal or intestinal epithelial precursor cells. In another embodiment, the administration of the rejuvenated stem cells may restore pancreatic β -cell function in the subject. According to certain embodiments, the stem cells enriched for functional mitochondria can rejuvenate hepatocytes. According to other embodiments, functional mitochondria-enriched stem cells can delay deterioration of renal function. According to still other embodiments, functional mitochondrial-enriched stem cells can reduce macular degeneration.

In certain embodiments, the stem cells are allogeneic to the subject having the debilitating condition. As used herein, the terms "allogeneic to the subject," "from a donor," and "from a healthy donor" are used interchangeably and refer to stem cells or mitochondria from different donor individuals. If possible, the donor stem cells are preferably HLA matched or at least partially HLA matched to the patient's cells. According to certain embodiments, the donor is matched to the patient based on the identification of a specific haplotypic population of mitochondrial DNA.

As used herein, the term "HLA-matched" refers to a requirement that the patient and donor of stem cells be HLA-matched as closely as possible, at least to the extent that the patient does not develop an acute immune response against the stem cells of the donor. Prevention and/or treatment of such immune responses may be achieved with or without acute or chronic use of immunosuppressive agents. In certain embodiments, the stem cells from the donor are HLA-matched to the patient to an extent where the patient does not reject the stem cells.

In certain embodiments, the patient is further treated by immunosuppressive therapy to prevent immune rejection of the stem cell transplant.

In certain embodiments, the mitochondria are from a single, uniform population.

In other embodiments, the mitochondria are from different haplotypes.

In certain embodiments, the above method further comprises the preliminary step of administering a pre-transplant conditioner to the patient prior to administering the pharmaceutical composition. As used herein, the term "pre-transplant conditioner" refers to any agent capable of killing bone marrow cells in the bone marrow of a human subject. In certain embodiments, the pre-transplant conditioner is busulfan.

In certain embodiments, the pharmaceutical composition is administered systemically. In certain embodiments, administration of the pharmaceutical composition to a subject is by a route selected from intravenous, intraarterial, intramuscular, subcutaneous, intravitreal, and direct injection into a tissue or organ. Each possibility represents a separate embodiment of the invention. According to certain embodiments, the pharmaceutical composition is injected directly into tissues and organs affected by the debilitating conditions described herein. Specific tissues or organs known to exhibit functional impairment associated with decreased mitochondrial quality and activity include, but are not limited to, the eye, kidney, liver, pancreas, brain, and heart.

In certain embodiments, the functional mitochondria are obtained from a human cell or human tissue selected from the group consisting of a placenta, placental cells grown in culture, and blood cells. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, the functional mitochondria have undergone a freeze-thaw cycle. Without wishing to be bound by any theory or mechanism, mitochondria that have undergone a freeze-thaw cycle exhibit a comparable rate of oxygen consumption after thawing as compared to control mitochondria that have not undergone a freeze-thaw cycle.

According to certain embodiments, the freeze-thaw cycle comprises freezing the functional mitochondria for at least 24 hours prior to thawing. According to other embodiments, the freeze-thaw cycle comprises freezing the functional mitochondria for at least 1 month, several months, or longer prior to thawing. Each possibility represents a separate embodiment of the invention. According to another embodiment, the oxygen consumption of the functional mitochondria after the freeze-thaw cycle is equal to or higher than the oxygen consumption of the functional mitochondria prior to the freeze-thaw cycle.

As used herein, the term "freeze-thaw cycle" refers to freezing the functional mitochondria to a temperature below 0 ℃, maintaining the mitochondria at a temperature below 0 ℃ for a defined period of time, and thawing the mitochondria to room or body temperature or any temperature above 0 ℃ that is capable of treating the stem cells with the mitochondria. Each possibility represents a separate embodiment of the invention. As used herein, the term "room temperature" generally refers to a temperature between 18 ℃ and 25 ℃. As used herein, the term "body temperature" refers to a temperature between 35.5 ℃ and 37.5 ℃, preferably 37 ℃. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are functional mitochondria.

In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are frozen at a temperature of-70 ℃ or less. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are frozen at a temperature of-20 ℃ or less. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are frozen at a temperature of-4 ℃ or less. According to another embodiment, the freezing of the mitochondria is gradual. According to some embodiments, the freezing of mitochondria is performed by flash freezing. As used herein, the term "flash freezing" refers to the rapid freezing of the mitochondria by subjecting them to ultra-low temperatures.

In another embodiment, the mitochondria undergoing a freeze-thaw cycle are frozen for at least 30 minutes prior to thawing. According to another embodiment, the freeze-thaw cycle includes 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 invention. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are 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 represents a separate embodiment of the present invention. In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are frozen for at least 4, 5, 6, 7, 30, 60, 120, 365 days prior to thawing. Each freezing time represents a separate embodiment of the present invention. According to another embodiment, the freeze-thaw cycle includes freezing the functional mitochondria for at least 1, 2, 3 weeks prior to thawing. Each possibility represents a separate embodiment of the 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 invention.

In another embodiment, the mitochondria that have undergone a freeze-thaw cycle are frozen at-70 ℃ for at least 30 minutes prior to thawing. Without wishing to be bound by any theory or mechanism, the possibility of freezing mitochondria and thawing them after a long period of time enables easy storage and use of the mitochondria and reproducible results even after long storage times.

According to a certain embodiment, the thawing is performed at room temperature. In another embodiment, the thawing is performed at body temperature. According to another embodiment, the thawing is performed at a temperature that enables administration of said mitochondria according to the method of the invention. According to another embodiment, the thawing is performed gradually.

According to another embodiment, the mitochondria undergoing a freeze-thaw cycle are frozen in a freezing buffer. According to another embodiment, the mitochondria undergoing a freeze-thaw cycle are frozen in a separation buffer. As used herein, the term "isolation buffer" refers to the buffer in which the mitochondria of the invention are isolated. In a non-limiting example, the separation buffer is a sucrose buffer. Without wishing to be bound by any mechanism or theory, freezing mitochondria in the isolation buffer saves time and isolation steps, as there is no need to replace the isolation buffer with a freezing buffer prior to freezing or replace the freezing buffer after thawing.

According to another embodiment, the freezing buffer comprises a cryoprotectant. According to some embodiments, the cryoprotectant is a sugar, an oligosaccharide or a polysaccharide. Each possibility represents a separate embodiment of the invention. According to another embodiment, the sugar concentration in the freezing buffer is a sugar concentration sufficient for protecting mitochondrial function. According to another embodiment, the separation buffer comprises a sugar. According to another embodiment, the sugar concentration in the separation buffer is a sugar concentration sufficient for protecting mitochondrial function. According to another embodiment, the sugar is sucrose.

In certain embodiments, the method further comprises the following preliminary steps: (a) freezing the human stem cells enriched for healthy functional human exogenous mitochondria, (b) thawing the human stem cells enriched for healthy functional human exogenous mitochondria, and (c) administering the human stem cells enriched for healthy functional human exogenous mitochondria to the patient.

In certain embodiments, the healthy, functional, exogenous mitochondria comprise at least 3% of total mitochondria in a mitochondria-enriched cell. In certain embodiments, the healthy, functional, exogenous mitochondria comprise at least 10% of total mitochondria in a mitochondria-enriched cell. In certain embodiments, the healthy, functional, exogenous mitochondria comprise at least about 3%, 5%, 10%, 15%, 20%, 25%, or 30% of total mitochondria in a mitochondria-enriched cell. Each possibility represents a separate embodiment of the invention.

The degree to which the stem cells are enriched for functional mitochondria can be determined by functional and/or enzymatic assays, including but not limited to oxygen (O)₂) Consumption rate, level of citrate synthase or activity, rate of Adenosine Triphosphate (ATP) production. In the alternative, enrichment of the stem cells with healthy donor mitochondria can be confirmed by detection of the donor's mitochondrial DNA. According to certain embodiments, the degree to which the stem cells are enriched for functional mitochondria can be determined by the level of heterogeneity variation and/or by the mtDNA copy number per cell. Each possibility represents a separate embodiment of the invention.

TMRM (tetramethylrhodamine methyl ester) or related TMRE (tetramethylrhodamine ethyl ester) is a cell-penetrating fluorogenic dye commonly used to assess mitochondrial function in living cells by identifying changes in mitochondrial membrane potential. According to certain embodiments, the level of enrichment may be determined by staining with TMRE or TMRM.

According to certain embodiments, the integrity of the mitochondrial membrane may be determined by any method known in the art. In a non-limiting example, integrity of mitochondrial membranes is measured using tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE) fluorescent probes. Each possibility represents a separate embodiment of the invention. The TMRM or TMRE stained mitochondria were observed under the microscope and shown to have an intact outer mitochondrial membrane. As used herein, the term "mitochondrial membrane" refers to a mitochondrial membrane selected from the group consisting of the inner mitochondrial membrane, the outer mitochondrial membrane, and both.

In certain embodiments, the level of mitochondrial enrichment in a mitochondrially enriched human stem cell is determined by sequencing at least a statistically representative portion of total mitochondrial DNA in the cell and determining the relative levels of host/endogenous mitochondrial DNA and exogenous mitochondrial DNA. In certain embodiments, the level of mitochondrial enrichment in the mitochondria-enriched human stem cells is determined by Single Nucleotide Polymorphism (SNP) analysis. In certain embodiments, the largest population of mitochondria and/or the largest population of mitochondrial DNA is the host/endogenous population of mitochondria and/or the host/endogenous population of mitochondrial DNA; and/or the second large population of mitochondria and/or the second large population of mitochondrial DNA is the exogenous population of mitochondria and/or the exogenous population of mitochondrial DNA. Each possibility represents a separate embodiment of the invention.

According to certain embodiments, enrichment of the stem cells with healthy functional mitochondria can be determined by conventional assays well known in the art. In certain embodiments, the level of mitochondrial enrichment in the mitochondria-enriched human stem cell is determined by: (i) levels of host/endogenous mitochondrial DNA and exogenous mitochondrial DNA; (ii) (ii) a level of a mitochondrial protein selected from the group consisting of Citrate Synthase (CS), cytochrome C oxidase (COX1), succinate dehydrogenase complex flavoprotein subunit a (sdha), and any combination thereof; (iii) (ii) a level of CS activity; or (iv) any combination of (i), (ii), and (iii). Each possibility represents a separate embodiment of the invention.

In certain embodiments, the level of mitochondrial enrichment in the mitochondria-enriched human stem cell is determined by at least one of: (i) levels of host mitochondrial DNA and exogenous mitochondrial DNA in the case of allogeneic mitochondria; (ii) the level of citrate synthase activity; (iii) removing succinic acid

Levels of the catalase complex flavoprotein subunit a (sdha) or cytochrome C oxidase (COX1) protein; (iv) oxygen (O)₂) A consumption rate; (v) phosphorus trioxideThe rate of production of adenosine monophosphate (ATP); or (vi) any combination thereof. Each possibility represents a separate embodiment of the invention. Methods for measuring these various parameters are well known in the art.

In certain aspects, the invention provides a pharmaceutical composition for treating or alleviating the debilitating effects of a condition in a subject comprising human stem cells enriched for healthy functional mitochondria, wherein the debilitating effects of the condition are selected from, but not limited to, aging, age-related diseases and sequelae of anti-cancer therapy.

In certain embodiments, the present invention provides a method of treating or alleviating the debilitating effects of a condition in a subject, said method comprising administering to said subject a pharmaceutical composition comprising human stem cells enriched in healthy functional mitochondria, wherein the debilitating effects of said condition are selected from, but not limited to, aging, age-related diseases and sequelae of anti-cancer therapy. In a particular embodiment, the anti-cancer treatment is selected from radiation, chemotherapy, immunotherapy with monoclonal antibodies, or any combination thereof.

According to some embodiments, the healthy functional mitochondria are isolated from donors selected from a specific mitochondrial haplotype group according to a debilitating condition of the subject. For example, for aging subjects, administration of stem cells enriched in functional mitochondria from J mitochondrial haplotypes is suitable because it is associated with longevity and lower blood pressure (De Benedictis et al, FASEB J.1999; 13(12): 1532-6; Rea et al, AGE 2013; 34(4): 1445-56). The H and N haplotypes are associated with better muscle function and strength (Larsen et al, Biochim Biophys acta.2014; 1837(2): 226-31; Fuku et al, Int J Sports Med.2012; 33(5): 410-4). The D4b haplotype group might provide protection against stroke (Yang et al, Mol Genet genomics.2014; 289(6):1241-6), K, U, H and V haplotype groups might provide protection against cognitive impairment (Colicino et al, Environ health.2014; 13(1):42), and it has been shown that the R haplotype group provides a better prognosis for recovery from septic encephalopathy (Yang et al, Intensive Care Med.2011; 37(10): 1613-9). Haplotype group N9a provides resistance to Diabetes (Fuku et al, Am J Hum Genet.2007; 80(3):407-15) and metabolic syndrome (Tanaka et al, Diabetes 2007; 56(2): 518-21). The H haplotype group provides protection against the development of eye diseases, including age-related macular degeneration (AMD) (Mueller et al, PloS one 2012; 7(2): e 30874).

According to some embodiments, the stem cells of the first composition are from donors selected from a particular mitochondrial haplotype group according to a debilitating condition of the subject. For example, for subjects with debilitating effects of anti-cancer therapy, the J, K2 and U haplotypes can be considered, as they have been shown to be better donors for allogeneic hematopoietic stem cell transplantation, eliciting less GVHD and/or relapse (Ross et al, Biol Blood Marrow Transplant 2015; 21: 81-88).

As used herein, the term "haplotype group" refers to a group of human genetic populations that share a common ancestor on maternal descent. Mitochondrial haplotypes were determined by sequencing.

In some cases, we may wish to match haplotypes between the donor and the recipient.

As used herein, the term "about" means a range of from 10% lower to 10% higher than the indicated integer, number or amount. For example, the phrase "about 1X10⁵"means" 1.1X 10⁵To 9X 10⁴”。

While the invention has been described with reference to certain embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from its scope. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

The following examples are provided to provide a more complete understanding of the invention. The specific techniques, conditions, materials, proportions and reported data set forth to illustrate the principles of the invention are exemplary and should not be construed as limiting the scope of the invention.

Examples

Example 1. isolated human mitochondria: preparation and ultra-low temperature preservation

Mitochondria can be isolated and stored as previously disclosed in WO 2013/035101 and WO 2016/135723.

The following is the separation of mitochondria (MNV-BLD) from peripheral blood cells and CD34⁺Exemplary procedure for enrichment of cells (MNV-BM-BLD):

first stage-MNV-BLD production: the buffy coat is isolated from peripheral blood (500mL) obtained from the patient or donated from a donor. Then the buffy coat is applied to Lymphoprep^TMThe layers were separated on top and centrifuged. Collection of leukocytes (Lymphoprep)^TMThe top buffy coat) and then centrifuged. The cell pellet (lymphocytes) was washed, frozen and suspended in ice-cold 250mM sucrose buffer (250mM sucrose, 10mM Tris, 1mM EDTA) at pH 7.4. The cell suspension was collected and passed through a 30G needle 3 times, followed by homogenization. The homogenate was centrifuged. The supernatant was collected and kept on ice, and the sediment was washed with sucrose solution, homogenized and centrifuged. A second supernatant from the washed sediment is collected and combined with the previous supernatant. The combined supernatants were filtered through a 5 μm filter and centrifuged at 8000 g. The sediment was washed with sucrose solution and resuspended in 1ml of cold 250mM sucrose buffer solution at pH 7.4. The resulting mitochondrial solution (referred to herein as MNV-BLD) was cryogenically stored in a gaseous nitrogen tank until use.

Second stage-MNV-BM-BLD Generation: CliniMACS was used after mobilization of bone marrow cells to peripheral blood^TMSystem for separating patient or donor CD34 from collected blood by leukapheresis⁺A cell. Will CD34⁺Cell sediment was suspended in 0.9% NaCl solution containing 4.5% HSA to a final concentration of 1X10⁶Individual cells/ml. MNV-BLD (mitochondrial suspension) was thawed at room temperature and at 4.4 milliunits Citrate Synthase (CS) activity/ml cell suspension (1X 10)⁶Individual cells) to the CD34⁺A cell. Mixing MNV-BLD and CD34⁺Cells in 2mL tubesMix and centrifuge at 7000g for 5 minutes at 4 ℃. After centrifugation, the cells were suspended with the same 0.9% NaCl solution containing 4.5% HSA, combined, seeded in shake flasks and incubated at room temperature for 24 hours. After incubation, enriched CD34 was added⁺Cells were washed twice with 4.5% HSA solution and centrifuged at 300g for 10 min. The cell pellet was resuspended in 100ml of 0.9% NaCl containing 4.5% HSA and loaded into an infusion bag.

Example 2 isolated mitochondria can enter fibroblasts

Mouse fibroblasts expressing Green Fluorescent Protein (GFP) in their mitochondria (3T3) (left panel) were incubated for 24 hours with Red Fluorescent Protein (RFP) -labeled mitochondria isolated from mouse fibroblasts expressing RFP in their mitochondria (3T3) (middle panel). As previously described in WO2016/135723, fluorescence confocal microscopy was used to identify fibroblasts marked with both GFP and RFP (right panel) that appeared yellow (fig. 1).

The results presented in figure 1 indicate that mitochondria can enter fibroblast cells.

Example 3 mitochondrial increase of ATP production in cells with inhibited mitochondrial Activity

Mouse fibroblasts (10)⁴3T3) was left untreated (control) or treated with 0.5 μ M rotenone (rotenone, mitochondrial complex I irreversible inhibitor, CAS No. 83-79-4) for 4 hours, washed, and further treated with 0.02mg/ml mouse placenta mitochondria (rotenone + mitochondria) for 3 hours. The cells were washed and ATP levels were determined as previously shown in WO2016/135723 using the Perkin Elmer ATPlite kit (figure 2). As can be seen in figure 2, ATP production was completely rescued in cells incubated with mitochondria compared to controls.

The results presented in figure 2 clearly show that although rotenone alone reduced ATP levels by about 50%, the addition of mitochondria was able to substantially eliminate the inhibitory effect of rotenone, reaching ATP levels of control cells. The experiments provide evidence that mitochondria are able to increase mitochondrial ATP production in cells with impaired or damaged mitochondrial activity.

Example 4 mitochondria can enter murine bone marrow cells

Mouse bone marrow cells (10)⁵) Incubated for 24 hours with GFP-labeled mitochondria isolated from mouse myeloma cells. Fluorescence confocal microscopy was used to identify GFP-labeled mitochondria inside the bone marrow cells as previously described in WO2016/135723 (figure 3).

The results presented in figure 3 indicate that mitochondria can enter bone marrow cells.

Bone marrow cells from wild type (ICR) and mitochondrial mutant (FVB/N with a mutation in ATP 8) mice were separated from isolated mitochondria of different origin at 37 ℃ and 5% CO₂Incubate for 24 hours in DMEM under an atmosphere to increase their mitochondrial content and activity. Table 1 depicts representative results of the mitochondrial enhancement process, as determined by the relative increase in CS activity of the cell after the process compared to the CS activity of the cell before the process.

Table 1.

To investigate the in vivo effects of mitochondrion-enhancing therapy, FVB/N bone marrow cells enriched with C57/BL placental mitochondrion with 4.4 milliunits of CS activity (1X 10)⁶) IV was injected into FVB/N mice. Bone marrow was collected from mice 1 day, 1 week, 1 month and 3 months after treatment and WT mtDNA levels were measured using dPCR. As can be seen in fig. 4, significant amounts of WT mtDNA were detected in the bone marrow 1 day after treatment.

Example 5 entry of mitochondria into bone marrow cells in a concentration-dependent manner

Mouse bone marrow cells (10)⁶) Untreated or incubated for 15 hours with varying amounts of GFP-labeled mitochondria isolated from mouse myeloma cells. Mitochondria were mixed with the cells and either left to stand for 5 minutes at room temperature ((-) centrifugation) or centrifuged at 8,000g at 4 ℃ before plating the cells5 min ((+) centrifugation). The cells were then plated in 24 wells (10)⁶Individual 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 as previously described in WO2016/135723 using the CS0720 Sigma kit (fig. 5). The levels of CS activity measured under the conditions specified above are summarized in table 2.

Table 2.

The results demonstrated in fig. 5 show that the added mitochondria increase CS activity of the cells in a dose-dependent manner, and that increasing the concentration and thus presumably increasing the contact between the mitochondria and the cells, e.g. by centrifugation, results in a further increase in CS activity.

Mouse bone marrow cells (10)⁶) GFP-labeled mitochondria (17 or 34 milliunits, indicating the level of citrate synthase activity as a marker of mitochondrial content) were left untreated or incubated for 24 hours with mouse myeloma cells. The cells were mixed with mitochondria, centrifuged at 8000g and resuspended. After 24 hours incubation, the cells were washed twice with PBS and the levels of Citrate Synthase (CS) activity (fig. 6A) and cytochrome C reductase activity (fig. 6B) were measured using CS0720 and CYOIOO kit (Sigma), respectively, as previously described in WO 2016/135723.

FVB/N bone marrow cells (carrying mutations in mtDNA ATP 8) were incubated with C57/BL Wild Type (WT) mitochondria isolated from placenta at various doses (0.044, 0.44, 0.88, 2.2, 4.4, 8.8, 17.6 milliunits of CS activity per 1M cells in 1 mL). As can be seen in fig. 7A, dPCR using WT specific sequences showed that WT mtDNA increased in a dose dependent manner for most doses. The enriched cells also showed dose-dependent increases in mtDNA-encoded (COX1) (fig. 7B) and nuclear-encoded (SDHA) (fig. 7C) content.

Example 6 mitochondria can enter human bone marrow cells

Human CD34⁺Cells (1.4X 10)⁵ATCC PCS-800-012) were not treated or incubated for 20 hours with GFP-labeled mitochondria isolated from human placental cells. Mitochondria were mixed with the cells, centrifuged at 8,000g and resuspended prior to plating the cells. After incubation, the cells were washed twice with PBS and CS activity was measured using CS0720 Sigma kit (fig. 8A). ATP content was measured using ATPLite (Perkin Elmer) (FIG. 8B). The levels of CS activity measured under the conditions specified above (fig. 8A) are summarized in table 3.

Table 3.

The results demonstrated in figure 8 (see table 3) clearly show that by interacting and co-incubating with isolated human mitochondria, the mitochondrial content of human bone marrow cells can be increased many-fold, to an extent that exceeds the capacity of human or murine fibroblasts or murine bone marrow cells.

The cell populations depicted in fig. 8B were further evaluated by FACS analysis. CD34 incubated in non-GFP labelled mitochondria⁺Only a small fraction (0.9%) of the cells fluoresced (FIG. 9A), while CD34 incubated with GFP-labeled mitochondria⁺Cells were significantly fluorescent (28.4%) after centrifugation (fig. 9B), as previously shown in WO 2016/135723.

⁺Example 7 mitochondria can enter human CD34 bone marrow cells

Isolation of human CD34 by apheresis from healthy donors treated with GCS-F⁺Cells, purified using the CliniMACS system and frozen. The cells were thawed and treated with blood-derived mitochondria (MNV-BLD) (4.4 milliunits of mitochondrial CS activity/1 x10⁶Individual cells) treated or Not (NT), centrifuged at 8000g and incubated for 24 h. Cells were then washed with PBS and measured for CS activity (fig. 10B) and ATP content (fig. 10A) (CS 0720 Sigma kit and ATPlite Perkin Elmer, respectively).

CD34 treated with blood-derived mitochondria⁺The cells showed a significant increase in mitochondrial activity as measured by CS activity (fig. 10B) and ATP content (fig. 10A).

CD34 from healthy donors⁺Cells were treated with Mitotracker orange (MTO) and washed, then MAT was performed using mitochondria isolated from HeLa-TurboGFP-mitochondrial cells (CellTrend GmbH). Cells were fixed with 2% PFA for 10min and DAPI. The cells were scanned using a confocal microscope equipped with a 60X/1.42 oil immersion objective.

As can be seen in fig. 11, the exogenous mitochondria entered CD34 as soon as 0.5 hours after MAT⁺Cells (bright, almost white spots inside the cells) and continued entry at 8 and 24 hours tested.

⁺Example 8 incubation of CD34 cells with saline at room temperature increases their survival

Will CD34⁺Cells were either untreated (NT) or incubated with blood-derived mitochondria (MNV-BLD). The cells were cultured at Room Temperature (RT) or 37 ℃ in medium (CellGro) containing 4.5% Human Serum Albumin (HSA)^TM) Or saline (Zenalb)^TM) Culturing in medium.

Cell viability under different culture conditions is summarized in table 4.

Table 4.

	Survival rate%
		CellGroTM 37℃NT	55.3
CellGroTM 37℃MNV-BLD	59.6
		CellGroTM RT NT	72.5
CellGroTM RT MNV-BLD	78.2
		ZenalbTM RT NT	93.9
ZenalbTM RT MNV-BLD	94.7

The results presented in table 4 show that CD34 was obtained when cultured in saline instead of medium using human serum albumin at RT⁺The survival rate of the cells is improved.

Example 9 bone marrow from NSGS mice engrafted with human umbilical cord blood contained more 2 months after MAT Human mtDNA

Cord blood cells from patients with Pearson syndrome were incubated with 0.88mU of human mitochondria for 24hr, then the medium was removed, and the cells were washed and resuspended in 4.5% HSA. The enriched cells IV were injected into NSGS mice (100,000 CD34 per mouse)⁺A cell).

Fig. 12A is a graphical illustration of mtDNA deletion in umbilical cord blood cells of a patient with pearson syndrome, showing the 4978kb deleted UCB mtDNA region (left panel), and showing southern blot analysis of the deletion (right panel).

Bone marrow was collected from mice 2 months after MAT and the copy number of WT mtDNA without deletion was analyzed in dPCR using primers and probes identifying WT mtDNA sequence without deletion of UCB.

As can be seen in fig. 12B, 2 months after mitochondrial potentiation therapy, the bone marrow of the mice contained-100% more human mtDNA than bone marrow of mice injected with non-enhanced cord blood cells.

Example 10 in vivo safety and biodistribution animal models

Mitochondria were introduced into bone marrow cells from two different backgrounds of control healthy mice: the source of mitochondria is from mice with different mtDNA sequences (Jenuth JP et al, Nature Genetics,1996, Vol.14, p. 146-.

Mitochondria were isolated from wild type mouse (C57BL) placenta. Bone marrow cells were isolated from FVB/N mice. The mutated FVB/N bone marrow cells (10)⁶) The healthy functional C57BL mitochondria (4.4mU) were loaded and IV administered to FVB/N mice.

The method comprises the following steps: (1) mitochondria were isolated from the placenta of C57BL mice, frozen and thawed at-80 ℃, or used freshly; (2) obtaining bone marrow cells from mtDNA-mutated FVB/N mice; (3) contacting the mitochondria with bone marrow cells, centrifuging at 8000g for 5 minutes, resuspending and incubating for 24 hours; (4) the bone marrow cells were washed twice with PBS and injected into the tail vein of FVB/N mice. At various time points after transplantation, e.g., 24 hours, one week, one month, and 3 months, tissues (blood, bone marrow, lymphocytes, brain, heart, kidney, liver, lung, spleen, skeletal muscle, eye, ovary/testis) were collected and DNA was extracted for further sequence analysis.

The reduction in FVB/N levels in bone marrow 1 month post-transplantation is depicted in FIG. 13A. As seen in fig. 13B, the level of mtDNA in the liver of FVB/N mice also decreased 3 months after transplantation.

Bone marrow harvested from female FVB/N was treated with C57BL/6 placental mitochondria (4.4mU CS activity/1X 10)⁶Individual cells) were enriched. Recipient mice underwent IV administration of 1 million enhanced cells per animal. The C57BL/6 specific SNP was detected using digital PCR. FIG. 14A demonstrates the presence of C57BL/6mtDNA in bone marrow of FVBN mice 1 day after MAT, and that some mice show persistence until 3 post-treatmentAnd (4) month. Fig. 14B and 14C show the presence of C57 BL/6-derived mtDNA in the heart and brain of mice at 3 months after MAT.

Example 11 in vivo preclinical animal study: effect of premodulation on foreign mitochondrial Implantation

Mitochondria were isolated from the liver of wild type mice (C57 BL). Bone marrow cells were isolated from mice with mutated mitochondria (FVB/N mice). Loading the mutated FVB/N bone marrow cells with the healthy functional C57BL mitochondria. Untreated FVB/N mice (control), FVB/N mice administered the enriched mitochondria, FVB/N mice treated with a chemotherapeutic agent (busulfan) prior to administration of the enriched mitochondria, and FVB/N mice subjected to systemic irradiation (TBI) prior to administration of the enriched mitochondria were compared.

The method comprises the following steps: (1) mitochondria were isolated from the liver of C57BL mice, frozen and thawed at-80 ℃, or used freshly; (2) obtaining bone marrow cells from mtDNA-mutated FVB/N mice; (3) contacting the mitochondria with bone marrow cells, centrifuging at 8000g for 5 minutes, resuspending and incubating for 24 hours; (4) washing the bone marrow cells twice with PBS; (5) administering busulfan or systemic irradiation (TBI) to a predetermined group; (6) the FVB/N mouse bone marrow cells enriched in C57BL mouse healthy mitochondria were injected into the tail vein of FVB/N mice. 1 month after transplantation, tissues (blood, bone marrow, lymphocytes, brain, heart, kidney, liver, lung, spleen, pancreas, skeletal muscle, eye, ovary/testis) were collected and DNA was extracted for further sequence analysis.

The reduction of FVB/N levels in the brain of mitochondria, TBI and busulfan treated mice at 1 month post-transplantation is depicted in figure 15.

Example 12 Effect of mitochondrial enrichment on aging mice

Mitochondria were isolated from C57BL murine placenta. Bone marrow cells were obtained from 12-month-old C57BL mice. Mitochondria-enriched bone marrow cells (MNV-BM-PLC, 1X 10)⁶Individual cells), bone marrow cells alone (BM, 1x 10)⁶Individual cells) or control medium solution (medium, 4.5% albumin in 0.9% w/v NaCl) was injected IV at the start of the experimentTail veins of 12-month-old C57BL mice were injected again at approximately 15, 18, and 21 months of age. BUN blood tests were performed 1, 3, 4, and 6 months after the first IV injection. Open field trials were performed 9 months after the first IV injection. BUN blood tests were performed 2, 4 and 6 months after IV injection.

As can be seen in fig. 16A-16D, aging mice (12 months of age) transplanted with bone marrow cells enriched for healthy mitochondria (MNV-BM-PLC) exhibited improved physical activity and exploratory behavior compared to age-matched mice transplanted with bone marrow not enriched for mitochondria (BM control) and mice that were not transplanted at all (control). MNV-BM-PLC treated mice showed: greater travel distance (fig. 16A), more time spent in the center of the cage (fig. 16B), and less time spent near the cage walls (fig. 16C), a typical behavior pattern for younger mice. Administration of bone marrow enriched with functional mitochondria to aging mice also stopped renal function deterioration, as depicted in fig. 16D.

The increase in time spent in the central area of the sport area indicates a strongly exploratory behavior of mice undergoing mitochondrial-enhanced therapy. It is combined with the decrease in thigmotaxis associated with anxiety-like behavior, demonstrating a mitochondrially enhanced anxiolytic effect.

Total motor performance and coordination capacity was also assessed in these mice using a rotarod device.

As shown in fig. 16E-16F, the vehicle and BM control groups showed a decrease in latency of drop from the rotarod at 1 month post-dosing (-2.82% and-2.18% compared to baseline, ns), which was a further decrease of 14.15% and 21.79% from baseline at 3 months post-dosing (═ 0.0008). MNV-BM-PLC mice showed a 16.17% reduction in latency of dropping from the rotarod 1 month after mitochondrial enrichment therapy (═ p ═ 0.0464), stopping the reduction 3 months after enrichment (-8.72% reduction compared to baseline, ns).

The results demonstrate that motor function impairment is more mild in mitochondrially enriched middle-aged mice relative to age-matched controls, suggesting that mitochondrion-enrichment therapy can attenuate age-related motor function deterioration.

Skeletal muscle function was also assessed in these mice by the forelimb grip test.

As shown in fig. 16G-16H, MNV-BM-PLC mice maintained their grip scores constant at 1 and 3 months post-mitochondrial potentiation therapy (-1.29% and-1.40% of baseline, respectively), and exhibited a slower decline in grip time from 3 months post-dose (wait time to release grip (+ 6.07% and-0.69% of baseline, respectively, 1 and 3 months post-dose).

As shown in figures 16I-16J, the observed-4.80% and-0.9% decrease from baseline 1 month after dosing compared to vehicle and BM controls was further exacerbated after 2 months (-15.3% and-6.35%, ns, respectively, of baseline). Baseline grip was increased in vehicle and BM control mice 1 month after dosing (+ 6.01% and + 4.06%, ns compared to baseline), and decreased to-6.03% (. p. ═ 0.0084) and-17.77% (. p. ═ 0.0404) of baseline by 2 months, respectively.

These results show a slowing/reduction of deterioration of grip strength and residence time in mitochondria-enriched treated mice, suggesting that mitochondria-enriched therapy can ameliorate age-related impaired muscle function.

Example 13 mitigating the debilitating effects of aging and age-related disorders in a human subject

The steps of the method for alleviating the effects of frailty in an aging human subject or a subject suffering from one or more age-related disorders are: (1) administering G-CSF to the aging subject or donor at a dose of 10-16 μ G/kg for 5 days; (2) on day 5, consider administering Mozobil to the subject for 1-2 days; (3) on day 6, apheresis of the subject's blood was performed to obtain bone marrow cells. If the amount of stem cells is insufficient, apheresis may be performed again on day 7; (4) in parallel, functional mitochondria were isolated from blood samples or placenta of healthy donors. The functional mitochondria can also be isolated prior to this process by storing the frozen mitochondria at-80 ℃ (at least) and thawing prior to use; (5) incubating bone marrow cells with functional mitochondria for 24 hours; (6) washing the bone marrow cells; and (7) infusing mitochondria-enriched bone marrow cells into the aging subject. Throughout the procedure, the patients were assessed for changes in food consumption, body weight, lactic acidosis, blood counts and biochemical blood markers.

Another method for reducing the debilitating effects of an aging human subject or a subject suffering from one or more age-related conditions is: (1) obtaining adipose tissue of the aging subject using a surgical procedure, such as liposuction; (2) isolating Mesenchymal Stem Cells (MSCs), propagating the cells in culture and optionally cryopreserving the cells; (3) in parallel, functional mitochondria were isolated from blood samples or placenta of healthy donors. The functional mitochondria can also be isolated prior to this process by storing the frozen mitochondria at-80 ℃ (at least) and thawing prior to use; (5) incubating MSCs with functional mitochondria for 24 hours; (6) washing the MSC; and (7) infusing the mitochondrially enriched MSCs into the subject. Throughout the procedure, the patients were assessed for changes in food consumption, body weight, lactic acidosis, blood counts and biochemical blood markers.

Example 14 treatment of human patients with nonhematopoietic tumor diseases

The method steps for the therapy of a human patient suffering from a non-hematopoietic neoplastic disease are: (1) administering G-CSF to a patient suffering from a neoplastic disease at a dose of 10-16 μ G/kg for 5 days; (2) apheresis of the patient's blood on day 6 to obtain bone marrow cells; (3) in parallel, functional mitochondria were isolated from blood samples of healthy donors; (4) incubating bone marrow cells with functional mitochondria for 24 hours; (5) washing the bone marrow cells; and (6) infusing the mitochondria-loaded bone marrow cells into the patient. Throughout the procedure, the patients were assessed for changes in food consumption, body weight, lactic acidosis, blood counts and biochemical blood markers.

Example 15 use of MNV-BLD enriched (blood-derived) for young patients with Pearson Syndrome (PS) ⁺Mitochondria) homeopathic treatment of autologous CD34 cells

A6.5 year old male patient (patient 1) was diagnosed with Pearson syndrome having a deletion in its mtDNA at nucleotide 5835-9753. Before mitochondrial enhanced therapy (MAT), he was 14.5KG in weight and was unable to walk more than 100 meters or climb stairs. His growth was significantly delayed for 3 years prior to treatment, and his weight at baseline was-4.1 Standard Deviation Score (SDS), height was-3.2 SDS (relative to the population), and was not improved despite feeding through the gastrostomy tube (G-tube) for more than one year. He had renal failure (GFR 22ml/min) and proximal glomerulopathy, requiring electrolyte supplementation. He had hypoparathyroidism requiring calcium supplementation and had incomplete right bundle branch block (ICRBB) on the electrocardiogram.

Mobilization of Hematopoietic Stem and Progenitor Cells (HSPC) was performed by subcutaneous administration of GCSF alone (10. mu.g/kg) for 5 days. Leukocyte apheresis was performed through the peripheral venous interface using the Spectra Optia system (TerumoBCT) according to institutional guidelines. Mobilized peripheral blood-derived cells were positively selected for CD34 using CliniMACS CD34 reagent according to the manufacturer's instructions. Mitochondria were isolated from maternal Peripheral Blood Mononuclear Cells (PBMCs) by differential centrifugation using 250mM sucrose buffer, pH 7.4. For mitochondrial potentiation therapy (MAT), autologous CD34 was administered⁺Cells were incubated with healthy mitochondria from the patient's mother (mitochondria at 1X10 with an amount of 4.4 milliunits Citrate Synthase (CS) per serving⁶Individual cells) resulted in a 1.56-fold increase in the mitochondrial content of the cells (a 56% increase in mitochondrial content as evidenced by CS activity). Incubation with mitochondria was performed for 24 hours at RT in saline containing 4.5% HSA. The enriched cells were suspended in a saline solution containing 4.5% human serum albumin. 1.1X 10 infusion by time line IV as shown in FIG. 17A⁶Autologous CD34 enriched with healthy mitochondria⁺Cells per kilogram body weight, the patient receiving a single round of treatment.

As can be seen in fig. 17B, the aerobic task Metabolic Equivalent (MET) score of the patient increased 4 months after transplantation of the mitochondria-enriched cells, and this effect remained unchanged 8 months after transplantation. The data demonstrate that the aerobic MET score of the patient increased significantly over time after therapy, from 5 (moderate intensity activities such as walking and cycling) to 8 (high intensity activities such as running, jogging and skipping). The MET is a physiological metric representing the energy cost of physical activity. The ability of enriched cell transplantation to improve this parameter is encouraging for aging subjects, as the aerobic MET score declines with aging.

Figure 17C illustrates lactate levels found in the patient's blood as a function of time after i.v. injection. Blood lactate, which is lactic acid that appears in the blood as a result of anaerobic metabolism at the time of mitochondrial damage or when the delivery of oxygen to tissues is insufficient to support normal metabolic demand, is one of the hallmarks of mitochondrial dysfunction. As can be seen in fig. 4C, patient 1 had decreased blood lactate levels to normal after MAT. Lactate is oxidized in mitochondria, which in part causes turnover of lactate in the human body. Lactate levels rise as mitochondrial quality and activity decline with aging. Thus, the ability of enriched bone marrow stem cells to reduce lactate levels suggests a potential effect on aging subjects.

Table 5 shows the change in the pediatric mitochondrial disease scale (IPMDS) -quality of life (QoL) questionnaire results of the patients over time after cell therapy. In both categories, "complaints and symptoms" and "physical examination", 0 means the relevant attribute "normal", and the aggravated condition is scored 1 to 5 according to severity.

Table 5.

	Before treatment	+6 months
			Complaints and symptoms	24	11
Physical examination	13.4	4.6

It should be noted that the patient did not gain weight in the 3 years prior to treatment, i.e. there was no weight gain since the age of 3.5 years. The data presented in figure 17D shows growth measured by standard deviation scores of the patient's weight and height, starting 4 years before MAT and covering the follow-up period. The data indicate that there is an increase in weight and height of the patient at about 15 months after a single treatment.

Another evidence of growth in the patient is from his alkaline phosphatase level. The alkaline phosphatase level test (ALP test) measures the amount of alkaline phosphatase in the blood stream. Having a lower than normal level of ALP in the blood may indicate malnutrition, which may be caused by a deficiency of certain vitamins and minerals. The data presented in figure 17E indicate that a single treatment was sufficient to increase the alkaline phosphatase level of the patient from 159 to 486IU/L in only 12 months. The tendency to weight loss reverses and ALP increases are associated with both aging and anti-cancer treatments that may lead to weight loss and malnutrition.

As can be seen in fig. 17F-H, treatment resulted in significant improvements in red blood cell levels (fig. 17F), hemoglobin levels (fig. 17G), and hematocrit levels (fig. 17H). These results show that a single treatment is sufficient to ameliorate the symptoms of anemia.

Figure 17I demonstrates the cessation of kidney deterioration as depicted by the creatinine levels of urine following cell transplantation. As can be further seen in fig. 17J and 17K, cell therapy also resulted in significant improvements in bicarbonate (fig. 17J) and base excess (fig. 17K) levels without supplementation of bicarbonate. Figure 17L shows the level of magnesium in the blood of the patient as a function of magnesium supplementation and time following cell therapy. The data indicate that the patient's blood magnesium levels increase significantly over time such that magnesium supplementation is no longer required. Achieving high magnesium levels without magnesium supplementation is evidence of improved magnesium absorption and reabsorption in the renal proximal tubule. As can be seen in fig. 17M-17P, a single treatment also resulted in significant reductions in the levels of several indicators of renal tubule disease, such as glucose levels in urine (fig. 17M) and levels of certain salts (fig. 17N-potassium; fig. 17O-chloride; fig. 17P-sodium). FIGS. 17I-17P are all related to aging subjects, as renal function worsens with aging.

A genetic indicator of success of the therapy used is the occupancy of normal mtDNA per cell compared to total mtDNA. As shown in fig. 18A (pt.1), the occupancy of total normal mtDNA in the peripheral blood of the patients increased from about 1 at baseline to as high as 1.6(+ 60%) in only 4 months, and to 1.9(+ 90%) after 20 months from treatment and above baseline levels at most time points. Notably, normal mtDNA levels are above baseline levels at most time points.

Another indication of the effectiveness of transplanting cells enriched with healthy functional mitochondria is presented in fig. 18B. There was a slight decrease in heterogeneity (less mtDNA missing) following MAT in patient 1, with a relatively high level of heterogeneity at baseline. This persists throughout the follow-up.

According to the reports of the neurologist in the hospital, neurological improvement was confirmed after autologous cell transplantation with healthy mitochondria without deletion mutations; the patient improves his walking skills, ability to climb stairs and ability to use scissors and painting. Substantial improvements were also noted in his ability to execute commands, reaction time, and motor and language skills. In addition, the patient's mother reports an improvement in the patient's memory. These findings are particularly relevant and important for aging subjects, since neurological deterioration of motor skills and memory usually occurs in the elderly.

As indicated by the data presented above, a single round of treatment with bone marrow stem cells enriched in functional mitochondria successfully treated a number of debilitating conditions caused by aging.

Example 16 use of MNV-BLD enriched (blood-derived line) in adolescents with Pearson Syndrome (PS) ⁺Mitochondria) homeopathic treatment of autologous CD34 cells

A 7 year old female patient (patient 2) was diagnosed with pearson syndrome with a 4977 nucleotide deletion in her mtDNA. The patient also suffers from anemia, endocrine pancreatic insufficiency, and is diabetic (HbA1C 7.1.1%). Patient 2 had high lactate levels (>25mg/dL), low body weight, and eating and weight gain problems. The patient also suffers from hypermagnesias (high levels in urine and low levels of magnesium in blood). The patient had memory and learning problems, astigmatism and low mitochondrial activity in peripheral lymphocytes, as measured by TMRE, ATP levels and O₂Consumption rate (relative to a healthy mother).

Bone marrow mobilization Using G-CSF (10. mu.g/kg) and 1 dose of plexafom Mozobil^TM(0.24 mg/ml). The patient started with 1.8 x10 according to the presented timeline⁶Autologous CD34 enriched for healthy mitochondria isolated from its mother for individual cells/kg⁺Cell therapy, in which HSPC mobilization, leukapheresis, and CD34 positive selection were performed similarly to patient 1 (example 18), except that plerixafor (n-2) was added 1 day prior to leukapheresis. Mitochondria were isolated from maternal Peripheral Blood Mononuclear Cells (PBMCs) by differential centrifugation using 250mM sucrose buffer, pH 7.4. For MAT, autologous CD34 was added⁺Cells were incubated with healthy mitochondria from the patient's mother (mitochondria at 1X10 with an amount of 4.4 milliunits Citrate Synthase (CS) per serving⁶Individual cells) resulted in a 1.62-fold increase in the mitochondrial content of the cells (a 62% increase in mitochondrial content as evidenced by CS activity). Incubation with mitochondria was performed for 24 hours at RT in saline containing 4.5% HSA. It should be noted that onlineCD34 from the patient after mitochondrial enrichment⁺The colony formation rate of the cells is improved by 26 percent.

1.8X 10 infusion by time line IV as shown in FIG. 19A⁶Autologous CD34 enriched with healthy mitochondria⁺Patient 2 (15 KG on the day of treatment) was treated per KG of body weight of cells.

Figure 19B depicts the effect of mitochondrially enriched cell transplantation on blood lactate levels, which decreased 5 months after treatment.

Muscle strength and mass are known to deteriorate with aging. Figures 19C-19E demonstrate the significant effect of enriched cell transplantation on these parameters in a series of functional experiments. Fig. 19C shows the sitting test results. Elderly people who cannot stand up from a chair without support run the risk of becoming more inactive and thus further impairing their mobility. The test subjects were invited to perform as many sitting-standing cycles as possible within a 30 second time frame. Patient 2 was able to perform more sitting up and standing cycles 5 months after transplantation. Fig. 19D depicts a 6 minute walk test (6MWT) and measures the distance in meters that the subject passes within the assigned 6 minutes. Patient 2 traveled a normal distance 5 months after transplantation. Figure 19E shows the improvement in muscle strength 5 months after cell transplantation, as evidenced by the increase in dynamometer units even after a third successive repeat of the test against the resistance of the dynamometer.

Figures 19F, 19G and 19H demonstrate improved renal function, illustrated by the ratio of magnesium, potassium and calcium, respectively, found in the urine of the patient as a function of time after i.v. injection, compared to creatinine.

Figure 19I shows the ratio between ATP8 and 18S in the urine of the patient as a function of time after i.v. injection. The immune system deteriorates with aging. Among the immune system components, the most affected by aging are T lymphocytes. In young people, naive T cells can metabolize glucose, amino acids, and lipids to catabolically promote ATP production in mitochondria. Since mitochondrial function is also known to be impaired with aging, a possible link between T cells and mitochondrial decline has been proposed and is being investigated. Figure 19J shows an increase in ATP content in lymphocytes of the patient.

Fig. 18A (pt.2) shows the occupancy of normal mtDNA as a function of time after i.v. injection. As can be seen in fig. 18A (pt.2), the occupancy of normal mtDNA increased from baseline of about 1 to as high as 2(+ 100%) for only 1 month and remained relatively high until 10 months post-treatment. Notably, normal mtDNA levels were above baseline levels at all time points.

Fig. 18B (pt.2) shows the level of heterogeneity as a function of time after MAT. It can be seen that in patient 2 there is a reduction in heterogeneity (less mtDNA present missing) after MAT. This persists throughout the follow-up.

Example 17 treatment of younger patients with Pearson Syndrome (PS) and PS-associated Vanconi syndrome (FS) ⁺Homeopathic treatment with autologous CD34 cells enriched with MNV-BLD (blood-derived mitochondria)

A female patient aged 10.5 years (patient 3) was diagnosed with Pearson syndrome having a deletion of nucleotide 12113-14421 in its mtDNA. The patient also suffers from anemia and fanconi syndrome that progresses to stage 4 renal insufficiency. The patient was treated with dialysis three times a week. Recently, patients also suffer from severe visual impairment, narrowing of the visual field and loss of near vision. The patient is completely unable to perform any physical activity (unable to walk, sitting in a stroller).

The patient has high lactate levels (>50mg/dL) and pancreatic disorders treated with insulin. Brain MRI shows many lesions and atrophic areas. The patient only eats through gastrostomy. The patient has memory and learning problems. The patient has low mitochondrial activity in peripheral lymphocytes, as measured by tetramethylrhodamine ethyl ester (TMRE), ATP levels, and O₂Consumption rate (relative to healthy mothers) determined experimentally.

Mobilization of Hematopoietic Stem and Progenitor Cells (HSPC) and leukapheresis and CD34 positive selection were performed similarly to patient 1 (example 3), except for the differences inPlerixafor (n-1) was added on day-1 prior to leukapheresis. Leukemia is performed by permanent dialysis catheters. Mitochondria were isolated from maternal Peripheral Blood Mononuclear Cells (PBMCs) by differential centrifugation using 250mM sucrose buffer, pH 7.4. For MAT, autologous CD34 was added⁺Cells were incubated with healthy mitochondria from the patient's mother (mitochondria at 1X10 with an amount of 4.4 milliunits Citrate Synthase (CS) per serving⁶Individual cells) resulting in a 1.14-fold increase in the mitochondrial content of the cells (14% increase in mitochondrial content as evidenced by CS activity). Cells were incubated with mitochondria for 24 hours at r.t. in saline containing 4.5% HSA. It should be noted that CD34 from the patient was obtained after mitochondrial enrichment⁺The colony formation rate of the cells is improved by 52 percent.

Infusion of 2.8X 10 by time line IV as shown in FIG. 20A⁶Autologous CD34 enriched with healthy mitochondria from her mother⁺Patient 3(21KG) was treated per KG of body weight.

Fig. 20B depicts the beneficial effect of a mitochondrial cell-enriched transplant on blood lactate levels, which decreased at 2 and 3 months post-transplant. The line below 20mg/dl represents normal blood lactate levels.

Fig. 20C shows the level of AST and ALT liver enzymes in the blood of the patients before and after cell therapy as a function of time. Achieving low liver enzyme levels in the blood is evidence of reduced liver damage.

Figure 20D shows the change in the levels of triglycerides, total cholesterol and Very Low Density Lipoprotein (VLDL) cholesterol in the blood of the patient over time before and after cell therapy. Achieving low triglyceride, total cholesterol, and VLDL cholesterol levels in the blood is evidence of increased liver function and improved lipid metabolism.

Glycated hemoglobin (also sometimes referred to as hemoglobin A1c, HbA1c, A1C, Hb1c, Hb1c, or HGBA1C) is a form of hemoglobin that is measured primarily to identify a three month average plasma glucose concentration. Since the lifespan of red blood cells is 4 months (120 days), the test is limited to a three month average. Fig. 20E shows the results of the A1C test on the patients before and after therapy as a function of time.

Figures 20F and 20G show the results of the "sitting test" (20F) and the "6 min walking" (20G) tests of the patients as a function of time after i.v. injection, showing an improvement of both parameters at 5 months after treatment.

Fig. 18A (pt.3) shows the occupancy of normal mtDNA as a function of time after i.v. injection. As can be seen in figure 18A (pt.3), the occupancy of normal mtDNA increased by 50% at 7 months post-treatment. Notably, normal mtDNA levels are above baseline levels at most time points.

Fig. 18B (pt.3) shows the level of heterogeneity as a function of time after MAT. It can be seen that in patient 3, which had a relatively low level of heterogeneity at baseline, there was a reduction in heterogeneity (less mtDNA present missing) following MAT. This persists throughout the follow-up.

Example 18 teenagers with Kearns-Sayre syndrome (KSS) use MNV-BLD enriched (blood from) ⁺Mitochondria of origin) of autologous CD34 cells

Patient 4 was a 14 year old, 19.5kg female patient diagnosed with Kearns-Sayre syndrome, experiencing tubular vision, ptosis, ophthalmoplegia, and retinal atrophy. The patient has vision problems, CPEO, seizures, pathological EEG, severe myopathy that fails to sit or walk, arrhythmia. The patient has a deletion of 7.4Kb in their mitochondrial DNA, including the following genes: TK, NC8, ATP8, ATP6, CO3, TG, ND3, TR, ND4L, TH, TS2, TL2, ND5, ND6, TE, NC9 and CYB.

Mobilization of Hematopoietic Stem and Progenitor Cells (HSPCs) and leukapheresis and CD34 positive selection were performed similarly to patient 3 (example 5). For MAT, autologous CD34 was added⁺Cells were compared with healthy mitochondria (mitochondria in an amount of 4.4 milliunits Citrate Synthase (CS) per serving, 1X 10) from the patient's mother⁶Individual cells) were incubated in saline containing 4.5% HSA at r.t. for 24 hours. The enrichment guideResulting in a 1.03-fold increase in the mitochondrial content of the cell (a 3% increase in mitochondrial content as demonstrated by CS activity).

Patient 4 was used 2.2 × 10 by following the timeline shown in fig. 20A⁶Autologous CD34 enriched with healthy mitochondria⁺Cells/kg body weight were treated.

Surprisingly, CD34 enriched with only 3% healthy mitochondria was used⁺The patient's EEG showed significant improvement 4 months after the single treatment, with no seizures. At 5 months after treatment, the patient suffered a disease-related Atrioventricular (AV) block and was fitted with a pacemaker. The patient recovered and continued to improve. ATP levels in peripheral blood were measured 6 months after treatment and showed an increase of approximately 100% compared to pre-treatment, as shown in figure 21. At 7 months after treatment, the patient can sit up alone, walk with assistance, talk, have better appetite and gain weight of 3.6 KG.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for performing the various disclosed functions may take a variety of different alternatives without departing from the invention.