# US 11,441,124 B2 · Mammalian cells enriched with functional mitochondria

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- Number: US 11,441,124 B2
- Document ID: US11441124B2
- Status: Granted
- Kind: B2
- Application number: 16/445,680
- Filed: 2019-06-19
- Published: 2019-10-31
- Granted: 2022-09-13
- Earliest claimed: 2015-02-26
- Assignees: Minovia Therapeutics Ltd.
- Applicants: Minovia Therapeutics Ltd.
- Inventors: Natalie Yivgi-Ohana; Uriel Halavee
- Web page: https://patentgrep.com/patent/US11441124B2

## CPC

- C12N 5/0669: {Bone marrow stromal cells; Whole bone marrow (isolated stem cells from bone marrow C12N5/0647, C12N5/0663)}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61P 7/06: Antianaemics
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/12: Antihypertensives
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 21/00: Drugs for disorders of the muscular or neuromuscular system
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 25/08: Antiepileptics; Anticonvulsants
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 27/02: Ophthalmic agents
- A61P 35/00: Antineoplastic agents
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- C12N 5/0656: {Adult fibroblasts}
- C12N 5/067: {Hepatocytes}
- C12N 5/12: Fused cells, e.g. hybridomas
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

The present invention provides human bone-marrow cells enriched with functional mitochondria, methods for their production, and therapeutic methods utilizing such cells.

## Claims

### Claim 1

An ex-vivo method for enriching human CD34+ bone-marrow cells with mitochondria, comprising:
(i) providing a first composition, comprising a plurality of intact human CD34+ bone-marrow cells obtained or derived from a patient afflicted with a mitochondrial disease or from a subject not afflicted with a mitochondrial disease;
(ii) providing a second composition, comprising a plurality of isolated intact functional human mitochondria obtained from a subject not afflicted with a mitochondrial disease;
(iii) co-incubating the human CD34+ bone-marrow cells of the first composition with the isolated intact functional human mitochondria of the second composition, thus forming a third composition; and
(iv) incubating the third composition under conditions allowing the human mitochondria to enter the human CD34+ bone-marrow cells thereby enriching said human CD34+ bone-marrow cells with said human intact functional mitochondria, thus forming a fourth composition;
wherein the mitochondrial content of the human CD34+ bone-marrow cells in the fourth composition is higher than the mitochondrial content of the human CD34+ bone-marrow cells in the first composition.

### Claim 2 (depends on 1)

The method of claim 1, further comprising determining the content or activity level of citrate synthase in the first composition and the fourth composition.

### Claim 3 (depends on 1)

The method of claim 1, wherein the bone-marrow cells in the first composition are mobilized from the bone marrow of the patient or the subject.

### Claim 4 (depends on 1)

The method of claim 1, further comprising concentrating the human CD34+ bone-marrow cells and the mitochondria in the third composition before or during incubation.

### Claim 5 (depends on 4)

The method of claim 4, further comprising centrifugation of the third composition before, during or after incubation.

### Claim 6 (depends on 1)

The method of claim 1, wherein the human CD34+ bone-marrow cells in the first composition are obtained from a patient afflicted with a mitochondrial disease, and have:
(i) a sub-normal rate of oxygen (O2) consumption;
(ii) a sub-normal content or activity level of citrate synthase;
(iii) a sub-normal rate of adenosine triphosphate (ATP) production; or
(iv) any combination of (i), (ii) and (iii).

### Claim 7 (depends on 6)

The method of claim 6, wherein the heteroplasmy level of the bone-marrow cells in the fourth composition is at least 50% lower than the heteroplasmy level of the bone-marrow cells in the first composition.

### Claim 8 (depends on 1)

The method of claim 1, wherein the human CD34+ bone-marrow cells in the first composition are obtained from a subject not afflicted with a mitochondrial disease, and have:
(i) a normal rate of oxygen (O2) consumption;
(ii) a normal content or activity level of citrate synthase;
(iii) a normal rate of adenosine triphosphate (ATP) production; or
(iv) any combination of (i), (ii) and (iii).

### Claim 9 (depends on 1)

The method of claim 1, wherein the isolated human mitochondria in the second composition are obtained from a subject not afflicted with a mitochondrial disease, and have:
(i) a normal rate of oxygen (O2) consumption;
(ii) a normal content or activity level of citrate synthase;
(iii) a normal rate of adenosine triphosphate (ATP) production; or
(iv) any combination of (i), (ii) and (iii).

### Claim 10 (depends on 1)

The method of claim 1, further comprising measuring the total amount of mitochondrial proteins in the second composition, wherein the total amount is between 20%-80% of the total amount of cellular proteins within the composition.

### Claim 11 (depends on 1)

The method of claim 1, comprising measuring the fourth composition for cytochrome C reductase or cytochrome C reductase activity, wherein cytochrome C reductase or cytochrome C reductase activity is not enriched as compared to the first composition.

### Claim 12 (depends on 1)

The method of claim 1, wherein the mitochondrial disease is a mitochondrial respiratory chain disease (MRCD).

### Claim 13 (depends on 1)

The method of claim 1, wherein the mitochondrial disease is selected from the group consisting of LHON (Leber's hereditary optic neuropathy); MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms); Pearson syndrome; Leigh syndrome; NARP (neuropathy, ataxia, retinitis pigmentosa, and ptosis); MERRF (myoclonic epilepsy with ragged red fibers); KSS (Kearns-Sayre Syndrome); MNGIE (myoneurogenic gastrointestinal encephalopathy); Friedreich Ataxia; and Alpers' disease.

### Claim 14 (depends on 13)

The method of claim 13, wherein the mitochondrial disease is selected from the group consisting of LHON, MELAS, Pearson syndrome, Leigh syndrome, NARP, MERRF, and KSS.