Brain-tropic stem cells

Abstract

The present invention relates to a population of cells overexpressing cytochrome c oxidase subunit VIIb (COX7B) protein, and a pharmaceutical composition or kit comprising the same, for use in treating and / or preventing brain diseases or disorders in a subject in need thereof. The present invention further relates to a method for inducing brain tropism of cells, preferably stem cells, by utilizing overexpression of COX7B.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present invention relates to the use of a population of cells overexpressing cytochrome c oxidase subunit VIIb (COX7B) protein for use in treating and / or preventing brain diseases. [Background technology]

[0002] Neurodegeneration is a pathological condition that leads to neuronal cell death. Although the causes of neurodegeneration are diverse and not always identifiable, numerous brain diseases share neurodegeneration as a common pathological condition. For example, Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS) all cause chronic neurodegeneration, which is characterized by slowly progressive neuronal cell death over several years. Acute neurodegeneration is characterized by the sudden onset of neuronal cell death as a result of ischemia, such as stroke, trauma, such as traumatic brain injury, or as a result of demyelination or traumatic axonal severance, for example, due to spinal cord injury or multiple sclerosis. Regardless of the underlying cause, mounting evidence suggests that once neurodegeneration is initiated, the outcome for all these diseases is always the same: the ultimate death of neurons.

[0003] Neurodegeneration is a particularly challenging biological environment for cell therapy. One of the problems in cell therapy is the low survival rate of transplanted cells (less than 5%), because transplanted cells tend to undergo significant cell death immediately after in vivo injection. However, the most important issue is targeting the transplanted cells: the brain is relatively isolated from other organs (especially by the blood-brain barrier, BBB). This means that cells must be injected into the brain, which is a particularly heavy and difficult medical intervention. In fact, there are two major problems with injecting stem cells into the brain. The first problem is the transplantation procedure itself, which raises questions about safety, ethics, and efficacy. It is invasive and ideally should be repetitive (i.e., chronic treatment with multiple injections at different time points), and spatial administration depends on the ability to detect lesions / degenerated areas in the brain. The second problem is the limited ability of transplanted cells to migrate from the injection site to widespread diseased areas, which is crucial for therapeutic efficacy in the central nervous system (see, for example, De Gioia, R., et al., Neural Stem Cell Transplantation for Neurodegenerative Diseases. Int J Mol Sci, 2020, 21(9)). Typically, injected cells aggregate and form clumps at the injection site in the brain parenchyma, and the transplantation efficiency / survival rate of injected cells is estimated to be less than 5%. Stem cells can also be delivered by lumbar puncture into the cerebrospinal fluid. Currently, this method is more efficient than intravenous administration, especially because more cells are found at the injury site.

[0004] Thus, there is an unmet need to target cells to the brain in cell therapy for brain diseases.

[0005] The Applicant has surprisingly provided a solution for increasing the brain tropism of cells. Summary of the Invention

[0006] The present invention relates to a population of cells that overexpress the cytochrome c oxidase subunit VIIb (COX7B) protein for use in treating and / or preventing a brain disease or disorder in a subject in need thereof.

[0007] In one embodiment, the cell is a stem cell, preferably a mesenchymal stem cell or a neural stem cell, hi one embodiment, the cell is a mammalian cell, preferably a human cell.

[0008] In one embodiment, the COX7B protein is overexpressed at a level that induces brain tropism in the cells. In one embodiment, the cells stably overexpress the COX7B protein.

[0009] In one embodiment, the COX7B protein is a mammalian COX7B protein, preferably a human COX7B protein.

[0010] In one embodiment, the cells further express at least one surface receptor that binds to a brain-specific ligand.

[0011] In one embodiment, the brain disease or disorder is selected from the group consisting of neurodegenerative diseases, neurological diseases, brain injuries, and cerebrovascular diseases. In one embodiment, the brain disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, diffuse Lewy body dementia, frontotemporal dementia (FTD), Lewis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, basal ganglia degeneration, Parkinson-ALS-dementia complex. and / or selected from the group consisting of Guam-Pick disease, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, mild cognitive impairment, syphilis, attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, bipolar disorder, stress disorders, spinal cord injury, myelitis, rheumatic diseases, traumatic brain injury (TBI), concussion, vascular dementia, high blood pressure, toxic brain injury such as hypoxia or carbon monoxide poisoning, encephalitis, stroke, brain tumor, brain abscess, autism spectrum disorder, and the like.

[0012] In one embodiment, the population of cells is administered to a subject in need thereof in a therapeutically effective amount, in one embodiment, the therapeutically effective amount is about 1,000 to about 100,000 billion of said cells per kg of body weight.

[0013] In one embodiment, the population of cells is administered to the subject systemically, preferably by intravenous injection.

[0014] The present invention also relates to a pharmaceutical composition comprising a population of cells that overexpress COX7B protein and a pharmaceutically acceptable vehicle for use in the prevention and / or treatment of a brain disease or disorder.

[0015] Another object of the present invention is a combination kit comprising (i) a population of cells overexpressing COX7B protein or a pharmaceutical composition containing the same and (ii) another therapeutic agent for use in the prevention and / or treatment of brain diseases or disorders.

[0016] The present invention further relates to a method for inducing brain tropism in cells, preferably stem cells, comprising the step of overexpressing COX7B protein in the cells. definition

[0017] In the present invention, the following terms have the following meanings:

[0018] The term "about," when preceding a numerical value, means plus or minus 10% of that numerical value.

[0019] The term "identity" refers to a measure of the identity of nucleotide or amino acid sequences. Generally, sequences are aligned to obtain the highest order match. "Identity" itself has a meaning recognized by those of skill in the art and can be calculated using published techniques. Methods for determining identity and similarity are incorporated into computer programs. Computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package and the GAP program. By way of example, a polynucleotide having a nucleotide sequence that is at least, e.g., 95%, "identity" to a reference nucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may contain an average of up to 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with alternative nucleotides, and multiple nucleotides, up to 5% of the total number of nucleotides in the reference sequence, may be inserted into the reference sequence. These variations in the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, and may be interspersed individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

[0020] The term "pharmaceutically acceptable excipient" refers to an excipient that does not cause adverse, allergic, or other untoward reactions when administered to animals, preferably humans. This includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type. For human administration, preparations must meet sterility, pyrogenicity, general safety, and purity standards as required by EMA or FDA biologics standards.

[0021] The terms "treating" or "treatment" or "alleviation" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or delay (alleviate) a disease or condition, preferably a brain disease or disorder. Those in need of treatment include those already suffering from a disease or condition, preferably a brain disease or disorder, as well as those who are predisposed to a disease or condition, preferably a brain disease or disorder, or those in whom a disease or condition, preferably a brain disease or disorder, is to be prevented. A subject or mammal is successfully "treated" for a disease or condition, preferably a brain disease or disorder, if, after receiving a therapeutic amount of a population of cells for use in accordance with the present invention, the patient shows an observable and / or measurable decrease or elimination in one or more symptoms associated with the disease or condition, preferably a brain disease or disorder, and / or reduced morbidity and mortality, and / or improvement in quality of life issues. The above parameters for assessing successful treatment and improvement in a disease or condition, preferably a brain disease or disorder, can be readily measured by routine procedures familiar to physicians.

[0022] A "therapeutically effective amount" refers to a level or amount of an agent intended to (1) delay or prevent the onset of a disease or condition, (2) delay or arrest the progression, worsening, or deterioration of one or more symptoms of a disease or condition, (3) bring about an improvement in the symptoms of a disease or condition, (4) reduce the severity or incidence of a disease or condition, or (5) cure a disease or condition, without causing significant negative or adverse side effects to the target. A therapeutically effective amount may be administered prior to the onset of a disease or condition for prophylactic or preventative measures. Alternatively, or in addition, a therapeutically effective amount may be administered after the onset of a disease or condition for therapeutic action.

[0023] The term "stem cell" refers to a progenitor cell capable of self-renewal, i.e., proliferation without differentiation, such that the progeny of the stem cell, or at least some thereof, substantially retain the specialized or less specialized phenotype, differentiation potential, and proliferation potential of the parent stem cell. The term encompasses stem cells that are capable of self-renewal substantially indefinitely, i.e., the ability of the progeny cell, or some portion thereof, for further proliferation is not substantially reduced compared to the parent cell, and stem cells that exhibit limited self-renewal, i.e., the ability of the progeny cell, or some portion thereof, for further proliferation is significantly reduced compared to the parent cell.

[0024] The term "subject" or "individual" refers to an individual animal, preferably an individual mammal, more preferably a human individual. In some embodiments, the individual may be a mammal. Mammals include, but are not limited to, all primates (human and non-human), cattle (including cows), horses, pigs, sheep, goats, dogs, cats, and other mammals awaiting medical treatment, undergoing medical treatment, or have been / will be / will be the subject of medical treatment, or are being monitored for the development of a disease or condition, preferably a brain disease or disorder. In some embodiments, the individual may be a "patient," i.e., a warm-blooded animal, more preferably a human, awaiting medical treatment, undergoing medical treatment, or have been / will be the subject of medical treatment, or are being monitored for the development of a disease or condition, preferably a brain disease or disorder. In some embodiments, the individual is an adult (e.g., an individual over the age of 18). In some embodiments, the individual is a child (e.g., an individual under the age of 18). In some embodiments, the individual is male. In some embodiments, the individual is female. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a population of cells that overexpress the cytochrome c oxidase subunit VIIb (COX7B) protein for use in treating and / or preventing a brain disease or disorder in a subject in need thereof.

[0026] In some embodiments, the cells are stem cells.

[0027] Stem cells are known in the art to be undifferentiated cells with a very high proliferation potential that can differentiate into multiple cell types. Stem cells can be totipotent, pluripotent, or multipotent. Within the scope of the present invention, stem cells are preferably multipotent, i.e., cells can differentiate into multiple cell types in a specific cell lineage.

[0028] In preferred embodiments, the stem cells are mesenchymal stem cells or neural stem cells. In preferred embodiments, the stem cells are neural stem cells. In certain embodiments, neural stem cells give rise to neurons.

[0029] As used herein, the terms "neural stem cell" and "neural progenitor cell" are used interchangeably.

[0030] In another embodiment, the stem cells are mesenchymal stem cells.

[0031] In some embodiments, the stem cells are adult stem cells or embryonic stem cells. In preferred embodiments, the stem cells are adult stem cells. In certain embodiments, the stem cells are induced pluripotent stem cells (iPSCs) or are derived from iPSCs.

[0032] In some embodiments, the cells are mammalian cells, preferably human cells. In some embodiments, the cells are obtained from a mammalian donor, preferably a human donor.

[0033] In some embodiments, the stem cells are neuronal precursors, oligodendrocyte precursors, astrocyte precursors, or microglial precursors. In some embodiments, the stem cells are neuronal precursors. In some embodiments, the stem cells are oligodendrocyte precursors. In some embodiments, the stem cells are astrocyte precursors. In some embodiments, the stem cells are microglial precursors.

[0034] Those skilled in the art will appreciate that at least some aspects of harvesting cells from donor subjects, particularly human subjects, must comply with respective legal and ethical regulations. By way of example and not limitation, harvesting cells from a living human donor may need to be compatible with the further survival of the donor.

[0035] Therefore, only a small portion of the cell population is usually removed from the tissue or organ of a living human donor, for example, by biopsy or resection, so that an appropriate level of physiological function is maintained in the donor. On the other hand, the collection of cells from non-human animals is also contemplated, but does not necessarily have to be consistent with the further survival of the non-human animal. For example, the non-human animal may be humanely killed after the collection of cells. These and similar considerations are clear to those skilled in the art and reflect legal and ethical standards.

[0036] The cells can be obtained from a donor, preferably a human donor, who maintains circulatory function, e.g., a beating heart, and respiratory function, e.g., pulmonary respiration or artificial ventilation. Harvesting cells from such donors is advantageous because the tissue does not experience the significant hypoxia (lack of oxygen supply) that normally occurs with ischemia (cessation of circulation).

[0037] In a preferred embodiment, the cells are derived from the subject to be treated.It should be understood that the purpose of autologous transplantation (i.e., administering cells from the same organism into an organism) is to minimize or eliminate the risk of immune reaction to the transplant, which induces rejection of the transplant.Those skilled in the art will understand the safety of transplanted cells.

[0038] In another embodiment, the cells are from an individual different from the subject, preferably an individual of the same species as the subject. In some embodiments, the cells are ReNcell cells.

[0039] Populations of cells for use in accordance with the present invention may be homogeneous or heterogeneous, preferably homogeneous.

[0040] In one embodiment, a population of cells for use according to the present invention is homogeneous. In some embodiments, a population of cells for use according to the present invention is substantially free of other cell types. In some embodiments, a population of cells for use according to the present invention is substantially free of cells that do not overexpress COX7B. As used herein, "substantially free" means 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01%, 0.001% or less.

[0041] In another embodiment, the population of cells for use according to the invention is heterogeneous. In a particular embodiment, the population of cells for use according to the invention comprises at least two cell types.

[0042] In some embodiments, populations of cells for use according to the present invention may be frozen (i.e., cryopreserved) for long-term storage. In some embodiments, populations of cells for use according to the present invention may be cryopreserved in liquid nitrogen or at any temperature, preferably about 0°C to about -196°C, more preferably about -20°C to about -196°C, and even more preferably about -80°C to about -196°C, so long as the cells are usable as stem cells after thawing. In some embodiments, populations of cells for use according to the present invention are contacted with at least one cryoprotectant, such as glycerol, prior to freezing. In one embodiment, populations of cells for use according to the present invention may be thawed and further expanded to obtain fresh cells.

[0043] In some embodiments, populations of cells for use according to the present invention are cultured, i.e., seeded and grown in vitro. General methods for maintaining populations of cells in culture are well known in the art. The environment in which cells are cultured includes at least a cell culture medium, typically a liquid medium, which supports the survival and / or growth of the cells. The terms "cell culture medium" or "cell culture medium" or "medium" refer to an aqueous liquid or gel-like substance containing nutrients that can be used for the maintenance or growth of cells. Cell culture medium may contain serum or may be serum-free.

[0044] Typically, the medium comprises a basal medium formulation well known in the art. Non-limiting examples of basal medium formulations include Eagle's Minimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), alpha modified minimal essential medium (α-MEM), Basal Medium (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJb Medium, F-12 Nutrient Mixture (Ham), Liebovitz L-15, DMEM / F-12, Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199, Waymouth's MB 752 / 1, Miltenyi's StemMacs™, Prime-XV from FUJIFILM Irvine Scientific, or Williams Medium E, and variations and / or combinations thereof. The compositions of the above basal media are generally known in the art, and it is within the skill of a person skilled in the art to appropriately modify or adjust the concentrations of the medium and / or medium supplements required for culturing cells. In a preferred embodiment, the medium is a commercially available serum-free medium that supports the growth of stem cells.

[0045] Such basal medium formulations contain components necessary for the growth of mammalian cells, which are known per se. For example, but not limited to, these components may include inorganic salts (particularly salts containing Na, K, Mg, Ca, Cl, P, and optionally Cu, Fe, Se, Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides, nucleosides and / or nucleic acid bases, ribose, deoxyribose, amino acids, vitamins, antioxidants (e.g., glutathione), and carbon sources (e.g., glucose, pyruvic acid, e.g., sodium pyruvate, acetic acid, e.g., sodium acetate). It will also be apparent that many media are available as low-glucose formulations, with or without sodium pyruvate.

[0046] For use in culture, basal media may be supplemented with one or more additional components. For example, additional supplements can be used to provide cells with trace elements and substances necessary for optimal growth and proliferation. Such supplements include insulin, transferrin, selenium salts, and combinations thereof. These components may be included in salt solutions such as Hank's Balanced Salt Solution (HBSS) and Earle's Salt Solution, but are not limited to these. Additionally, antioxidant supplements, such as β-mercaptoethanol, may be added. While many basal media already contain amino acids, some amino acids, such as L-glutamine, which are known to be less stable in solution, may be supplemented later. The medium may further be supplemented with antibiotic and / or antifungal compounds, typically mixtures of penicillin and streptomycin, and / or compounds such as, but not limited to, amphotericin, ampicillin, gentamicin, bleomycin, hydromycin, kanamycin, mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin, paromomycin, polymyxin, puromycin, rifampin, spectinomycin, tetracycline, tylosin, and zeocin.

[0047] In some embodiments, the cells are maintained under culture conditions that do not induce differentiation of the cells. In some embodiments, the cells are maintained under culture conditions that do not induce commitment of the cells.

[0048] In some embodiments, the COX7B protein is a mammalian COX7B protein, preferably a human COX7B protein.

[0049] Indeed, COX7B, having Entrez Gene ID No. 1349, also refers, without limitation, to cytochrome C oxidase subunit 7B; mitochondrial cytochrome C oxidase subunit 7B; cytochrome C oxidase polypeptide VIIb; cytochrome C oxidase subunit VIIb; cytochrome-C oxidase chain VIIb; LSDMCA2 or APLCC.

[0050] Cytochrome c oxidase subunit 7B (COX7B) typically has the amino acid sequence shown in SEQ ID NO:1 in humans.

[0051] In some embodiments, the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO:1.

[0052] In some embodiments, the COX7B protein is wild-type human COX7B. In some embodiments, the COX7B protein has the amino acid sequence of SEQ ID NO:1.

[0053] In some embodiments, the COX7B protein is a mutant COX7B. In some embodiments, the COX7B protein comprises at least one amino acid mutation compared to the sequence of SEQ ID NO: 1. As used herein, "at least one" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20. In some embodiments, "amino acid mutation" includes substitutions, deletions, insertions, inversions, and combinations thereof.

[0054] In some embodiments, the mutations in COX7B do not have any adverse effects on the cells, i.e., the mutations do not alter the cell's ability to survive, proliferate, and / or differentiate. In some embodiments, the mutations are not tumorigenic.

[0055] The present inventors have surprisingly found that the COX7B protein is involved in controlling the brain tropism of cells (see Examples).

[0056] In some embodiments, the COX7B protein is overexpressed at a level that induces brain tropism of the cells.

[0057] As used herein, "inducing brain tropism" means that, upon administration to a subject, preferably in the subject's systemic circulation, an increased proportion of a population of cells according to the present invention migrates to the subject's brain compared to the same type of cells without overexpression of COX7B.

[0058] In some embodiments, the brain tropism of a population of cells according to the present invention is increased by at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1,000-fold, 10,000-fold, 100,000-fold, or more compared to cells of the same type without COX7B overexpression. In some embodiments, overexpression of COX7B is required to induce brain tropism, i.e., cells of the same type without COX7B overexpression have 0% brain localization.

[0059] In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the population of cells according to the invention are localized to the brain of the subject, hi some embodiments, 100% of the population of cells according to the invention are localized to the brain of the subject.

[0060] In some embodiments, the cells overexpress COX7B protein by at least 1.2-fold. It should be understood that an increase of at least 1.2-fold is relative to the COX7B expression level prior to overexpression or relative to the COX7B expression level in an identical population of cells that do not overexpress COX7B. As used herein, "at least 1.2 times" means 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, 200 times, 300 times, 400 times, 500 times, 600 times, 700 times, 800 times, 900 times, 1000 times, 10,000 times, 100,000 times or more.

[0061] Overexpression of COX7B can be controlled and / or measured by methods well known in the art. In some embodiments, overexpression of COX7B in a population of cells is measured prior to administration to a subject.

[0062] In some embodiments, COX7B overexpression is measured at the protein level. Non-limiting examples of methods that allow for the quantification of specific proteins (i.e., COX7B proteins) include Western blotting, immunofluorescence, quantitative flow cytometry, and liquid chromatography-mass spectrometry (LC-MS).

[0063] In some embodiments, COX7B overexpression is measured at the RNA level, preferably at the messenger RNA (mRNA) level.Non-limiting examples of methods that allow the quantification of specific RNA (i.e., COX7B RNA, preferably COX7B mRNA) include RNA-seq, quantitative reverse transcription polymerase chain reaction (RT-qPCR), quantitative polymerase chain reaction (or "real-time polymerase chain reaction", qPCR), and fluorescent nucleic acid probes.

[0064] In some embodiments, the cells stably or transiently overexpress the COX7B protein. In a preferred embodiment, the cells stably overexpress the COX7B protein. In another embodiment, the cells transiently overexpress the COX7B protein.

[0065] In some embodiments, the COX7B protein is encoded by an exogenous or endogenous nucleic acid. In some embodiments, the exogenous or endogenous nucleic acid comprises a COX7B gene, preferably a mammalian COX7B gene, more preferably a human COX7B gene. In some embodiments, the COX7B protein is encoded by an exogenous nucleic acid. In some embodiments, the COX7B protein is encoded by an endogenous nucleic acid, typically the COX7B protein is encoded by at least two copies of the COX7B gene in the genome of the cell.

[0066] To achieve overexpression, it is conventional in the art to place the gene to be overexpressed under the control of a promoter, preferably a "strong" promoter. In some embodiments, the foreign or endogenous nucleic acid further comprises a promoter, preferably a strong promoter. Non-limiting examples of strong promoters include the cytomegalovirus (CMV) promoter, the phosphoglycerokinase (PGK) promoter, and the cytomegalovirus / actin / β-globin (CAG) promoter. To achieve overexpression, it is also conventional in the art to incorporate the gene to be overexpressed into a vector containing a selection cassette, such as a selection cassette that confers resistance to a certain antibiotic, such as hygromycin.

[0067] In some embodiments, overexpression of COX7B is achieved by contacting a population of cells with a vector suitable for overexpressing COX7B. In some embodiments, the vector comprises at least one nucleic acid as defined herein. In some embodiments, the vector comprises a human COX7B ORF cDNA sequence.

[0068] In some embodiments, the vector is an integrating vector or a non-integrating vector.

[0069] In some embodiments, the vector is an integrating vector. In some embodiments, the integrating vector comprises or is selected from the group consisting of an integrating virus, an integrating plasmid, an enzyme comprising modified transposase or modified integrase, or a genome editing method comprising CRISPR-Cas9.

[0070] In some embodiments, the integrating vector is a virus selected from the group consisting of viruses from the viral families Retroviridae, Adenoviridae, Flaviviridae, Herpesviridae, Hepadnaviridae, Papillomaviridae, Polyomaviridae, Parvoviridae, Arenaviridae, Bornaviridae, Bunyaviridae, Filoviridae, and Paramyxoviridae, preferably an integrating virus selected from the group consisting of viruses from the families Retroviridae, Adenoviridae, and Flaviviridae. In some embodiments, the integrating virus belongs to the Retroviridae family. In some embodiments, the integrating virus is a lentivirus.

[0071] In some embodiments, genome editing method is used to insert at least one copy of COX7B gene into the genome of cell.Non-limiting examples of genome editing method include CRISPR-Cas9, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN) etc.The means for carrying out these methods are well known in the art.

[0072] In some embodiments, overexpression of COX7B does not affect cell function and / or viability.

[0073] In some embodiments, the vector is a non-integrating vector. In some embodiments, the non-integrating vector is a viral vector or a non-viral vector.

[0074] In some embodiments, the vector is a non-integrating viral vector including or selected from the group consisting of adenoviral vectors, adeno-associated viral (AAV) vectors, integration-defective retroviral vectors (IDLVs), poxvirus vectors, and herpes simplex viral vectors.

[0075] In some embodiments, the vector is a non-integrating, non-viral vector selected from the group comprising or consisting of a plasmid, a fosmid, a cosmid, an artificial chromosome (e.g., a human artificial chromosome), a nanoparticle (e.g., a polymer-based nanoparticle, a proteoliposome, or a lentivirus-like particle), a minicircle DNA, etc. In some embodiments, the non-integrating, non-viral vector is a plasmid or a nanoparticle.

[0076] In some embodiments, the non-integrating, non-viral vector is a plasmid. In some embodiments, the plasmid is a pCMV3 plasmid. In some embodiments, the plasmid is a pCMV3 that encodes a human COX7B ORF cDNA sequence.

[0077] In another embodiment, overexpression of COX7B is achieved by contacting a population of cells with a naked nucleic acid that expresses COX7B. In some embodiments, the naked nucleic acid enters the cells by a means including or selected from the group consisting of electroporation, sonoporation, ballistic propulsion of DNA-coated particles, and injection.

[0078] Those skilled in the art will understand techniques for transient or stable expression of nucleic acid sequences (e.g., with non-integrating vectors) and other important parameters such as confluence (e.g., transfection at 30-60% confluence) or the time period to reach appropriate expression levels (7 days, 14 days, or longer).

[0079] In some embodiments, the cells further express at least one surface receptor that binds to a brain-specific ligand, hi some embodiments, expression of the at least one surface receptor further enhances the brain tropism induced by overexpression of COX7B.

[0080] As used herein, "brain disease or disorder" refers to any type of disease or condition that occurs in the brain of an animal, preferably the human brain. As used herein, "degenerative brain disease" or "neurodegenerative disease" is a subset of brain diseases and refers to any brain disease that results from degenerative changes in nerve cells of the nervous system. The causes of degenerative brain diseases are largely unknown, and these diseases have a slow onset and progress continuously by selectively invading the associated nervous system.

[0081] In some embodiments, the brain disease or disorder comprises or is selected from the group consisting of neurodegenerative diseases, neurological diseases, brain injury, and cerebrovascular diseases. In some embodiments, the brain disease or disorder comprises neuronal cell death. In some embodiments, the brain disease or disorder comprises or consists of increased neuronal cell death, where "increased neuronal cell death" refers to an increase of 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1,000-fold, 10,000-fold, or more compared to healthy subjects.

[0082] In some embodiments, the brain disease or disorder is Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, diffuse Lewy body dementia, frontotemporal dementia (FTD), Lewis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, corticobasal degeneration, Parkinson's-ALS- and / or selected from the group consisting of dementia complex (Guam-Pick disease), amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, mild cognitive impairment, syphilis, attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, bipolar disorder, stress disorders, spinal cord injury, myelitis, rheumatic diseases, traumatic brain injury (TBI), concussion, vascular dementia, hypertension, toxic brain injury such as hypoxia or carbon monoxide poisoning, encephalitis, stroke, brain tumor, brain abscess, autism spectrum disorder, and the like.

[0083] In some embodiments, the brain disease or disorder comprises or is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, diffuse Lewy body dementia, frontotemporal dementia (FTD), Lewis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, corticobasal degeneration, Parkinson-ALS-Guam dementia-Pick complex, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, mild cognitive impairment, syphilis, attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, bipolar disorder, stress disorders, and the like.

[0084] In some embodiments, the brain disease or disorder comprises or is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, diffuse Lewy body dementia, frontotemporal dementia (FTD), Lewis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, corticobasal degeneration, Parkinson-ALS-Guam dementia complex, and Pick's disease, amyloidosis, Pick's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, and the like.

[0085] In some embodiments, the brain disease or disorder comprises or is selected from the group consisting of traumatic brain injury (TBI), concussion, vascular dementia, hypertension, toxic brain injury such as hypoxia or carbon monoxide poisoning, encephalitis, stroke, brain tumor, brain abscess, autism spectrum disorder, and the like.

[0086] In some embodiments, the brain disease or disorder involves increased permeability of the blood-brain barrier (BBB).

[0087] In some embodiments, the population of cells compensates for neuronal cell death, ie, compensates for at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of neuronal cell death induced by a brain disease or disorder.

[0088] In some embodiments, administering the population of cells in a subject suffering from a brain disease or disorder increases the number of neuronal cells by at least 1.1 fold, 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 1.6 fold, 1.7 fold, 1.8 fold, 1.9 fold, 2 fold, 3 fold, 4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 20 fold or more compared to an untreated subject with the same brain disease or disorder.

[0089] In some embodiments, the population of cells restores normal brain function in a subject suffering from a brain disease or disorder.

[0090] In some embodiments, the population of cells is administered to a subject in need thereof in a therapeutically effective amount.

[0091] In some embodiments, the therapeutically effective amount is about 1,000 to about 100,000 billion cells per kg of body weight.

[0092] In some embodiments, a therapeutically effective amount is about 2,000 to about 100,000 billion cells per kg of body weight, about 3,000 to about 100,000 billion cells per kg of body weight, about 4,000 to about 100,000 billion cells per kg of body weight, about 5,000 to about 100,000 billion cells per kg of body weight, about 6,000 to about 100,000 billion cells per kg of body weight, about 7,000 to about 100,000 billion cells per kg of body weight, about 8,000 to about 100,000 billion cells per kg of body weight, about 9,000 to about 100,000 billion cells per kg of body weight, or about 11,000 to about 120,000 billion cells per kg of body weight. 100,000 billion cells, approximately 10,000 to approximately 100,000 billion cells per kg of body weight, approximately 20,000 to approximately 100,000 billion cells per kg of body weight, approximately 30,000 to approximately 100,000 billion cells per kg of body weight, approximately 40,000 to approximately 100,000 billion cells per kg of body weight, approximately 50,000 to approximately 100,000 billion cells per kg of body weight, approximately 60,000 to approximately 100,000 billion cells per kg of body weight, approximately 70,000 to approximately 100,000 billion cells per kg of body weight, approximately 80,000 to approximately 100,000 billion cells per kg of body weight Approximately 100,000 billion cells, approximately 90,000 to approximately 100,000 billion cells per kg of body weight, approximately 100,000 to approximately 100,000 billion cells per kg of body weight, approximately 200,000 to approximately 100,000 billion cells per kg of body weight, approximately 300,000 to approximately 100,000 billion cells per kg of body weight, approximately 400,000 to approximately 100,000 billion cells per kg of body weight, approximately 500,000 to approximately 100,000 billion cells per kg of body weight, approximately 600,000 to approximately 100,000 billion cells per kg of body weight, approximately 7 00,000 to approximately 100,000 billion cells per kg of body weight, approximately 800,000 to approximately 100,000 billion cells per kg of body weight, approximately 900,000 to approximately 100,000 billion cells per kg of body weight, approximately 1,000,000 to approximately 100,000 billion cells per kg of body weight, approximately 2,000,000 to approximately 100,000 billion cells per kg of body weight, approximately 3,000,000 to approximately 100,000 billion cells per kg of body weight, approximately 4,000,000 to approximately 100,000 billion cells per kg of body weight, approximately 5,000,000 to approximately 100,00 billion cells, about 6,000,000 to about 100,000 billion cells per kg of body weight, about 7,000,000 to about 100,000 billion cells per kg of body weight, about 8,000,000 to about 100,000 billion cells per kg of body weight, and about 9,000,000 to about 100,000 billion cells per kg of body weight.

[0093] In some embodiments, a therapeutically effective amount is from about 1,000 to about 9,000 billion cells per kg of body weight, from about 1,000 to about 8,000 billion cells per kg of body weight, from about 1,000 to about 7,000 billion cells per kg of body weight, from about 1,000 to about 6,000 billion cells per kg of body weight, from about 1,000 to about 5,000 billion cells per kg of body weight, from about 1,000 to about 4,000 billion cells per kg of body weight, from about 1,000 to about 3,000 billion cells per kg of body weight, or from about 1,000 to about 2,000 billion cells per kg of body weight. 0 to approximately 2,000 billion cells, approximately 1,000 to approximately 1,000 billion cells per kg of body weight, approximately 1,000 to approximately 900 billion cells per kg of body weight, approximately 1,000 to approximately 800 billion cells per kg of body weight, approximately 1,000 to approximately 700 billion cells per kg of body weight, approximately 1,000 to approximately 600 billion cells per kg of body weight, approximately 1,000 to approximately 500 billion cells per kg of body weight, approximately 1,000 to approximately 400 billion cells per kg of body weight, approximately 1,000 to approximately 3,000 billion cells, approximately 1,000 to approximately 200 billion cells per kg of body weight, approximately 1,000 to approximately 100 billion cells per kg of body weight, approximately 1,000 to approximately 90 billion cells per kg of body weight, approximately 1,000 to approximately 80 billion cells per kg of body weight, approximately 1,000 to approximately 70 billion cells per kg of body weight, approximately 1,000 to approximately 60 billion cells per kg of body weight, approximately 1,000 to approximately 50 billion cells per kg of body weight, approximately 1,000 to approximately 40 billion cells per kg of body weight, approximately 1,000 to approximately 300 billion cells, about 1,000 to about 20 billion cells per kg of body weight, about 1,000 to about 10 billion cells per kg of body weight, about 1,000 to about 9 billion cells per kg of body weight, about 1,000 to about 8 billion cells per kg of body weight, about 1,000 to about 6 billion cells per kg of body weight, about 1,000 to about 5 billion cells per kg of body weight, about 1,000 to about 4 billion cells per kg of body weight, about 1,000 to about 3 billion cells per kg of body weight, and about 1,000 to about 2 billion cells per kg of body weight.

[0094] In some embodiments, the therapeutically effective amount is from about 1,000 to about 10,000,000 cells per kg of body weight.

[0095] In some embodiments, a therapeutically effective amount is about 2,000 to about 10,000,000 cells per kg of body weight, about 3,000 to about 10,000,000 cells per kg of body weight, about 4,000 to about 10,000,000 cells per kg of body weight, about 5,000 to about 10,000,000 cells per kg of body weight, about 6,000 to about 10,000,000 cells per kg of body weight, about 7,000 to about 10,000,000 cells per kg of body weight, about 8,000 to about 10,000,000 cells per kg of body weight, about 9,000 to about 10,000,000 cells per kg of body weight, about 10,000 to about 10,000,000 cells per kg of body weight, about 11,000 to about 11,000,000 cells per kg of body weight, about 12,000 to about 12,000,000 cells per kg of body weight, about 13,000 to about 13,000,000 cells per kg of body weight, about 14,000 to about 14,000,000 cells per kg of body weight, about 15,000 to about 15,000,000 cells per kg of body weight, about 16,000 to about 16,000,000 cells per kg of body weight, about 17,000 to about 17,000,000 cells per kg of body weight, about 18,000 to about 18,000,000 cells per kg of body weight, about 19,000 to about 19,000,000 cells per kg of body weight, about 20, Approximately 9,000 to 10,000,000 cells per kg of body weight, approximately 10,000 to 10,000,000 cells per kg of body weight, approximately 20,000 to 10,000,000 cells per kg of body weight, approximately 30,000 to 10,000,000 cells per kg of body weight, approximately 40,000 to 10,000,000 cells per kg of body weight, approximately 50,000 to 10,000,000 cells per kg of body weight, approximately 60,000 to 10,000,000 cells per kg of body weight, approximately 70,000 to 10,000,000 cells per kg of body weight 00 cells, about 80,000 to about 10,000,000 cells per kg of body weight, about 90,000 to about 10,000,000 cells per kg of body weight, about 100,000 to about 10,000,000 cells per kg of body weight, about 200,000 to about 10,000,000 cells per kg of body weight, about 300,000 to about 10,000,000 cells per kg of body weight, about 400,000 to about 10,000,000 cells per kg of body weight, about 500,000 to about 10,000,000 cells per kg of body weight Approximately 600,000 to 10,000,000 cells per kg of body weight, approximately 700,000 to 10,000,000 cells per kg of body weight, approximately 800,000 to 10,000,000 cells per kg of body weight, approximately 900,000 to 10,000,000 cells per kg of body weight, approximately 1,000,000 to 10,000,000 cells per kg of body weight, approximately 2,000,000 to 10,000,000 cells per kg of body weight, approximately 3,000,000 to 10,000,000 cells per kg of body weight, approximately 4,,000,000 to about 10,000,000 cells per kg of body weight, about 5,000,000 to about 10,000,000 cells per kg of body weight, about 6,000,000 to about 10,000,000 cells per kg of body weight, about 7,000,000 to about 10,000,000 cells per kg of body weight, about 8,000,000 to about 10,000,000 cells per kg of body weight, and about 9,000,000 to about 10,000,000 cells per kg of body weight.

[0096] In some embodiments, a therapeutically effective amount is from about 1,000 to about 9,000,000 cells per kg of body weight, from about 1,000 to about 8,000,000 cells per kg of body weight, from about 1,000 to about 7,000,000 cells per kg of body weight, from about 1,000 to about 6,000,000 cells per kg of body weight, from about 1,000 to about 5,000,000 cells per kg of body weight, from about 1,000 to about 4,000,000 cells per kg of body weight, from about 1,000 to about 3,000,000 cells per kg of body weight, or from about 1,000 to about 2,000,000 cells per kg of body weight. 0 to about 2,000,000 cells, about 1,000 to about 1,000,000 cells per kg of body weight, about 1,000 to about 900,000 cells per kg of body weight, about 1,000 to about 800,000 cells per kg of body weight, about 1,000 to about 700,000 cells per kg of body weight, about 1,000 to about 600,000 cells per kg of body weight, about 1,000 to about 500,000 cells per kg of body weight, about 1,000 to about 400,000 cells per kg of body weight, about 1,000 to about 300,000 cells per kg of body weight cells, about 1,000 to about 200,000 cells per kg of body weight, about 1,000 to about 100,000 cells per kg of body weight, about 1,000 to about 90,000 cells per kg of body weight, about 1,000 to about 80,000 cells per kg of body weight, about 1,000 to about 70,000 cells per kg of body weight, about 1,000 to about 60,000 cells per kg of body weight, about 1,000 to about 50,000 cells per kg of body weight, about 1,000 to about 40,000 cells per kg of body weight, about 1,000 to about 50,000 cells per kg of body weight, about 1,000 to about 60,000 cells per kg of body weight, about 1,000 to about 70,000 cells per kg of body weight, about 1,000 to about 80,000 cells per kg of body weight, about 1,000 to about 90,000 cells per kg of body weight, about 1,000 to about 100,000 cells per kg of body weight, about 1,000 to about 120,000 cells per kg of body weight, about 1,000 to about 140,000 cells per kg of body weight, about 1,000 to about 160,000 cells per kg of body weight, about 1,000 to about 180,000 cells per kg of body weight, about 1,000 to about 200,000 cells per kg of body weight, about 1,000 to about 240,000 cells per kg of body weight, about 1,000 to about 260,000 cells per kg of body weight, about 1,000 to about 280,00 0 to about 30,000 cells per kg of body weight, about 1,000 to about 20,000 cells per kg of body weight, about 1,000 to about 10,000 cells per kg of body weight, about 1,000 to about 9,000 cells per kg of body weight, about 1,000 to about 8,000 cells per kg of body weight, about 1,000 to about 7,000 cells per kg of body weight, about 1,000 to about 6,000 cells per kg of body weight, about 1,000 to about 5,000 cells per kg of body weight, about 1,000 to about 4,000 cells per kg of body weight, about 1,000 to about 3,000 cells per kg of body weight, or about 1,000 to 2,000 cells per kg of body weight.

[0097] In some embodiments, the therapeutically effective amount is from about 1,000 to about 100,000,000 cells.

[0098] In some embodiments, a therapeutically effective amount is about 2,000 to about 100,000,000 cells, about 10,000 to about 100,000,000 cells, about 100,000 to about 100,000,000 cells, about 1,000,000 to about 100,000,000 cells, or about 10,000,000 to about 100,000,000 cells. In some embodiments, a therapeutically effective amount is about 1,000 to about 10,000,000 cells, about 1,000 to about 1,000,000 cells, about 1,000 to about 100,000 cells, or about 1,000 to about 10,000 cells.

[0099] In some embodiments, the population of cells is administered to a subject systemically, i.e., injected into the subject's bloodstream. In a preferred embodiment, the population of cells is administered by intravenous injection. In another embodiment, the population of cells is administered by intra-arterial or intracardiac injection. In another, less preferred embodiment, the population of cells is administered by intramuscular, intradermal, or subcutaneous injection.

[0100] In some embodiments, the population of cells is maintained in an appropriate medium.

[0101] In one embodiment, a suitable medium allows for the survival of the population of cells and is safe for administration to a mammal, preferably a human. In some embodiments, the medium provides nutrients to the population of cells. In some embodiments, the medium maintains a suitable osmolality for the population of cells.

[0102] In another embodiment, the population of cells is transferred to a separate medium, called an injection medium, prior to injection, which other medium allows for the survival of the population of cells and is safe for administration to mammals, preferably humans, but does not contain nutrients. In some embodiments, the injection medium is saline. In some embodiments, the injection medium is a biological buffer.

[0103] In some embodiments, the medium is sterilized by means known in the art, such as, for example, irradiation. In some embodiments, sterilization preferably removes 99.9%, 99.99%, 99.999%, or 100%, preferably 100%, of microorganisms. In some embodiments, the medium is sterile. As used herein, "sterile" means that the medium is free of bacteria, fungi, archaea, and / or protozoa.

[0104] In some embodiments, the population of cells is administered in combination with another therapeutic agent. Non-limiting examples of therapeutic agents include drugs, pharmaceuticals, peptides, cells (e.g., cells different from the cells of the present invention), etc. In some embodiments, the other therapeutic agent is for treating at least one brain disease. Such therapeutic agents are known to those skilled in the art and commonly used in medical practice. Non-limiting examples of brain disease treatments include, for example, acetylcholinesterase inhibitors for the treatment of Alzheimer's disease, anti-inflammatory molecules, antioxidants, etc.

[0105] In some embodiments, the other therapeutic agent is administered before, simultaneously with, or after the population of cells for use according to the present invention. In some embodiments, the other therapeutic agent is administered prior to the population of cells for use according to the present invention, preferably between about one month and about one minute prior to the population of cells for use according to the present invention. In some embodiments, the other therapeutic agent is administered simultaneously with the population of cells for use according to the present invention. In some embodiments, the other therapeutic agent is administered after the population of cells for use according to the present invention, preferably between about one minute and about one month after the population of cells for use according to the present invention.

[0106] The present invention further relates to a pharmaceutical composition comprising a population of cells overexpressing COX7B protein and a pharmaceutically acceptable vehicle for use in the prevention and / or treatment of a brain disease or disorder.

[0107] In some embodiments, the pharmaceutically acceptable vehicle comprises or is selected from the group consisting of a solvent, diluent, carrier, excipient, dispersion medium, coating agent, and any combination thereof. A carrier, diluent, solvent, or excipient must be "acceptable" in the sense of being compatible with the cell population for use according to the invention and not harmful when administered to an individual. Typically, a vehicle will not cause adverse, allergic, or other untoward reactions when administered to an individual, preferably a human individual.

[0108] For the specific purpose of administration to humans, pharmaceutical compositions should meet general safety and purity standards required by regulatory authorities, such as the Food and Drug Administration (FDA) or the European Medicines Agency (EMA).

[0109] The present invention further relates to a combination kit comprising (i) a population of cells overexpressing COX7B protein or a pharmaceutical composition comprising the same and (ii) another therapeutic agent for use in the prevention and / or treatment of a brain disease or disorder.

[0110] The present invention also relates to a combination kit comprising (i) a means for overexpressing COX7B protein in cells and (ii) another therapeutic agent, for use in the prevention and / or treatment of brain diseases or disorders. Accordingly, the present invention relates to (i) a cell population overexpressing COX7B protein, or a pharmaceutical composition comprising the same, or a combination kit comprising a means for overexpressing COX7B protein in cells and (ii) another therapeutic agent, for use in the prevention and / or treatment of brain diseases or disorders.

[0111] The means and methods for overexpressing protein in cells are known to those skilled in the art.Examples of the means for overexpressing COX7B protein in cells include, but are not limited to, the vector comprising at least one coding sequence for COX7B as described hereinabove, and the reagent for gene transfection of cells.In some embodiments, the cell in which COX7B is overexpressed is the cell (autologous cell) of the subject to be treated.

[0112] In some embodiments, the cells are stem cells. In preferred embodiments, the stem cells are mesenchymal stem cells or neural stem cells. In one embodiment, the stem cells are mesenchymal stem cells. In one embodiment, the stem cells are neural stem cells.

[0113] In some embodiments, the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO:1.

[0114] In some embodiments, the kit further comprises a means for administering the population of cells to a subject in need thereof.

[0115] In some embodiments, the kit comprises a means for introducing a nucleic acid molecule encoding a COX7B protein into a cell, preferably a stem cell.

[0116] The present invention further relates to a method for treating and / or preventing a brain disease or disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a population of cells overexpressing COX7B protein or a pharmaceutical composition containing same.

[0117] In some embodiments, the cells are stem cells. In preferred embodiments, the stem cells are mesenchymal stem cells or neural stem cells. In one embodiment, the stem cells are mesenchymal stem cells. In one embodiment, the stem cells are neural stem cells. Suitable cells are described herein above.

[0118] In some embodiments, the method includes (i) harvesting a population of stem cells from a subject, (ii) overexpressing COX7B protein in the population of stem cells, and (iii) administering the population of stem cells overexpressing COX7B protein to the subject. Means for overexpressing COX7B in cells are known in the art and are described above.

[0119] In some embodiments, the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO:1.

[0120] In some embodiments, the therapeutically effective amount is about 1,000 to about 100,000 billion cells per kg of body weight.

[0121] In some embodiments, the methods comprise administering the population of cells to the subject by systemic injection, preferably by intravenous injection. In some embodiments, the cells are co-administered with a suitable carrier or solute.

[0122] In some embodiments, the method further comprises administering to the subject an additional therapeutic agent, preferably a therapeutic agent for treating a brain disease or disorder.

[0123] Brain diseases and disorders are described herein above.

[0124] The present invention further relates to a method for inducing brain tropism in a cell, preferably a stem cell, comprising the step of overexpressing COX7B protein in the cell. The cell and overexpression of COX7B are described herein above.

[0125] In some embodiments, the COX7B protein has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity to SEQ ID NO:1.

[0126] The present invention further relates to a population of cells overexpressing COX7B protein for the manufacture of a medicament for the treatment and / or prevention of a brain disease or disorder in a subject in need thereof. [Brief explanation of the drawings]

[0127] [Figure 1A] ~ [Figure 1F] This is a combination of schemes, dot plots, histograms, and graphs demonstrating the validation of brain-seeking variants derived from MDA-MB-231. Figure 1A shows a schematic of the in vivo experiment. Six-week-old female mice were intracardially injected with 100,000 cancer cells expressing luciferase and green fluorescent protein (GFP) on day 0 and imaged weekly to track metastasis. Mice were sacrificed at week 4 for organ harvest, followed by ex vivo bioluminescence imaging and immunohistochemistry. Figure 1B shows ex vivo bioluminescence imaging of mouse brains at the end of the protocol shown in Figure 1A. The graph shows the brain bioluminescence intensity in mice receiving parental MDA-MB-231 cancer cells, 231-BR, or the brain-seeking variant 231-BR-2 (n = 3–9). In Figures 1C–1E, migration of MDA-MB-231, 231-BR, and 231-BR-2 cells was assayed in transwells against 1% FBS (n = 2–3, Figure 1C), mouse astrocytes (n = 6, Figure 1D), or human astrocytes (n = 6, Figure 1E). Figure 1F shows cell counts (%) measured over time on a SpectraMax i3 spectrophotometer equipped with a MiniMax Imaging Cytometer (Molecular Devices) after seeding 5,000 cells per well in a 96-well plate (n = 18–19). Data are presented as individual values ​​and median (Figure 1B) or as mean ± SEM (Figure 1C–1F). *P<0.05, **P<0.01, ***P<0.005, ns:P>0.05; compared with MDA-MB-231 (Figure 1B-F); using Mann-Whitney test, one-way ANOVA with Dunnett's post-hoc test, or two-way ANOVA with Dunnett's post-hoc test. [Figure 2A] ~ [Figure 2M] This is a combination of histograms, graphs, and photographs showing that the brain-tropic variants are more oxidative than the parental MDA-MB-231 human breast cancer cells. In Figure 2A-2C, mitochondrial fuel utilization in MDA-MB-231 (Figure 2A, n = 14), 231-BR (Figure 2B, n = 14-16), and 231-BR-2 (Figure 2C, n = 14-15) cancer cells was measured using a Fuel Flex test kit (Agilent) on a Seahorse XF96 bioenergetics analyzer. Data are presented as pie charts and bar graphs, with total fuel utilization = 100%. In Figures 2D–2F, glucose consumption (Figure 2D, n = 11–16), lactate production (Figure 2E, n = 16–17), and lactate / glucose ratio (Figure 2F, n = 13–16) were determined from measurements in deproteinized cell supernatants using an enzymatic CMA600 analyzer. In Figures 2G–2J, mitochondrial basal (Figure 2H, n = 29–32) and maximal (Figure 2I, n = 28–32) oxygen consumption rates (mtOCR), and the cellular oxygen consumption rate (OCR) associated with mitochondrial ATP production (Figure 2J, n = 29–32) were measured with a Seahorse XF96 bioenergetics analyzer using the XF Cell Mito Stress Test Kit (Agilent). A representative Seahorse output is shown in Figure 2G. Transmission electron micrographs of cells are shown in Figure 2K (bar = 1 µm). Figure 2L shows the mitochondrial density quantified on a graph (n = 6–12). Figure 2M shows the mitochondrial DNA / nuclear DNA (mtDNA / nDNA) intracellular content measured using RT-qPCR (n = 5). All data are shown as mean ± SEM. *P < 0.05, ***P < 0.005, ns: P > 0.05 compared with the first column; one-way ANOVA with Dunnett's post-hoc test. [Figure 3A] ~ [Figure 3H]Figure 3A shows a series of histograms demonstrating the identification of cyclooxygenase 7b (COX7B) as a candidate protein for brain tropism of human breast cancer. Figure 3A shows ALDH9A1 mRNA expression normalized to β-actin (n=5-6) in MDA-MB-231, 231-BR, and 231-BR-2 cancer cells, and Figure 3B shows ALDH9A1 protein expression normalized to vinculin (n=6) in the same cancer cells. Figure 3C shows FH mRNA expression normalized to β-actin (n=5-6), and Figure 3D shows FH protein expression normalized to β-actin (n=9). Figure 3E shows NDUFB8 mRNA expression normalized to β-actin (n=6), and Figure 3F shows NDUFB8 protein expression normalized to β-actin (n=3). Figure 3G shows COX7B mRNA expression normalized to β-actin (n = 5–6), and Figure 3H shows COX7B protein expression normalized to β-actin (n = 9). All data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.005, ns: P > 0.05 compared to MDA-MB-231 cells; one-way ANOVA with Dunnett's post-hoc test. [Figure 4A] ~ [Figure 4Q]This is a combination of histograms and photographs showing the causal relationship between COX7B expression and selective migration of human breast cancer cells toward astrocytes. In Figure 4A-4E, migration was assayed in transwells using the 231-BR brain-tropic variant, expressing or not expressing COX7B (KO using the CRISP-Cas9 strategy), toward 1% FBS (Figure 4A, n = 9), human astrocytes (Figure 4B, n = 3), mouse astrocytes (Figure 4C, n = 5), human hepatocytes (Figure 4D, n = 6), or human bronchial cells (Figure 4E, n = 6). In Figures 4F–4J, migration was assayed in transwells using 231-BR-2 brain-tropic variants with or without COX7B expression for 1% FBS (4F, n = 9), human astrocytes (Figure 4G, n = 5–6), mouse astrocytes (Figure 4H, n = 7), human hepatocytes (Figure 4I, n = 6), or human bronchial cells (Figure 4J, n = 6). In Figures 4K–4O, migration was assayed in transwells using MDA-MB-231 parental cancer cells with or without COX7B overexpression for 1% FBS (Figure 4K, n = 7), human astrocytes (Figure 4L, n = 9), mouse astrocytes (Figure 4M, n = 6), human hepatocytes (Figure 4N, n = 6), or human bronchial cells (Figure 4O, n = 6). All data were normalized to the control (first column) and presented as mean ± SEM. *P<0.05, **P<0.01, ***P<0.005, ns:P>0.05; Student's t-test was used. Figure 4P shows COX7B silencing in 231-BR and 231-BR-2 cells using CRISPR-Cas9. Figure 4Q shows COX7B overexpression in MDA-MB-231 cells using the pCMV3 vector. Western blots reporting COX7b expression in triplicate are shown. β-actin served as a loading control. [Figure 5A] ~ [Figure 5N]This is a combination of graphs and histograms showing that COX7B expression drives the oxidative switch in brain-tropic variants. Figures 5A–5D show 231-BR brain-tropic variant cells expressing or not expressing COX7B (KO using the CRISP-Cas9 strategy). Basal (Figure 5B) and maximal (Figure 5C) mitochondrial oxygen consumption rates (mtOCR), as well as mitochondrial OCR (Figure 5D), which is associated with mitochondrial ATP production, were measured using the XF Cell Mito Stress Test Kit (Agilent) on a Seahorse XF96 bioenergetics analyzer (n = 20 total). A representative Seahorse trace is shown in Figure 5A. Figures 5E–5H show the same results as Figures 5A–5D, but using 231-BR-2 brain-tropic variant cells expressing or not expressing COX7B (n = 15–20 total). Figures 5I–5L show the same results as Figures 5A–5D, but using MDA-MD-231 parental cancer cells (n = 23–32 total) with or without COX7B overexpression. All data are normalized to the control (dotted line) and shown as mean ± SEM. **P < 0.01, ***P < 0.005, ns: P > 0.05; Student's t-test was used. Figures 5M–5N show cell counts for 231-BR cells (Figure 5M) and 231-BR-2 cells (Figure 5N) with or without COX7B silencing. COX7B silencing does not alter the number of brain-tropic variant cells in vitro. 231-BR and 231-BR-2 cells were wild-type or COX7B silenced using a CRISPR-Cas9 strategy. The graph shows the percentage of cells measured over time on a SpectraMax i3 spectrophotometer equipped with a MiniMax Imaging Cytometer after seeding 5,000 cells per well in 96-well plates (n=3-4). Data are shown as mean ± SEM. ns: p>0.05 compared to MDA-MB-231 cells; 2-way ANOVA with Dunnett's post-hoc test. [Figure 6A] ~ [Figure 6L]This is a combination of dot plots, histograms, and graphs demonstrating that COX7B promotes brain tropism of metastatic human breast cancer cells in mice. Figures 6A–6I show the brain tropism of the 231-BR and 231-BR-2 brain-tropic variants, which express or do not express COX7B (KO using the CRISP-Cas9 strategy), and the parental human breast cancer cell line, MDA-MB-231, which overexpresses or does not overexpress COX7B, as assessed using the protocol shown in Figure 1A. The cells constitutively expressed luciferase and GFP. Figures 6A–6C show the ex vivo bioluminescence intensity of mouse brains (n ​​= 4–10) performed 4 weeks after intracardiac injection of cancer cells. Figures 6D–6I show the number of detected metastases per mouse (Figures 6D, 6F, 6H) and the metastasis-positive surface area (Figures 6E, 6G, 6I) (n = 4–10). Figures 6J–6L show overall survival for patients with breast cancer (Figure 6J, total of all types, 1,090 patients), lung cancer (Figure 6K, total of all types, 1,925 patients), and renal cell carcinoma (RCC, Figure 6L, 530 patients). Data are presented as individual values ​​and median (Figure 6A–6C), mean ± SEM (Figure 6D–6I), or individual values ​​(Figure 6J–6L). *P<0.05, **P<0.01, ns:P>0.05; using the Mann-Whitney test (Figure 6A–6C), Student's t-test (Figure 6D–6I), or log-rank test (Figure 6J–6L). [Figure 7A] ~ [Figure 7C]These combined histograms show that silencing COX7B does not alter metabolic plasticity in brain-targeting variant cells. 231-BR and 231-BR-2 cells were wild-type or had COX7B silenced using the CRISPR-Cas9 strategy. (Figures 7A-7C) Mitochondrial fuel utilization in parental MDA-MB-231 cells, 231-BR cells expressing or not expressing COX7B, and 231-BR-2 cells expressing or not expressing COX7B. Bar graphs show mitochondrial utilization of glucose (Figure 7A; n = 7-15), glutamine (Figure 7B; n = 12-16), and fatty acids (Figure 7C; n = 14-16). Total fuel utilization (glucose + glutamine + fatty acids) for each cell line is 100%. Data are shown as mean ± SEM. *P<0.05, **P<0.01, ***P<0.005, ns:P>0.05 compared with MDA-MB-231 cells; using one-way ANOVA with Dunnett's post-hoc test. [Figure 8] This photograph demonstrates that ReNcell VM human neural progenitor cells can be engineered to overexpress human COX7B. ReNcell VM human neural progenitor cells were transfected or not with the pCMV3 plasmid encoding the human COX7B cDNA ORF. The parental cells were Rencell VM, and the COX7B-overexpressing cells were designated ReNcell VM COX7B OE cells. Cells were allowed to recover in growth medium for 24 hours after transfection. Cells were then selected with 10 μg / mL hygromycin for 14 days to generate ReNcell VM COX7B OE clones, which were individually analyzed for COX7B protein expression by Western blotting (detected at 9 kD using Western blotting). β-actin expression (detected at 42 kD) was used as a loading control. Clones (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, and 24) that overexpressed COX7B compared to the parental cells were pooled together to generate the ReNcell COX7B OE cell line. [Figure 9A] ~ [Figure 9H]This is a combination of histograms and photographs showing that COX7B-overexpressing human stem cells have better homing to injured brains after injection into the bloodstream than parental stem cells that do not express COX7B. Brain injury was induced by injecting 100,000 ReNcell VM human neural stem cells constitutively expressing GFP, 100,000 ReNcell VM COX7B OE human neural stem cells constitutively expressing GFP, or no cells (control) into the bloodstream of nude mice pretreated with LPS. Mice were sacrificed 3 days later by cervical dislocation under terminal anesthesia, and their brains were harvested, sectioned along the separation between the left and right hemispheres, and embedded in paraffin. (Figures 9A-9F) show representative images of brain sections immunostained for GFP. For visualization, each positive cell is indicated by a black dot. (Figure 9A) Representative image of two brain hemispheres from a control mouse (scale bar = 1 mm). (Figure 9B) A representative image of the cerebral cortex of a control mouse at a higher magnification (scale bar = 0.2 mm). (Figure 9C) A representative image of two cerebral hemispheres of a mouse injected with ReNcell VM cells (scale bar = 1 mm). (Figure 9D) A representative image of the cerebral cortex of a mouse injected with ReNcell VM cells at a higher magnification (scale bar = 0.2 mm). (Figure 9E) A representative image of two cerebral hemispheres of a mouse injected with ReNcell VM COX7B OE cells (scale bar = 1 mm). (Figure 9F) A representative image of the cerebral cortex of a mouse injected with ReNcell VM COX7B OE cells at a higher magnification (scale bar = 0.2 mm). (Figure 9G) A graph showing the number of GFP-positive cells per cerebral hemisphere in control mice, mice injected with ReNcell VM cells in the bloodstream, and mice injected with ReNcell VM COX7B OE cells in the bloodstream. (FIG. 9H) Graph showing the number of GFP-positive cells per whole brain in control mice, mice injected with ReNcell VM cells in the bloodstream, and mice injected with ReNcell VM COX7B OE cells in the bloodstream.

[0128] Example The present invention is further illustrated by the following examples.

[0129] Example 1: material and method Cells and cell culture Parental MDA-MB-231 human triple-negative breast adenocarcinoma cells were obtained from Caliper (catalog number #119369). MDA-MB-231-derived brain-tropic variants 231-BR and 231-BR-2 were obtained from the National Cancer Institute, Bethesda, and Indiana University School of Medicine, Indianapolis, respectively. Cells were routinely cultured in DMEM (ThermoFisher; catalog number #61965026) containing glutaMAX and 4.5 g / L glucose supplemented with 10% FBS and maintained at 37°C in a 5% CO2 humidified atmosphere. Cells were authenticated using short tandem repeat (STR) profiling (Eurofins Genomics).

[0130] Human astrocytes (T0281, expressing hTERT), mouse astrocytes (T0289, expressing SV40 large T antigen), human hepatocytes (T0063, expressing HPV E6 / E7, hTERT, and MycT58A), and human bronchial epithelial cells (T0753, expressing hTERT and Cdk4) used in the migration assay were immortalized cells obtained from Applied Biological Materials Inc. (ABM). Astrocytes were routinely cultured in DMEM (ThermoFisher; catalog number #61965026) containing glutaMAX and 4.5 g / L glucose, supplemented with 10% FBS and 5% astrocyte growth factor (Sanbio; catalog number #1852; for human astrocytes only). Hepatocytes were maintained in PriGrow IX Medium (ABM; Catalog No. TM019) supplemented with 10% FBS and then sequentially transferred to the same medium as the cancer cells. Bronchial cells were maintained in PriGrow X Medium (ABM; Catalog No. TM0753) and then sequentially transferred to Bronchial Epithelial Cell Growth Medium (BEGM; Lonza; Catalog No. CC-3170).

[0131] Genetic manipulation For constitutive expression of luciferase and GFP, cells were infected with a lentivirus (Amsbio; catalog number #LPV020) carrying luciferase and GFP sequences along with a puromycin resistance gene. Briefly, cells at 70-80% confluence in a 24-well plate were transduced with 2 μL / well of lentivirus solution in 1 mL of medium containing 10 μL / mL of polybrene. Cells were selected by culturing with 1 μg / mL of puromycin (InvivoGen) for 48-72 hours and then sorted for GFP expression by FACS using a Becton Dickinson FACSAria III system.

[0132] Silencing of the COX7B gene was performed using the CRISPR-Cas9 strategy according to the Zhang lab's protocol using the pSpCas9(BB)-2A-Puro (pX459; Addgene; catalog number #62988, puromycin selection) or pU6-(BbsI)_CBh-Cas9-T2A-mCherry (Addgene; catalog number #64324, red fluorescent selection) plasmids. These plasmids contain both Cas9 and gRNA expression cassettes with a BbsI restriction site for inserting the gRNA sequence. Pre-validated guide RNA (gRNA) sequences were selected from the GenScript genome-wide database as a non-overlapping gRNA pair of 5'-AGCGCACTAAATCGTCTCCA-3' (SEQ ID NO: 15) and 5'-GAGTTACCCCAAAGGAATGG-3' (SEQ ID NO: 16). Cohesive ends were created for insertion into the vector plasmid with CACCG at the 5' end of the gRNA sense sequence and AAAC at the 5' end and C at the 3' end of the gRNA antisense sequence. The gRNA oligonucleotides (Eurogentec) were annealed to the double-stranded DNA using 1 μL of a stock solution containing 100 μM of each sense and antisense oligonucleotide in 2 μL of 5X T4 ligase buffer (ThermoFisher, catalog number #46300018), 0.5 μL of T4 PNK (BIOKE, catalog number #M0201), and 5.5 μL of DNase / RNase-free distilled water. The incubation time was 37°C for 30 minutes, followed by 95°C for 5 minutes, then decreased at 5°C / min until 25°C was reached.The Golden Gate DNA Assembly protocol was then used to insert 1 μL of annealed gRNA at 1 μM into 100 ng of vector plasmid in a solution containing 5 μL of 10X Fast Digest buffer (ThermoFisher; Catalog No. #B64), 0.5 μL of ATP 0.1 M (ThermoFisher; Catalog No. #R1441), 0.5 μL of BSA 10 mg / mL (Promega; Catalog No. #R396D), 1 μL of restriction enzyme BpiI (ThermoFisher; Catalog No. #FD1014), and 2 μL of T4 ligase 5 U / μL (ThermoFisher; Catalog No. #EL0014) in a total volume of 50 μL completed with water. The mixture was incubated for 20 cycles at 37°C for 5 minutes and 20°C for 5 minutes, followed by 80°C for 20 minutes. Five microliters of the resulting solution was used to transform TOP10 bacteria (ThermoFisher; Catalog No. #C404003) using prewarmed LB agar plates (ThermoFisher; Catalog No. #22700-025) containing 100 μg / mL ampicillin, according to the manufacturer's instructions. A single colony was inoculated into LB medium (ThermoFisher; Catalog No. #12780-052) containing 50 μg / mL ampicillin and incubated overnight at 37°C. Plasmid DNA was then recovered using the PureYield Plasmid Miniprep System (Promega; Catalog No. #A1223), and its concentration was obtained using a NanoDrop instrument (ThermoFisher). The sequence was verified by Sanger sequencing (Genewiz, Leipzig, Germany). Cells at 70-80% confluence were transfected using the Lipofectamine LTX / Plus transfection kit (ThermoFisher; catalog number #15338100) or the jetOPTIMUS kit (Westburg; catalog number #117-01).The first kit was used with 0.25 μg of each gRNA plasmid for a total of 0.5 μg of DNA in 100 μL of OptiMEM containing 0.5 μL of Plus and 2.25 μL of Lipofectamine LTX for 15 and 30 minutes of incubation, respectively. The second kit was used with 0.5 μg of each gRNA plasmid for a total of 1 μg of DNA in 200 μL of buffer, with 1 μL of reagent per well of a 6-well plate. Cells were selected in medium containing puromycin (1 μg / mL) for 48–72 hours or FACS-sorted by mCherry fluorescence 48 hours post-transfection using a Becton Dickinson FACSAria III system.

[0133] A human untagged COX7B cDNA ORF clone in the expression vector pCMV3 (Bio-Connect; catalog number #HG20762-UT) was used to overexpress COX7b in MDA-MB-231 cancer cells. Cells at 70-80% confluence were transfected with 10 μg of plasmid in a 10 cm dish using Lipofectamine 3000 (ThermoFisher; catalog number #L3000001). The cells were allowed to recover the following day in fresh DMEM (ThermoFisher; catalog number #61965026) containing glutaMAX and 4.5 g / L glucose supplemented with 10% FBS and selected for 10 days in hygromycin (400 μg / ml)-containing medium. The medium was refreshed every 3-4 days. Colonies were individually selected, expanded, and tested for COX7b expression by Western blotting.

[0134] Metastasis rate in mice On day 0, 6-week-old female NMRI nude mice (Janvier) underwent image-guided intraventricular injection of 100,000 cells using a Vevo 2100 imaging system (FUJIFILM VisualSonics) equipped with a 30 MHz transducer. Briefly, mice were anesthetized (ketamine 80 mg / kg, xylazine 8 mg / kg), secured supine on an animal platform, and their chests were shaved. Two-dimensional (2D) parasternal left ventricular long-axis ultrasound images of the left ventricle were acquired to identify the optimal apical location for intraventricular injection. A microinjector system equipped with a 26G hypodermic needle was used to perform precise echocardiographically guided intraventricular injection of 100,000 cancer cells constitutively expressing luciferase and GFP. After injection, blood flow within the left ventricle was closely monitored (and images were recorded) to confirm successful intraventricular injection of the cells. All mice were followed for several minutes by ultrasound to ensure that the injections did not cause any injury and were carefully observed until recovery. Metastatic development was monitored using a Xenogen IVIS 50 bioluminescence imaging system (PerkinElmer) and quantified using Living Image software (PerkinElmer). Mice were intraperitoneally injected weekly with 0.15 mg / g body weight of luciferin (PerkinElmer). After a 10-minute incubation period, mice were anesthetized using isoflurane. Chemiluminescence was detected with an acquisition time of 1–12 seconds. Mice were sacrificed by cervical dislocation under terminal anesthesia after 4 weeks, and organ chemiluminescence was acquired ex vivo before fixation in 4% paraformaldehyde (PFA).

[0135] Cell migration and invasion The migration and invasion abilities of cells were measured using transwell inserts (Corning; Catalog No. #353097). Cells (astrocytes, hepatocytes, and bronchial cells) seeded in the lower chamber were used as chemoattractants, either with 1% FBS (general migration / invasion) or with cells (astrocytes, hepatocytes, and bronchial cells). Invasion was assessed by coating the inserts with 250 μg / mL Matrigel (Corning; Catalog No. #356231) for 2 hours at 37°C, whereas migration was assessed without Matrigel coating. Fifty thousand cells were seeded in 500 μL of serum-free medium in the upper chamber of each transwell, while the lower chamber contained either 1% FBS (general migration / invasion) or confluent non-tumor astrocytes, hepatocytes, or bronchial cells as a chemoattractant. Test cells were allowed to migrate / invade for 24 hours at 37°C in a humidified atmosphere of 5% CO2. At the end of the assay, cells were fixed with 4% PFA for 10 minutes, washed three times with PBS, and non-migrating cells (upper compartment of the insert) were wiped off. Migratory cells were stained with DAPI for 30 minutes, washed three times with PBS, and imaged at 5x magnification on an AxioVert microscope equipped with an AxioCam-MRc camera (Zeiss). Two images were taken per well, including the upper and lower halves, respectively. Nuclei were counted using QuPath software version 0.1.2 using the Positive Cell Detection analysis tool.

[0136] number of cells Cell numbers were measured over time using a SpectraMax i3 spectrophotometer with a MiniMax Imaging Cytometer (Molecular Devices) after seeding 5,000 cells per well in a 96-well plate. Results were normalized to the initial cell number.

[0137] Metabolic assays Oxygen consumption rate (OCR) was measured using a Seahorse XF96 bioenergetics analyzer (Agilent Technologies) with the XF Cell Mito Stress Test Kit and Fuel Flex Test Kit according to the manufacturer's instructions. Glucose and lactate concentrations were measured in the supernatants of 150,000 (for the 24-hour assay) and 250,000 (for the 48-hour assay) cells seeded in exactly 1 mL of medium using an enzymatic CMA600 analyzer (Aurora Borealis) according to the manufacturer's instructions. Wells containing medium alone served as controls for the calculation of glucose consumption and lactate production. ATP content of 10,000 cells per well of a 96-well plate was measured using Promega's Cell titer Glo assay (catalog number #G7570). All metabolic measurements were normalized to total protein content determined after overnight incubation with 0.5 M NaOH using the Bio-Rad protein assay (catalog no. #5000006) on a SpectraMax i3 spectrophotometer equipped with a MiniMax imaging cytometer.

[0138] electron microscope Cells were collected and resuspended in 400 μL of 2.5% glutaraldehyde solution containing 0.1 M sodium cacodylate (pH 7.4) in pyramidal BEEM capsules (Agar Scientific, Cat. #G360). Samples were then processed as previously described. Images were taken using TECNAI G 2 20 Acquired with a LaB6 transmission microscope (Field Electron and Ion Company).

[0139] Mitochondrial abundance and mitochondrial DNA content Mitochondrial DNA (mtDNA) content was measured using RT-qPCR as previously described. Total DNA was isolated using the QIAmp DNA kit (Qiagen, Antwerp, Belgium). The 12S-rRNAA mitochondrial gene (forward primer: 5′-GTA CCC ACG TAA AGA CGT TAG G-3′ (SEQ ID NO: 12); reverse primer: 3′-TAC TGC TAA ATC CAC CTT CG-5′ (SEQ ID NO: 13); labeled probe: 5′-CCC ATG AGG TGG CAA GAA AT-3′ FAM (SEQ ID NO: 14)) was then analyzed by RT-qPCR (50 ng of sample and 1 μL of each primer pair [10 μM]) using TaqMan universal master mix II with UNG (ThermoFisher) in parallel with nuclear gene RNase P (RNase P VIC-labeled probe; ThermoFisher Scientific; catalog no. #4401631). For presentation, mtDNA content was normalized to nuclear DNA (nDNA) content.

[0140] Microarray database analysis According to Gene Expression Omnibus (GEO) and references, database #GSE66495 reports whole-genome expression of parental MDA-MB-231 cells maintained in MEM containing 10% FBS and their derived tissue-specific metastatic variants (including 231-BR), as determined using an Illumina HumanHT-12 V4 Expression BeadChip. For database reanalysis, metabolic genes related to glycolysis, oxidative phosphorylation, and the TCA cycle were first extracted. Next, only genes differentially expressed (p<0.05, one-way ANOVA) in the brain (231-BR), adrenal gland (ADMD-231), bone (BMD-231), and / or lung (LMD-231) metastatic variants compared to parental MDA-MB-231 cells were retained. These genes are listed in Table 1. Genes that were differentially expressed in 231-BR versus ADMD-231, BMD-231, and LMD-231 cells (p<0.05 using one-way ANOVA) were then identified. Expression changes were independently confirmed by RT-qPCR using fresh lysates from the brain-targeting variants 231-BR and 231-BR-2, and parental MDA-MB-231 cells.

[0141] RT-qPCR Total RNA was collected using a NucleoSpin RNA kit (Filter Service; catalog number #740955.50), quantified on a NanoDrop 1000 spectrophotometer (Thermo Fisher), and reverse-transcribed to cDNA with the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher; catalog number K1621) using the same amount (500–1,000 ng) for all RNA samples and a 90-minute incubation time. cDNA was diluted 1:10 in DNase / RNase-free distilled water (Thermo Fisher), and 2 μL was used for RT-qPCR analysis (ViiA 7417 Real-Time Instrument, ThermoFisher) with 5 μL of 2X Takyon qPCR Master Mix, 0.2 μL of each primer (10 μM), and made up to 10 μL with water. Primers were: ALDH9A1 forward 5'-AAG GAG CAG GGT GCT AAA GT-3' (SEQ ID NO:2) and reverse 5'-TCG TCT CTG CAA TTA GTT AAT ACA C-3' (SEQ ID NO:3); FH forward 5'-TGC CAA CCC CAG TTA TTA AAG C-3' (SEQ ID NO:4) and reverse 5'-CTT CAG CTA CCT CAT CTG CTG-3' (SEQ ID NO:5); NDUFB8 forward 5'-CGG ATG ATG GCA TGG GGT A-3' (SEQ ID NO:6) and reverse 5'-GGT GCC AGT GCA TCG GTT-3' (SEQ ID NO:7); and COX7B forward 5'-TAC CTG AAG CGA ATT GGC AC-3' (SEQ ID NO:8) and reverse 5'-GCT TCG AAC TTG GAG ACG AT-3' (SEQ ID NO:9), and β-actin forward 5'-CCC GCG AGC ACA GAG C-3' (SEQ ID NO: 10) and reverse 5'-TCA TCA TCC ATG GTG AGC TGG-3' (SEQ ID NO: 11). All gene expression data were normalized to β-actin gene expression.

[0142] Western blotting Western blotting was performed after protein collection in RIPA buffer containing phosphatase (PhosSTOP) and protease (protease inhibitor cocktail) inhibitors. Membranes were incubated overnight with rabbit primary antibodies against ALDH9A1 (Proteintech; Catalog No. #26621), FH (BIOKE; Catalog No. #4567S), NDUFB8 (Proteintech; Catalog No. #14794), COX7B (Abcam; Catalog No. #ab137094), and vinculin (BIOKE; Catalog No. #4650S); or mouse antibody against β-actin (Sigma-Aldrich; #A5441). Staining was developed using an Amersham Imager 600 (GE Healthcare). All data are normalized to vinculin or β-actin expression.

[0143] immunohistochemistry Brains were harvested, cut along the separation between the right and left hemispheres, and embedded in paraffin. Sections (5 μm thick) were made from the center of each hemisphere, producing 10 slides for each sample. Three slides from the beginning, middle, and end of each hemisphere were used for immunostaining, and this procedure was repeated up to three times to analyze sections representative of the whole brain. Brain sections were immunostained for GFP (Bio-Techne; Catalog No. #600-308; BIOKE; Catalog No. #2956) with the secondary antibody Envision anti-rabbit antibody conjugated to HRP (Agilent; Catalog No. #K4003) and hematoxylin and eosin counterstaining. Slides were scanned at 20x magnification using an SCN400 bright-field slide scanner (Leica Biosystems). The number and surface area of ​​metastases were determined using Cytomine (cytomine.org) and QuPath software version 0.1.2.

[0144] Clinical database analysis Overall survival (OS) curves were generated using Kaplan-Meier plotter (kmplot.com) using the best cutoff values ​​automatically selected for the RNA-seq mRNA datasets (pan-cancer breast cancer and renal clear cell carcinoma) and for the lung cancer gene chip mRNA dataset, 202110 Affy ID (COX7B). Database sources included GEO, EGA, and TCGA.

[0145] statistics Data are presented as mean ± SEM (error bars may be smaller than the symbol) or individual values ​​with median. "n" represents the total number of replicates per group / condition. Graphpad Prism version 9.2.0 was used for statistical analysis. Mann-Whitney, Student's t-test, one-way analysis of variance, and two-way analysis of variance were used as appropriate. P < 0.05 was considered statistically significant.

[0146] result Validation of a brain-tropic variant model derived from human MDA-MB-231 triple-negative breast cancer cells The goal of the series of experiments presented herein was to identify metabolic proteins responsible for brain tropism of human metastatic breast cancer. As models, we used MDA-MB-231 triple-negative breast cancer (TNBC) cells and two independently derived brain-tropic variant cell lines, 231-BR and 231-BR-2, generated by successive cycles of in vivo selection in mice. The selection protocol involved intracardiac injection of cancer cells into the left ventricle of female nude mice, surgical isolation, expansion of metastatic cancer cells recovered from the brain, and intracardiac injection of these cells into subsequent animals for several rounds until metastatic dissemination was restricted to the brain.

[0147] To identify metabolic drivers of brain-specific metastasis, we first validated the validity of two model cell lines in vitro and in vivo. Short tandem repeat (STR) profiling confirmed that all variants were genomically similar to the parental MDA-MB-231 cells (data not shown). For in vivo assays, cells were infected with lentivirus to constitutively express luciferase and green fluorescent protein (GFP). Intracardiac injection of 100,000 parental MDA-MB-231 cells (Figure 1A) did not produce brain metastases in female nude mice, whereas use of either the 231-BR or 231-BR-2 variants produced metastases in most animals (4 / 5 for 231-BR and 6 / 9 for 231-BR-2) 4 weeks after injection, as detected by ex vivo bioluminescence imaging of isolated brains (Figure 1B).

[0148] To validate the in vitro model, a transwell assay was developed to test the general and organ-parent-directed migration of human breast cancer cells in the top well toward 1% FBS in the bottom well or toward viable, immortalized, non-tumorous astrocytes, respectively. Because selection of the brain-tropic variants was performed in mice, both mouse and human astrocytes were tested as attractants. Parental MDA-MB-231 cells had a higher ability to migrate toward 1% FBS compared to the two brain-tropic variants; conversely, 231-BR and 231-BR-2 cells migrated more efficiently toward mouse or human astrocytes (Figures 1C-1E), thus confirming their preferential tropism for the brain. Notably, both brain-tropic variants were slightly but significantly more proliferative than the parental cells, as determined by direct cell counting over time (Figure 1F).

[0149] Brain-tropic variant of MDA-MB-231 undergoes an oxidative switch The oxidative metabolic preferences of parental and brain-targeted variant cells were determined using Seahorse oximetry and Agilent's proprietary Fuel Flex test kit. The assay involves sequential inhibition of glucose-fueled (using 2 μM of the mitochondrial pyruvate carrier inhibitor UK5099), glutamine-fueled (using 3 μM of the glutaminase 1 inhibitor BPTES), and lipid-fueled (using 4 μM of the carnitine palmitoyltransferase 1A inhibitor Etomoxir) oxidative phosphorylation. The results (Figures 2A–2M and 7A–7C) showed that oxidative phosphorylation in MDA-MB-231 cells was supported almost equally by glutamine (52%) and fatty acids (48%) but not at all by glucose, whereas oxidative phosphorylation in the two brain-targeting variants was supported not only by glutamine and fatty acids but also by glucose (5.1 ± 0.6% in 231-BR cells and 8.4 ± 1.4% in 231-BR-2 cells) (Figures 2A–2C). Glucose uptake, lactate release, and the resulting glycolysis ratio ([glucose] / [lactate]) were unchanged in complete medium (glutaMAX, DMEM containing 4.5 g / L glucose and 10% FBS) (Figures 2D–2F). Thus, the change in the ability to utilize fuels for oxidative phosphorylation reflected increased oxidative flexibility of the brain-targeting variants compared to the parental cells, rather than increased dependence on glucose. Accordingly, both the 231-BR and 231-BR-2 cell lines exhibited improved basal and maximal respiratory activity as well as improved oxidative ATP production compared to the parental cells (Figures 2G-J).

[0150] The oxidative switch evidenced in the brain-targeting variant was associated with qualitative and quantitative changes affecting mitochondria. Qualitatively, electron microscopy revealed enlarged mitochondria in 231-BR compared to parental cells, whereas they were smaller but more abundant in 231-BR-2 compared to MDA-MB-231 cells (Figures 2K-2M). The ratio of mitochondrial to nuclear DNA (mtDNA / nDNA) was determined using RT-qPCR and revealed significantly increased amounts of mtDNA in both 231-BR and 231-BR-2 compared to parental cells (Figure 2M). Collectively, these results support the conclusion that mitochondrial oxidative efficiency is increased in the brain-targeting variant of MDA-MB-231 cells.

[0151] Identification of four candidate metabolic genes that may be key contributors to brain tropism in human breast cancer cells Based on the working hypothesis of metabolic preferences of brain-tropic variants for brain-resident metabolites and the above evidence of metabolic differences in offspring, our next objective was to identify metabolic genes / proteins associated with the brain tropism of 231-BR and 231-BR-2 cells. We analyzed the publicly available microarray database GEO#GSE66495 (which reports on the whole-genome expression of MDA-MB-231 and 231-BR cells as well as MDA-MD-231-derived adrenal (ADMD-231), bone (BMD-231), and lung (LMD-231) metastatic variants), focusing on genes involved in glycolysis, the TCA cycle, and oxidative phosphorylation.

[0152] Using the two-step methodology described in Materials and Methods, we identified 22 metabolic genes that were differentially expressed in at least one metastatic variant compared with the parental MDA-MB-231 cells, six of which were also differentially expressed in 231-BR cells compared with all other metastatic variants (Table 1). The significantly upregulated genes were ALDH9A1 (Genbank ID 223, chromosome 1), encoding aldehyde dehydrogenase 9 family member A1; FH (Genbank ID 2271, chromosome 1), encoding fumarate hydratase; and COX7B (Genbank ID 1349, chromosome X), encoding cytochrome c oxidase subunit 7B. Significantly downregulated genes were ALDH1A3 (Genbank ID 220, chromosome 15), encoding aldehyde dehydrogenase 1 family member A3, NDUFB8 (Genbank ID 4714, chromosome 10), encoding NADH:ubiquinone oxidoreductase subunit B8, and PGM5 (Genbank ID 5239, chromosome 9), encoding phosphoglucomutase 5.

[0153] [Table 1]

[0154] Among the six genes, the expression of ALDH1A3 and PGM5 was not significantly different between 231-BR and parental MDA-MB-231 cells (p>0.5) (Table 1), and they were therefore excluded from further analysis.

[0155] Cytochrome c oxidase subunit 7b in mitochondrial complex IV is a candidate protein supporting brain tropism of human breast cancer cells Investigations were conducted on four genes from the shortlist: ALDH9A1, FH, NDUFB8, and COX7B. To avoid potential specificity associated with 231-BR cells, changes in gene expression were independently tested in both 231-BR and 231-BR-2 brain-targeting variants using RT-qPCR. It was further confirmed that changes in protein coincided with changes in mRNA expression.

[0156] ALDH9A1 encodes a cytosolic aldehyde hydrogenase that catalyzes the oxidation of polyamine-derived γ-aminobutyraldehyde and aminoaldehydes. It is involved in the biosynthesis of carnitine, which facilitates the transport of fatty acids across the inner mitochondrial membrane for β-oxidation and potentially participates in the critical pathway for the biosynthesis of the neurotransmitter γ-aminobutyric acid (GABA). Compared to parental MDA-MB-231 cells, ALDH9A1 mRNA expression was significantly increased in 231-BR cells, but it was slightly decreased in 231-BR-2 cells (Figure 3A). The corresponding protein was overexpressed in 231-BR but not in 231-BR-2 cells (Figure 3B). This disqualifies it as a common brain-targeted metabolic sensor in the model cell lines used herein.

[0157] FH encodes fumarate hydratase, the seventh enzyme in the TCA cycle that catalyzes the hydration of fumarate to L-malate. When mutated or inactivated, FH can cause a variety of diseases, including hereditary and sporadic cancers. In the context of brain-specific breast cancer metastasis, FH mRNA and protein expression were increased in 231-BR cells but not in 231-BR-2 cells (Figures 3C-3D), thus disqualifying this enzyme as a common brain-directed metabolic sensor.

[0158] NDUFB8 encodes an auxiliary subunit of NADH ubiquinone oxidoreductase, a large protein complex known as ETC complex I in the inner mitochondrial membrane. This subunit is bound to NADH dehydrogenase 5 (ND5) in the proton pump module of complex I. Similar to microarray data analysis, RT-qPCR showed significantly decreased NDUFB8 mRNA expression in 231-BR cells compared to MDA-MB-231 cells (Figure 3E). However, it was significantly increased in 231-BR-2 cells, and the change in protein expression did not coincide with the change in mRNA expression (Figure 3E-F). Overall, this disqualifies NDUFB8 as a metabolic sensor for brain-selective metastasis.

[0159] COX7B encodes subunit 7B of cytochrome c oxidase (COX), a large protein complex known as ETC complex IV, which catalyzes the transfer of electrons from reduced cytochrome c to molecular oxygen in the inner mitochondrial membrane. COX7B is a short 80-amino acid protein that stabilizes this complex and regulates COX activity. COX7B mRNA expression was increased in 231-BR but decreased in 231-BR-2 cells (Figure 3G). However, the corresponding protein phenotype was increased in both variants (Figure 3H). Considering that, of the four candidate proteins, only COX7B protein expression showed similar changes in both brain-targeting variants, COX7B was retained for further investigation. COX7B expression drives human breast cancer cell migration toward astrocytes

[0160] Transwell migration assays were used to establish a causal relationship between COX7B expression and chemotaxis to MDA-MB-231 brain, mimicking cell migration toward immortalized human and mouse astrocytes. Chemoattraction at other important metastatic sites was mimicked with immortalized human hepatocytes (T0063) and human bronchial epithelial cells (T0763). All four cell lines were nontumorigenic.

[0161] As expected, COX7B protein silencing in brain-tropic variant cells using the CRISPR-Cas9 strategy (Figure 4P) significantly reduced migration of 231-BR cells toward human and mouse astrocytes, but not toward human hepatocytes or human bronchial cells (Figures 4A-4E). The general migration phenotype toward serum (1% FBS), used as a control, was unaffected. Similarly, COX7B silencing significantly reduced migration of 231-BR-2 toward human and mouse astrocytes, but not toward human hepatocytes, human bronchial cells, or serum (Figures 4F-4J). Conversely, experimental overexpression of COX7B protein in parental MDA-MB-231 cells (Figure 4Q) increased cell migration toward human and mouse astrocytes, but did not alter cell migration toward human hepatocytes, human bronchial cells, or serum (Figures 4K–4O).

[0162] Collectively, these in vitro results support a causal relationship between COX7B protein expression and brain tropism of human metastatic breast cancer cells. In particular, overexpression of COX7B protein was sufficient to induce selective brain tropism of otherwise pan-metastatic wild-type MDA-MB-231 cells. COX7B expression promotes the oxidative phenotype of human metastatic breast cancer cells

[0163] The data shown in Figures 2A-2M indicate that increased oxidative phosphorylation is a key metabolic characteristic of the brain-targeted variants of 231-BR and 231-BR-2 compared to parental cells. Because COX7B is present in the ETC, its expression may regulate cellular OCR, which was measured using Seahorse oximetry.

[0164] Silencing COX7B reduced the basal OCR and the OCR associated with ATP production in 231-BR cells, but not the maximal OCR, which reflects respiratory reserve (Figures 5A-5D). In comparison, silencing COX7B reduced the basal OCR, maximal OCR, and OCR associated with ATP production in 231-BR-2 cells (Figures 5E-5H), indicating that loss of COX7B suppresses oxidative phosphorylation in this brain-tropic metastatic breast cancer variant. Notably, silencing COX7B did not reduce cell number (Figures 5M-5N), suggesting the existence of a metabolic pathway that prevents cell death. Furthermore, overexpression of COX7B in wild-type MDA-MB-231 cells induced the opposite effect, i.e., an oxidative switch characterized by increases in all basal OCR, maximal OCR, and OCR associated with ATP production (Figures 5I-5L).

[0165] Collectively, these experiments demonstrated that COX7B is an inducer of oxidative phosphorylation. They further demonstrated a positive correlation between the oxidative activity of human metastatic breast cancer cells and their preferential migration toward astrocytes.

[0166] COX7B expression is responsible for brain tropism of metastatic human breast cancer cells in mice To experimentally demonstrate the causal relationship between COX7B expression and breast cancer brain metastasis, a series of in vivo experiments were performed in nude mice (see Figure 1A). Briefly, this study investigated metastatic tropism related to metastatic engraftment (late metastatic events) rather than the spread of metastatic cells from the primary tumor (early metastatic events). Therefore, breast cancer cells were injected into the left ventricle, which in MDA-MB-231 cells generates systemic metastatic lesions to the bone, brain, ovaries, and adrenal glands. The constitutive and simultaneous expression of luciferase and GFP by the model cell line used in this study allowed us to confirm the bioluminescence data by immunohistochemistry.

[0167] Silencing COX7B using the CRISPR-Cas9 strategy in the brain-tropic variants 231-BR and 231-BR-2, following the protocol shown in Figure 1A, resulted in a near-complete loss of brain tropism after intracardiac injection (Figures 6A-6C). This was demonstrated using ex vivo luciferase bioluminescence imaging of brains isolated at the time of mouse sacrifice. Conversely, parental MDA-MB-231 cells, which did not produce detectable brain metastases at 4 weeks, had a significant increase in the development of brain metastases upon COX7b overexpression. For validation, brains were harvested at the end of the experiment, sliced, stained with an antibody against GFP, and counterstained with hematoxylin and eosin. Figures 6D-6I show the number of metastases per mouse and the metastatic surface area. Analysis revealed a significant reduction in the number of metastases per mouse and metastasis-positive tumor area per section in mice injected with COX7B-deficient 231-BR and 231-BR-2 cells compared with wild-type 231-BR and 231-BR-2 cells (Figures 6D-6G). The opposite effect was observed in mice implanted with COX7B-overexpressing MDA-MB-231 cells compared with wild-type MDA-MB-231 cells (Figures 6H-6I). Collectively, these in vivo data demonstrated a causal relationship between COX7B expression in mice and the brain tropism of human TNBC.

[0168] We analyzed publicly available gene chip and RNA-seq mRNA expression databases reporting on clinical human breast and lung cancers, as well as renal clear cell carcinoma (including all subtypes). High COX7B expression was identified as an independent prognostic factor for poor patient overall survival in all three cancer types (Figures 6J-6L).

[0169] The findings described herein have important implications for the field of brain rejuvenation therapy: overexpressing COX7B in neural stem cells or mesenchymal stem cells, thus enhancing their tropism after systemic injection, promotes the regeneration of neurons, oligodendrocytes, astrocytes, and / or microglia.

[0170] Example 2: Example 1 shows that high expression of COX7B is responsible for brain-specific metastasis of human breast cancer cells MDA-MB-231. Therefore, experimentally induced overexpression of COX7B is speculated to be useful for targeting cells to the brain in the context of cell therapy for neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and multiple sclerosis, as well as for brain trauma, including ischemic stroke, accidental trauma, and surgical trauma, and for central nervous system lymphoma. Cell therapy is indeed a promising strategy that can be used for neuroprotection (i.e., slowing the progression of neurodegenerative diseases) and / or brain repair (see, for example, Steffanoni, S., et al., Diagnosis and Treatment Using Autologous Stem-Cell Transplantation in Primary Central Nervous System Lymphoma: A Systematic Review. Cancers (Basel), 2023.15(2)).

[0171] A major research area in brain repair and regeneration therapy is the use of stem cells. Among stem cells, mesenchymal stem cells and neural stem cells are pluripotent cells capable of self-renewal and differentiation into various central nervous system neuronal and glial cells. Human mesenchymal stem cells can be isolated from various bodily sources, such as abdominal fat, bone marrow, and umbilical cord blood. Conversely, human neural stem cells can be obtained by direct isolation from brain tissue and in vitro expansion, differentiation of pluripotent stem cells (e.g., human embryonic stem cells [hESCs] and induced pluripotent stem cells [iPSCs]), or transdifferentiation of somatic cells in culture. For brain therapy, these cells are typically transplanted intrathecally at the precise location of the brain lesion.

[0172] Mesenchymal stem cells are used to suppress inflammation and utilize their secretome, which can provide neuroprotection and promote regeneration, while neural stem cells produce neurotrophic factors, reduce neuroinflammation, enhance neuroplasticity by integrating into the host neural circuitry, and replace damaged cells (Uchida, N., et al., Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA, 2000. 97(26): p. 14720-5).

[0173] Injecting stem cells into the brain poses two major problems. The first is the transplantation procedure itself, which raises safety, ethics, and efficacy concerns. It is invasive and ideally should be repeated (i.e., chronic treatment involving several injections at different time points), and spatial administration depends on the ability to detect brain lesions / degenerative regions. The second problem is the limited ability of transplanted cells to migrate from the injection site to widespread affected areas, which is crucial for therapeutic efficacy in the central nervous system (see, for example, De Gioia, R., et al., Neural Stem Cell Transplantation for Neurodegenerative Diseases. Int J Mol Sci, 2020, 21(9)). Typically, injected cells aggregate and form clumps at the injection site within the brain parenchyma, and the transplantation efficiency / survival rate of injected cells is estimated to be less than 5%. Stem cells can also be injected into the cerebrospinal fluid via lumbar puncture. Currently, this method is more efficient than intravenous administration, especially because more cells are found at the injury site.

[0174] For stem cell therapy, optimizing stem cell delivery to the brain is therefore a key challenge in translational research. Here, using ReNcell VM human neural progenitor cells as an example, we show that overexpressing COX7B in stem cells promotes their homing to the brain parenchyma after systemic delivery into the bloodstream.

[0175] material and method Cells and cell culture ReNcell VM human neural progenitor cells (Sigma-Aldrich, Catalog No. #SCC008) were routinely grown in flasks coated with 1% Matrigel (Corning, Catalog No. #356231) in growth medium DMEM F12 (Gibco, Catalog No. #11320-074) containing L-glutamine, supplemented with 20 ng / mL hEGF (Sigma-Aldrich, Catalog No. #E9644-.2MG), 20 ng / mL hFGF (Sigma-Aldrich, Catalog No. #GF003AF-100UG), 2 μg / mL heparin (StemCell, Catalog No. #07980), 2% B27 supplement (ThermoFisher, #17504044), and 1% antibiotic-antimycotic (ThermoFisher, #15240062) at 37°C in a humidified atmosphere of 5% CO2.

[0176] Genetic manipulation The expression vector pCMV3, encoding the human COX7B ORF cDNA sequence and carrying a hygromycin selection cassette (Sinobio, #HG20762-UT), was used to overexpress COX7B in ReNcell VM cells. Briefly, cells at 30–60% confluence in a 10 cm dish were transfected with 10 μg of plasmid using Lipofectamine Stem Transfection Reagent (ThermoFisher, #STEM00001). They were allowed to recover for 24 h after transfection. Cells were then selected with 10 μg / mL hygromycin (InvivoGen, #ant-hg-1) for 14 days. The medium was refreshed every 3–4 days. Colonies (clones) were then individually selected, expanded, and tested for COX7B protein expression by Western blotting.

[0177] For constitutive luciferase and GFP expression, cells were infected with a lentivirus (Amsbio, #LVP403) carrying luciferase and GFP sequences along with a neomycin resistance gene. Briefly, cells at 70-80% confluence in a 96-well plate were transduced with 20 viral particles. The next day, cells were selected by incubation with 400 μg / mL neomycin (InvivoGen, #ant-gn-1) for 10 days. After treatment, cells were FACS-sorted for GFP expression on a Becton Dickinson FACSAria III system (Erembodegem, Belgium).

[0178] Western blotting Western blotting was performed as previously described by Van Hee, VF, et al. (Van Hee, VF, et al., Lactate does not activate NF-kappaB in oxidative tumor cells. Front Pharmacol, 2015.6:228), which is incorporated herein by reference. Membranes were incubated overnight with primary rabbit antibodies against COX7b (Abcam, #ab137094) or primary mouse antibodies against β-actin (Sigma-Aldrich, #A5441). Secondary antibodies were peroxidase-affinity-purified goat anti-rabbit IgG (Jackson ImmunoResearch, #111-035-003) and peroxidase-affinity-purified goat anti-mouse IgG (Jackson ImmunoResearch, #115-035-003). Staining was detected with an Amersham Imager 600 (Diegem, Belgium). All data were normalized to the expression of β-actin.

[0179] In vivo assay All in vivo experiments were performed under the approval of the Universite catholique de Louvain (UCLouvain) authorities (Comite d'Ethique Facultaire pour l'Experimentation Animale) and in accordance with national animal care regulations. The specific approval ID for this study was 2020 / UCL / MD / 033.

[0180] Brain injury was induced by LPS injection. LPS was injected intraperitoneally at 1 μg / g body weight (gbw) for two consecutive days according to the protocol of Bodea, LG, et al., Neurodegeneration by activation of the microglial complement-phagosome pathway. J Neurosci, 2014. 34(25): p. 8546-56 (incorporated herein by reference).

[0181] Stem cells with or without COX7B overexpression were delivered into the bloodstream of 18-week-old female NMRI nude mice as described in Example 1 for the delivery of MDA-MB-231 cancer cells. All mice were sacrificed 3 days later by cervical dislocation under terminal anesthesia. Their brains were harvested, sectioned along the separation between the right and left hemispheres, and embedded in paraffin.

[0182] immunohistochemistry The harvested brains were processed for immunohistochemistry. Sections (5 μm thick) were prepared from the center of each hemisphere to create 10 slides per sample and fixed in 4% paraformaldehyde (PFA). Brain sections were then immunostained with an anti-GFP antibody (NovusBio, #NB-600 308) and an HRP-conjugated secondary Envision anti-rabbit antibody (Agilent, #K4003). All sections were counterstained with hematoxylin and eosin. Slides were scanned at 20x magnification using a bright-field Panoramic Scan II microscope (3DHISTECH Kft. Budapest, Hungary). Images were analyzed using SlideViewer (3DHISTECH Kft. Budapest, Hungary) and QuPath software version 0.1.2 (Belfast, UK).

[0183] result Overexpression of COX7B in human neural progenitor cells We therefore tested whether overexpression of COX7B in stem cells could promote their homing to the injured brain after systemic administration of the cells in the bloodstream. As a cellular model, we used the ReNcell VM human neural progenitor cell line (ReN), an immortalized cell line that can be maintained in culture for more than 45 passages and can differentiate into neurons and glial cells by simple growth factor withdrawal. This cell line has already been used by others to study brain engraftment after intrathecal injection in rats (see, e.g., Hovakimyan, M., et al., Survival of transplanted human neural stem cell line (ReNcell VM) into the rat brain with and without immunosuppression. Ann Anat, 2012, 194(5):429-35).

[0184] To study stem cell homing to the injured brain, ReNcell VM cells were first genetically modified to express high levels of COX7B protein. To induce COX7B overexpression, we used the pCMV3 plasmid, which contains the human untagged COX7B open reading frame cDNA sequence and a hygromycin resistance sequence. Post-transfection selection allowed us to obtain 14 ReNcell VM cell clones that expressed higher levels of COX7B protein compared to the wild-type parental cells (Figure 8). To maintain the diversity of the initial cell population, these 14 clones were pooled together to create the ReNcell VM COX7B OE stem cell line. Evidence that homing of human neural progenitor cells overexpressing COX7B in the injured brain is increased compared with human neural progenitor cells that do not overexpress COX7B

[0185] For in vivo cell tracking, ReNcell VM and ReNcell VM COX7B OE cells were modified to constitutively express luciferase and green fluorescent protein (GFP). Retroviruses carrying luciferase and GFP sequences along with a neomycin resistance cassette were used. After selection, ReNcell VM cells constitutively expressing luciferase and GFP were obtained, and ReNcell VM COX7B OE cells constitutively expressing luciferase and GFP were obtained. The two cell lines were used to compare their homing in injured mouse brains. NMRI nude mice treated with LPS (1 μg / gbw) were used because this strain is compatible with human cell transplantation and because LPS has previously been shown to induce neuroinflammation and brain injury and is now widely used as a model of brain injury and neurodegeneration (Trepanier, MO, et al., "Increased brain docosahexaenoic acid has no effect on the resolution of neuroinflammation following intracerebroventricular lipopolysaccharide injection." Neurochem Int, 2018, 118:115-126; Bodea, LG, et al., "Neurodegeneration by activation of the microglial complement-phagosome pathway." J Neurosci, 2014, 34(25):8546-8556).

[0186] Three weeks after LPS treatment, mice received a bolus injection of either 100,000 ReNcell VM cells or 100,000 ReNcell VM COX7B OE cells into the systemic circulation. All mice were euthanized by cervical dislocation under terminal anesthesia three days after stem cell injection, and their brains were harvested and embedded in paraffin. For each mouse, serial sections from two brain hemispheres were immunostained for GFP expression, which reliably detects injected human stem cells. The results, shown in Figures 9A–9H, clearly demonstrated that COX7B overexpression improved human neural stem cell homing to the injured brain of immunodeficient mice. Within the brain parenchyma, ReNcell VM COX7B OE cells were widely distributed in the cortex along areas known to be densely packed with brain blood vessels, and also stained positive for brain cell bodies.

[0187] conclusion The experimental data disclosed herein above support that intervention by overexpressing COX7B in human stem cells improves the homing of these cells to the injured brain. While mice were used for the in vivo demonstration in Example 2, Example 1 shows that overexpression of COX7B enables directional targeting of cells not only to mouse astrocytes but also to human astrocytes.

[0188] Thus, all experimental evidence provided herein demonstrates that the strategy of overexpressing COX7B in stem cells prior to injection into the bloodstream improves their engraftment into damaged brain tissue. In brain preventative / regenerative cell therapy, the present invention allows for the use of administration routes that are less invasive, safer, and potentially more efficient (in terms of spatial distribution) than stem cell transplantation in the brain or injection into the cerebrospinal fluid.

[0189] Importantly, it has been comprehensively demonstrated that engineering human cancer cell lines and human stem cell lines to increase COX7B expression improved their homing to the brain, which may generalize this finding by stating that overexpressing COX7B in human cells in general should increase their homing to the brain after injection into the bloodstream.

Claims

1. A population of cells overexpressing cytochrome c oxidase subunit VIIb (COX7B) protein for use in treating and / or preventing a brain disease or disorder in a subject in need thereof.

2. The population of cells for use according to claim 1 , wherein the cells are stem cells, preferably mesenchymal stem cells or neural stem cells.

3. 3. The population of cells for use according to claim 1 or 2, wherein the cells are mammalian cells, preferably human cells.

4. The population of cells for use according to any one of claims 1 to 3, wherein the cells are derived from the subject.

5. The population of cells for use according to any one of claims 1 to 4, wherein the COX7B protein is overexpressed at a level that induces brain tropism in the cells.

6. The population of cells for use according to any one of claims 1 to 5, wherein the cells stably overexpress COX7B protein.

7. The population of cells for use according to any one of claims 1 to 6, wherein the brain disease or disorder comprises or is selected from the group consisting of neurodegenerative diseases, neurological diseases, brain injuries and cerebrovascular diseases.

8. The brain disease or disorder may be Alzheimer's disease, Parkinson's disease, Huntington's disease, dementia, diffuse Lewy body dementia, frontotemporal dementia (FTD), Lewis body dementia, ataxia, motor neuron disease, amyotrophic lateral sclerosis (ALS), epilepsy and seizures, multiple system atrophy, multiple sclerosis, leukodystrophy, progressive supranuclear palsy, olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatonigral degeneration, corticobasal degeneration, Parkinson's disease, ALS, Guam dementia, Pick's disease, amyloidosis, 8. The population of cells for use according to any one of claims 1 to 7, comprising or selected from the group consisting of: neuroleptic malignancies, Pick's disease, Lou Gehrig's disease, Creutzfeldt-Jakob disease, mild cognitive impairment, syphilis, attention deficit hyperactivity disorder (ADHD), schizophrenia, depression, bipolar disorder, stress disorders, spinal cord injury, myelitis, rheumatic diseases, traumatic brain injury (TBI), concussion, vascular dementia, hypertension, toxic brain injury such as hypoxia or carbon monoxide poisoning, encephalitis, stroke, brain tumor, brain abscess, autism spectrum disorder, and the like.

9. The population of cells for use according to any one of claims 1 to 8, wherein the population of cells is administered to a subject in need thereof in a therapeutically effective amount.

10. 10. The population of cells for use according to claim 9, wherein said therapeutically effective amount is from about 1,000 to about 100,000 billion said cells per kg of body weight.

11. The population of cells for use according to any one of claims 1 to 10, wherein said population of cells is administered to said subject systemically, preferably by intravenous injection.

12. The population of cells for use according to any one of claims 1 to 11, wherein the population of cells is administered in combination with another therapeutic agent.

13. A pharmaceutical composition comprising a population of cells that overexpress COX7B protein and a pharmaceutically acceptable vehicle for use in the prevention and / or treatment of a brain disease or disorder.

14. A combination kit comprising (i) a population of cells that overexpresses COX7B protein or a pharmaceutical composition containing the same or a means for overexpressing COX7B protein in cells, and (ii) another therapeutic agent, for use in the prevention and / or treatment of a brain disease or brain disorder.

15. 1. A method for inducing brain tropism in cells, preferably stem cells, comprising the step of overexpressing COX7B protein in said cells.

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