Genetically modified cells enriched with mitochondria and their use

Abstract

The present invention is based on the discovery that mitochondria-enriched cells are useful for the treatment of diseases and disorders. Pharmaceutical compositions of mitochondrially-enriched genetically engineered T cells and therapeutic methods using mitochondrially-enriched genetically engineered T cells are disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of priority under § 119(e) of U.S. Patent Application No. 63 / 003,184, filed on 31 March 2020, which is incorporated herein by reference in its entirety.

[0002] Field of Invention The present invention relates, as a whole, to pharmaceutical compositions and therapeutic methods using genetically modified cells, and more specifically to genetically modified lymphocytes enriched with mitochondria. [Background technology]

[0003] Background information Despite significant advancements in medical and therapeutic approaches, cancer remains one of the most life-threatening diseases in developed industrial nations. Standard treatment strategies, such as a combination of surgery, chemotherapy, and radiation, can be effective in treating primary tumors in most cases, but these strategies often fail to successfully treat metastatic tumors and prevent disease progression via disseminated tumor cells. Recently, cell-based immunotherapy, particularly adoptive T lymphocyte transfer, has emerged as a highly promising alternative modality for cancer treatment, aiming to prevent the spread of disease metastasis and improve the quality of life for patients with terminal disease, including those refractory to standard therapies. T cell-based immunotherapy leverages the intrinsic ability of T cells to penetrate tissues, become activated, and eliminate target cells. Genetically modified T cells that specifically recognize antigen targets expressed on cancer cells (e.g., T cell receptor (TCR) and chimeric antigen receptor (CAR) transduced T cells) have been used in clinical trials and have achieved promising results.

[0004] The scope of T cell-based therapies is being further expanded to include autoimmune disorders. Preliminary results from preclinical and clinical studies support the application of CAR T therapy in autoimmunity, particularly in the modulation of adverse autoimmune responses.

[0005] T cell receptors (TCRs) are molecules found on the surface of T cells or T lymphocytes that recognize antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. The binding of TCRs to antigen peptides is of relatively low affinity and is degenerate; that is, many TCRs recognize the same antigen peptide, and many antigen peptides are recognized by the same TCR.

[0006] The transcatheter cytology chain (TCR) is composed of two distinct protein chains. In humans, in 95% of T cells, the TCR consists of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), while in 5% of T cells, the TCR consists of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). This ratio changes during individual development and in disease states (such as leukemia). It also differs among species. Orthologs of the four gene loci have been mapped across various species.

[0007] Mitochondria are membrane-bound organelles ranging in diameter from 0.5 to 1.0 μm that supply cellular energy and play a central role in cellular metabolic functions. Mitochondria are found in almost all eukaryotes, and their number and location vary depending on the cell type. Mitochondria contain their own DNA (mtDNA) and their own mechanisms for synthesizing RNA and proteins. Because mtDNA contains only 37 genes, most of the gene products in mammals are encoded by nuclear DNA.

[0008] The primary function of mitochondria is the production of energy as adenosine triphosphate (ATP) through the electron transport chain and oxidative phosphorylation system ("respiratory chain"). Furthermore, mitochondria perform numerous important tasks within eukaryotic cells, including pyruvate oxidation, the Krebs cycle, and the metabolism of amino acids, fatty acids, and steroids. Additional processes involving mitochondria include thermogenesis, calcium ion storage, calcium signaling, programmed cell death (apoptosis), and cell proliferation.

[0009] Intracellular ATP concentrations are typically between 1 and 10 mM. ATP can be produced by redox reactions using monosaccharides and complex sugars (carbohydrates) or lipids as energy sources. To synthesize complex fuels into ATP, they must first be broken down into smaller, simpler molecules. Complex carbohydrates are hydrolyzed into monosaccharides such as glucose and fructose. Fats (triglycerides) are metabolized to yield fatty acids and glycerol.

[0010] The overall process of oxidizing glucose to carbon dioxide is known as cellular respiration, and can produce approximately 30 molecules of ATP from a single molecule of glucose. ATP is produced by several different cellular processes. The three main pathways used to generate energy in eukaryotes are glycolysis and the citric acid cycle / oxidative phosphorylation (both components of cellular respiration) and beta-oxidation. The majority of this ATP production by non-photosynthetic eukaryotes occurs in mitochondria, which can account for nearly 25% of the total volume of a typical cell.

[0011] Attempts to induce mitochondria migration into host cells or tissues have been reported. Most methods require active mitochondria transport via injection. Mitochondria transport encapsulated within vehicles such as liposomes is also known. Further evidence suggests that mitochondrial migration can occur spontaneously between cells in vitro, although only mtDNA, rather than whole intact mitochondria, has been established. Mitochondrial transport in vitro via endocytosis or internalization has also been demonstrated. [Overview of the project]

[0012] This invention is based on the significant discovery that mitochondrial-enriched cells are useful in treating diseases and disorders. This invention provides pharmaceutical compositions of mitochondrial-enriched genetically modified T cells. This invention also provides therapeutic methods using mitochondrial-enriched genetically modified T cells.

[0013] In one embodiment, the present invention provides a pharmaceutical composition comprising mitochondrial-enriched genetically modified T cells and a pharmaceutically acceptable carrier, wherein the mitochondrial-enriched genetically modified T cells are enriched with exogenous mitochondria.

[0014] In one embodiment, mitochondrial-enriched genetically modified T cells are produced by a method comprising: obtaining T cells from a subject suffering from a disease or disorder, or from a donor; obtaining exogenous mitochondria; producing mitochondrial-enriched T cells by contacting T cells with exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the T cells; and producing mitochondrial-enriched genetically modified T cells by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the mitochondrial-enriched T cells, wherein the mitochondrial content of the mitochondrial-enriched genetically modified T cells is detected to be higher than that of T cells. In one embodiment, the conditions that allow exogenous mitochondria to enter the T cells are 10 6 This involves incubating T cells with exogenous mitochondria at a ratio of approximately 0.088 to 176 mU of citrate synthase (CS) activity per T cell.

[0015] In a further aspect, genetically engineered T cells enriched with mitochondria are obtained by obtaining T cells from a subject suffering from a disease or disorder or from a donor, genetically engineering the T cells by introducing a nucleic acid encoding a chimeric T cell receptor (TCR) or a chimeric antigen receptor (CAR), obtaining exogenous mitochondria, and contacting the genetically engineered T cells with the exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the genetically engineered T cells, thereby producing genetically engineered T cells enriched with mitochondria. The mitochondria content of the genetically engineered T cells enriched with mitochondria produced by this method is detectably higher than the mitochondria content of the genetically engineered T cells. In one aspect, the conditions that allow the exogenous mitochondria to enter the genetically engineered T cells include incubating the genetically engineered T cells with the exogenous mitochondria at a ratio of citrate synthase (CS) activity of about 0.088 - 176 mU per 10 6 T cells.

[0016] In certain embodiments, the genetically engineered T cells and the genetically engineered T cells enriched with mitochondria are CAR-T cells. In another embodiment, the genetically engineered T cells and the mitochondria-enriched T cells are TCR-T cells. In various embodiments, the exogenous mitochondria are derived from human cells. In some embodiments, the human cells are derived from human placenta, human blood cells, human stem cells or human somatic cells. In some embodiments, the human cells are cells grown in culture. In certain embodiments, the stem cells are induced pluripotent stem cells, embryonic stem cells or pluripotent cells. In another embodiment, the exogenous human mitochondria are syngeneic or allogeneic. In a further embodiment, the exogenous mitochondria are autologous.

[0017] In one aspect, the pharmaceutical composition has at least 1×10 5 -5×10 10 mitochondria-enriched T cells or genetically engineered T cells enriched with mitochondria per kg of body weight of the subject in need thereof. In some embodiments, dose escalation is carried out.

[0018] In certain embodiments, genetically engineered T cells enriched with mitochondria have an increased mitochondrial DNA content, an increased level of citrate synthase (CS) activity, an increased content of at least one mitochondrial protein selected from SDHA and COX1, an increased O2 consumption rate, an increased ATP production rate, or at least one of any combination thereof, as compared to the corresponding levels in T cells before mitochondrial enrichment.

[0019] In various embodiments, the conditions that allow exogenous mitochondria to enter T cells or genetically engineered T cells include incubating the T cells or genetically engineered T cells with exogenous mitochondria at a temperature in the range of about 16-37 °C for a time in the range of about 0.5-30 hours. In a further embodiment, the exogenous mitochondria constitute more than 1% of the total mitochondrial content in mitochondrial-enriched T cells or genetically engineered T cells enriched with mitochondria.

[0020] In one embodiment, the disease or disorder is cancer. In certain embodiments, the cancer is a blood cancer. In another embodiment, the T cells are autologous or allogeneic.

[0021] In additional embodiments, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject genetically engineered T cells enriched with mitochondria, wherein the genetically engineered T cells enriched with mitochondria are enriched with exogenous mitochondria.

[0022] In one embodiment, mitochondrial-enriched genetically engineered T cells are produced by a method comprising: obtaining T cells from a subject suffering from a disease or disorder, or from a donor; obtaining exogenous mitochondria; producing mitochondrial-enriched T cells by contacting T cells with exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the T cells; and producing mitochondrial-enriched genetically engineered T cells by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the mitochondrial-enriched T cells, wherein the mitochondrial content of the mitochondrial-enriched genetically engineered T cells is detected to be higher than that of T cells. In some embodiments, the mitochondrial content of the mitochondrial-enriched genetically engineered T cells is detected to be higher than that of T cells. In one embodiment, the conditions that allow the exogenous mitochondria to enter the T cells are 10 6 This involves incubating T cells with exogenous mitochondria at a ratio of approximately 0.088 to 176 mU of citrate synthase (CS) activity per T cell.

[0023] In a further embodiment, mitochondrial-enriched genetically modified T cells are produced by a method comprising: obtaining T cells from a subject suffering from a disease or disorder or from a donor; producing genetically modified T cells by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the T cells; obtaining exogenous mitochondria from a donor; and producing mitochondrial-enriched genetically modified T cells by contacting the genetically modified T cells with exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the genetically modified T cells, wherein the mitochondrial content of the mitochondrial-enriched genetically modified T cells is detected to be higher than that of the genetically modified T cells. In one embodiment, the conditions that allow the exogenous mitochondria to enter the genetically modified T cells are 10 6This involves incubating genetically modified T cells with exogenous mitochondria at a ratio of approximately 0.088 to 176 mU of citrate synthase (CS) activity per T cell.

[0024] In one embodiment, the subject is a human subject. In another embodiment, the disease or disorder is cancer. In a specific embodiment, the cancer is a blood cancer. In a particular embodiment, the administration is by intravenous, intraperitoneal, intra-arterial, intrathecal, and intramuscular administration. In one embodiment, the administration is by intravenous administration.

[0025] In a further embodiment, the treatment method also includes administering chemotherapy, radiotherapy, or other anti-cancer therapies to the subject. In a further embodiment, the pharmaceutical composition is administered concurrently with, in parallel with, or after the administration of chemotherapy, radiotherapy, or other anti-cancer therapies. In certain embodiments, exogenous mitochondria are derived from human cells. In some embodiments, human cells are derived from human placenta, human blood cells, stem cells, or somatic cells. In some embodiments, human cells are cells grown in culture. In certain embodiments, stem cells are induced pluripotent stem cells, embryonic stem cells, or pluripotent cells. In certain embodiments, exogenous mitochondria are allogeneic or autologous. In one embodiment, after enrichment with exogenous mitochondria, at least 1% of the total mitochondrial content in mitochondrial-enriched T cells or mitochondrial-enriched genetically engineered T cells contains exogenous mitochondria. In various embodiments, the conditions that allow exogenous mitochondria to enter T cells or genetically modified T cells include incubating the T cells or genetically modified T cells with exogenous mitochondria for a period of time ranging from 0.5 to 30 hours at a temperature ranging from approximately 16 to 37°C. [Brief explanation of the drawing]

[0026] [Figure 1A] Figure 1A shows mitochondrial-enriched CAR-T cells. It also shows ATP levels and citrate synthase activity in CAR-T cells enriched with mitochondria 5 days after cell activation. [Figure 1B] Figure 1B shows mitochondrial-enriched CAR-T cells. The ATP levels and citrate synthase activity of mitochondrial-enriched CAR-T cells 10 days after cell activation are shown. [Figure 1C] Mitochondrial-enriched CAR-T cells are shown. Figure 1C shows the percentage of exogenous mtDNA measured in CAR-T cells enriched with 4.4 mU of mitochondria on days 5 and 10 of cell activation. [Figure 2] This shows the proliferation of activated and inactivated T cells, as well as the CAR-T% after activation. A1: CAR-T cells enriched with 4.4 mU of placental mitochondria at 7000 g, A2: CAR-T cells enriched with 4.4 mU of placental mitochondria at 400 g, UnT: untransduced and unenriched cells, Tr: transduced cells not subjected to mitochondrial enrichment conditions, Ctrl: transduced cells subjected to mitochondrial enrichment conditions without introducing exogenous mitochondria into the enrichment process. [Modes for carrying out the invention]

[0027] Detailed description of the invention This invention is based on the significant discovery that mitochondrial-enriched cells are useful in treating diseases and disorders. This invention provides pharmaceutical compositions of mitochondrial-enriched genetically modified T cells. This invention also provides therapeutic methods using mitochondrial-enriched genetically modified T cells.

[0028] Before describing the compositions and methods of the present invention, it should be understood that the present invention is not limited to the specific compositions, methods, and experimental conditions described, and that such compositions, methods, and conditions may vary. It should also be understood that the terms used herein are for describing only specific embodiments, and are not intended to limit the scope of the present invention, as it is limited to the appended claims.

[0029] Where used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple references unless the context makes this obvious. Thus, for example, a reference to “the method” includes one or more methods and / or steps of the type described herein, which would be obvious to those skilled in the art upon reading this disclosure, etc.

[0030] All publications, patents, and patent applications referenced herein are incorporated by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated as being incorporated by reference.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but it should be understood that modifications and variations are included in the spirit and scope of this disclosure. Preferred methods and materials are described below.

[0032] The present invention provides pharmaceutical compositions for cancer immunotherapy and their use, the pharmaceutical compositions comprising human mature lymphocytes, including non-genetically modified lymphocytes and genetically modified lymphocytes (e.g., CART cells), which are ex vivo enriched with exogenous mitochondria.

[0033] As used herein, the term “lymphocyte” refers to white blood cells that play a major role in defending the body from disease, and includes T cells, natural killer cells (NK cells), B cells, and mixtures thereof. It will be understood by those skilled in the art that the above-mentioned immune cell types can be further divided into subsets. In some embodiments, the lymphocytes are mature lymphocytes. In some embodiments, the lymphocytes are non-genetically modified lymphocytes. In other embodiments, the lymphocytes are genetically modified lymphocytes.

[0034] As used herein, the term “mature lymphocyte” refers to fully differentiated or terminally differentiated lymphocytes that have undergone development leading to selection and maturation within central lymphoid tissue. After maturation, lymphocytes enter the circulating and peripheral lymphoid organs (e.g., the spleen and lymph nodes). According to some embodiments, the term “mature lymphocyte” does not include lymphoid progenitor cells or lymphocyte precursor cells. According to other embodiments, the term “mature lymphocyte” also includes lymphocyte precursor cells. As used herein, the term “lymphocyte precursor cell” usually refers to partially differentiated unipotent cells that constitute intermediate cells before differentiation into lymphocytes. As used herein, the term “unipotent” refers to cells that have the ability to differentiate into only one cell type. In contrast to precursor cells, progenitor cells are pluripotent and have the potential to differentiate into separate cell types.

[0035] Cell maturity can be assessed based on assays such as marker expression, function, integration, and non-mitotic cells. For example, with respect to T cells, mature T cells are characterized by the expression of either CD4 or CD8, but not both (i.e., they are single-positive), and by the expression of CD3. The expression of such markers can be determined by methods known in the art, such as FACS analysis or immunohistochemistry techniques.

[0036] The T cell receptor (TCR) is a protein complex found on the surface of T cells or T lymphocytes, and it plays a role in recognizing antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. The binding of TCRs to antigen peptides is of relatively low affinity and is degenerate. The TCR is composed of two different protein chains. In humans, in 95% of T cells, the TCR consists of alpha (α) and beta (β) chains (encoded by TRA and TRB, respectively), while in 5% of T cells, the TCR consists of gamma and delta (γ / δ) chains (encoded by TRG and TRD, respectively). This ratio changes during individual development and in disease states (such as leukemia). The antigen molecules that activate γδ T cells remain largely unknown. However, γδ T cells appear to be able to recognize whole proteins, rather than being limited by MHC and requiring the peptide to be presented by an MHC molecule on the APC. Human γδT cells using the Vγ9 and Vδ2 gene fragments constitute the major γδT cell population in peripheral blood.

[0037] This invention is based in part on the finding that T lymphocytes are receptive to being enriched with exogenous mitochondria, and that lymphocytes can experience increased mitochondrial content, cell viability, fatty acid oxidation (FAO), and ATP production through exogenous mitochondria enrichment. Without being constrained by any theory or mechanism, it is hypothesized that co-incubation of lymphocytes with exogenous mitochondria promotes the transfer of intact, functional mitochondria into lymphocytes. It is further hypothesized that improving mitochondrial function in lymphocytes through mitochondrial enrichment can improve the in vivo function of transplanted lymphocytes, thereby enhancing cell-based immunotherapy.

[0038] As used herein, the term “cell-based immunotherapy” refers to therapies that involve the application of genetically modified or non-genetically modified immune cells, such as T cells. This therapeutic approach can be used to treat a wide range of cancerous diseases as well as infectious diseases and autoimmune disorders.

[0039] The present invention provides a pharmaceutical composition comprising mitochondrial-enriched genetically modified T cells and a pharmaceutically acceptable carrier, wherein the genetically modified T cells are enriched with exogenous mitochondria. The present invention also provides a method for treating a target in need, comprising administering mitochondrial-enriched genetically modified T cells to the target, wherein the mitochondrial-enriched genetically modified T cells are enriched with exogenous mitochondria.

[0040] As used herein, “pharmaceutical composition” refers to a formulation comprising an active ingredient and optionally pharmaceutically acceptable carriers, diluents, or excipients. The term “active ingredient” can be synonymous with “active ingredient” and means any active substance capable of inducing the desired effect upon administration. Examples of active ingredients include, but are not limited to, compounds, drugs, therapeutic agents, and small molecules.

[0041] "Pharmacologically acceptable" means that a carrier, diluent, or excipient must be compatible with the other components of the formulation and not harmful to its recipient or to the activity of the active ingredient of the formulation. Pharmaceutically acceptable carriers, excipients, or stabilizers are well known in the art, for example, in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980). Pharmacopoecially acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the doses and concentrations used and include buffers such as phosphoric acid, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkylparabens such as methyl or propylparaben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; serum albumin This may include proteins such as methyl esters, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Examples of carriers include, but are not limited to, liposomes, nanoparticles, ointments, micelles, microspheres, microparticles, creams, emulsions, and gels.Examples of excipients include, but are not limited to, anti-adhesion agents such as magnesium stearate, binders such as sugars and their derivatives (sucrose, lactose, starch, cellulose, sugar alcohols, etc.), proteins and synthetic polymers such as gelatin, lubricants such as talc and silica, and antioxidants, vitamins A, E, C, retinyl palmitate, selenium, cysteine, methionine, citric acid, sodium sulfate, and parabens. Examples of diluents include, but are not limited to, water, alcohol, saline solution, glycol, mineral oil, and dimethyl sulfoxide (DMSO).

[0042] The term “treatment” is used herein interchangeably with the term “treatment method” and refers to 1) the cure, delay, reduction of symptoms, and / or cessation of progression of a diagnosed pathological condition or disorder, as well as 2) both prophylactic / preventive measures. Individuals requiring treatment may include individuals who already have a specific medical disorder, as well as individuals who may eventually acquire that disorder (i.e., individuals requiring preventive measures).

[0043] Terms such as “therapeutic effective dose,” “effective dose,” “therapeutic effective dose,” and “effective dose” refer to the amount of a target compound that elicits a biological or medical response in a tissue, system, animal, or human, as sought by researchers, veterinarians, physicians, or other clinicians. The effective dose may be determined as described herein.

[0044] The terms “administering” and / or “administering” should be understood to mean providing a therapeutically effective amount of the pharmaceutical composition to a subject in need of treatment. The route of administration may be enteral, topical, or parenteral. Therefore, routes of administration include, but are not limited to, intravenous, intraperitoneal, intra-arterial, intrathecal, and intramuscular administration. As used herein, the terms “parenteral administration” and “administered parenterally” mean a mode of administration other than enteral and topical administration. The pharmaceutical composition may be administered in various unit dosage forms depending on the method of administration. Preferred unit dosage forms include, but are not limited to, capsules, injections, implantable sustained-release formulations, and lipid complexes.

[0045] In a particular embodiment, mitochondrial-enriched genetically engineered T cells are produced by obtaining T cells from a diseased or impaired subject or from a donor, obtaining exogenous mitochondria, contacting the T cells with the exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the T cells, thereby producing mitochondrial-enriched T cells, and introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the mitochondrial-enriched T cells, thereby producing mitochondrial-enriched genetically engineered T cells, wherein the mitochondrial content of the mitochondrial-enriched genetically engineered T cells is detected to be higher than that of T cells. In a particular embodiment, the conditions that allow the exogenous mitochondria to enter the T cells are 10 6 This involves bringing T cells into contact with exogenous mitochondria at a ratio of approximately 0.088 to 176 mU of citrate synthase (CS) activity per T cell.

[0046] In another embodiment, genetically engineered T cells enriched with mitochondria are obtained by obtaining hematopoietic stem cells from a subject suffering from a disease or disorder or from a donor, obtaining exogenous mitochondria from a donor, producing mitochondria-enriched hematopoietic stem cells under conditions that allow the exogenous mitochondria to enter the hematopoietic stem cells, differentiating the mitochondria-enriched hematopoietic stem cells into mitochondria-enriched T cells, and introducing a nucleic acid encoding a chimeric T cell receptor (TCR) or chimeric antigen receptor (CAR) into the mitochondria-enriched T cells, and the exogenous mitochondrial content of the genetically engineered T cells enriched with mitochondria is detectably higher than the mitochondrial content of the hematopoietic stem cells. In certain embodiments, the conditions for allowing exogenous mitochondria to enter the hematopoietic stem cells are 10 6 by contacting the hematopoietic stem cells with exogenous mitochondria at a ratio of citrate synthase (CS) activity of about 0.088 to 176 mU per 10

[0047] In a further embodiment, genetically engineered T cells enriched with mitochondria are obtained by obtaining T cells from a subject suffering from a disease or disorder or from a donor, producing genetically engineered T cells by introducing a nucleic acid encoding a chimeric T cell receptor (TCR) or chimeric antigen receptor (CAR) into the T cells, obtaining exogenous mitochondria, and producing genetically engineered T cells enriched with mitochondria under conditions that allow the exogenous mitochondria to enter the genetically engineered T cells, and the mitochondrial content of the genetically engineered T cells enriched with mitochondria is detectably higher than the mitochondrial content of the T cells. In certain aspects, the conditions for allowing exogenous mitochondria to enter the T cells include contacting the T cells with exogenous mitochondria at a ratio of citrate synthase (CS) activity of about 0.088 to 176 mU per 10 6 T cells.

[0048] According to some embodiments, the term “mitochondrial content” refers to functional mitochondrial content and non-functional mitochondrial content. According to some embodiments, the term “mitochondrial content” refers to functional mitochondrial content.

[0049] The terms “T cell” and “T lymphocyte” are used interchangeably herein. T cells are a specific type of lymphocyte that plays a crucial role in regulating and shaping the immune response by providing a variety of immune-related functions. T cells can be distinguished from other lymphocytes by the presence of a T cell receptor (TCR) on their cell surface. As used herein, the term “T cell” includes cytotoxic T cells, T helper cells, regulatory T cells, and natural killer T cells (NKTs). According to some embodiments, T cells are T cell precursors. According to other embodiments, T cells are mature T cells. According to some embodiments, T cells are fully differentiated T cells. In the methods of the present invention, T cells obtained from a diseased or impaired subject or from a donor are not actively altered or modified (e.g., reduction of mtDNA or mitochondrial function) before enrichment with exogenous mitochondria. More specifically, mitochondrial function and / or mitochondrial DNA in T cells are not actively altered or modified before contact with exogenous mitochondria.

[0050] According to some embodiments, the T cells are genetically modified T cells. According to further embodiments, the genetically modified T cells are selected from T cell receptor (TCR) transduced T cells and chimeric antigen receptor (CAR) transduced T cells. Each possibility represents a distinct embodiment of the present invention. According to certain embodiments, the genetically modified T cells are CAR-T cells.

[0051] In some embodiments, the genetically engineered T cells are autologous to the subject. In some embodiments, the genetically engineered T cells are allogeneic to the subject. In specific embodiments, the allogeneic T cells are obtained from a donor that is at least partially HLA-matched to the subject. In certain embodiments, if the genetically engineered T cells are allogeneic to the subject, the method further includes the step of administering to the subject an agent that prevents, delays, minimizes, or neutralizes adverse immunogenic reactions between the subject and mitochondrial-enriched human stem cells. Each possibility represents a distinct embodiment of the invention. In certain embodiments, the adverse immunogenic reaction is graft-versus-host disease (GvHD).

[0052] As used herein, the term "T cells are of autogenous origin to the subject" refers to cells that are the subject's own cells. As used herein, the term "T cells are allogeneic to the subject" refers to cells that are from a different donor individual.

[0053] The terms “CAR-transduced T cells” and “CAR-T cells” are used interchangeably herein and refer to genetically modified T cells that express chimeric antigen receptors (CARs) that target specific antigens. According to certain embodiments, the antigens are present on the surface of cancer cells. According to some embodiments, the cancer cells are hematopoietic cancer cells. According to some embodiments, CAR-T cells express CARs that target antigens selected from, but not limited to, CD19, CD30, CD33, CD123, FLT3, and BCMA.

[0054] As is known to those skilled in the art, CAR-T cells are isolated from a subject and ex vivo genetically engineered using either a lentiviral or retroviral vector or a nonviral gene transfer system to express engineered CARs specific to a particular tumor target. These reprogrammed CAR-T cells are then grown, selected as needed, and injected into the subject after immunosuppressive pretreatment. According to the principles of the present invention, ex vivo genetic engineering of isolated T cells can be performed before or after the mitochondrial enrichment process.

[0055] After transplantation, CAR-T cells undergo antigen engagement, amplify in the peripheral blood, migrate to the tumor site, identify and kill tumor cells expressing the corresponding antigen. This can lead to widespread proliferation of CAR-T cells and release of tumor antigens, which activates the target immune system, mobilizing non-CAR-T immune cells and thus inducing a further antitumor response through a process known as cross-priming from epitope diffusion.

[0056] When the number of CAR-T cells decreases to undetectable levels after transplantation, disease relapse is frequently observed. It has been suggested that enriching CAR-T cells with exogenous mitochondria can enhance their survival and activity in vivo, thereby expanding and amplifying the therapeutic effects of the cells.

[0057] The terms “TCR-transduced T cells” and “TCR-T cells” are used interchangeably herein and refer to T cells that have been genetically modified to express the T cell receptor (TCR). Unlike CAR-T cells, which recognize proteins expressed on the surface, the T cell receptor (TCR) can recognize tumor-specific proteins located inside the cell. When tumor-specific proteins are broken down into fragments, they appear on the cell surface along with another protein called the major histocompatibility complex (MHC). TCRs are engineered to recognize the tumor-specific protein fragment / MHC combination.

[0058] According to some embodiments, genetically modified T cells are derived from mammalian subjects, preferably human subjects.

[0059] According to some embodiments, the T cells of the present invention are T cells having a lower mitochondrial membrane potential compared to the corresponding T cells.

[0060] As used herein, the term “mitochondrial-enriched genetically engineered T cell” refers to a T cell that has been genetically engineered (i.e., into which nucleic acids encoding a TCR or CAR have been introduced) and into which exogenous mitochondria have been inserted.

[0061] As used herein, the term “mitochondrial-enriched T cell” refers to a T cell into which exogenous mitochondria have been inserted.

[0062] As used herein, the term “genetically modified T cell” means a T cell that has been genetically modified (i.e., a nucleic acid encoding a TCR or CAR has been introduced into the cell).

[0063] As used herein, the term "mitochondrial-enriched hematopoietic stem cell" refers to a hematopoietic stem cell into which exogenous mitochondria have been inserted.

[0064] As used herein, the term “stem cell” generally refers to any mammalian stem cell. Stem cells are undifferentiated cells that can differentiate into other types of cells and can divide to produce nearly identical stem cells. Stem cells can be either totipotent or pluripotent.

[0065] As used herein, the term “human stem cells” generally refers to all stem cells naturally found in humans, and all stem cells produced or induced ex vivo that are compatible with humans. “Progenitor cells” like stem cells tend to differentiate into specific types of cells, but are already more specific than stem cells and can be differentiated into their “target” cells. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, while progenitor cells can divide only a limited number of times. As used herein, the term “human stem cells” further includes “progenitor cells” and “not fully differentiated stem cells.”

[0066] In certain embodiments, the stem cells are pluripotent stem cells (PSCs). In other embodiments, the PSCs are non-embryonic stem cells. According to some embodiments, embryonic stem cells are expressly excluded from the scope of the invention. In some embodiments, the stem cells are artificial PSCs (iPSCs). In certain embodiments, the stem cells are embryonic stem cells. In certain embodiments, the stem cells are derived from bone marrow cells. In certain embodiments, the stem cells are CD34+ cells. In certain embodiments, the stem cells are mesenchymal stem cells. In other embodiments, the stem cells are derived from adipose tissue. In yet another embodiment, the stem cells are derived from blood. In a further embodiment, the stem cells are derived from umbilical cord blood. In a further embodiment, the stem cells are derived from oral mucosa. In specific embodiments, the stem cells obtained from a subject suffering from a disease of impairment or from a healthy subject are bone marrow cells or bone marrow-derived stem cells.

[0067] As used herein, the term “pluripotent stem cells (PSCs)” refers to cells that can proliferate indefinitely and give rise to multiple cell types within the body. Totipotent stem cells are cells that can give rise to all other cell types within the body. Embryonic stem cells (ESCs) are totipotent stem cells, and induced pluripotent stem cells (iPSCs) are pluripotent stem cells.

[0068] As used herein, the term “induced pluripotent stem cells (iPSCs)” refers to one type of pluripotent stem cell that can be generated from human adult somatic cells. Some non-exclusive examples of somatic cells that can generate iPSCs as used herein include fibroblasts, endothelial cells, capillary hematopoietic cells, keratinocytes, and myeloid epithelial cells.

[0069] As used herein, the term “embryonic stem cell (ESC)” refers to a type of totipotent stem cell derived from the inner cell mass of a blastocyst.

[0070] As used herein, the term “bone marrow cells” generally refers to all human cells and all cell populations naturally found in human bone marrow. The terms “bone marrow stem cells” and “bone marrow-derived stem cells” refer to populations of stem cells derived from bone marrow.

[0071] In some embodiments, the autologous or allogeneic human stem cells are pluripotent stem cells (PSCs) or induced pluripotent stem cells (iPSCs). In further embodiments, the autologous or allogeneic human stem cells are mesenchymal stem cells.

[0072] According to some embodiments, human stem cells are derived from adipose tissue, oral mucosa, blood, umbilical cord blood, or bone marrow. Each possibility represents a distinct embodiment of the present invention. In specific embodiments, human stem cells are derived from bone marrow.

[0073] In certain embodiments, bone marrow-derived stem cells include bone marrow hematopoietic cells. As used herein, the term “bone marrow hematopoietic cells” refers to bone marrow hematopoiesis, for example, bone marrow and all cells arising therefrom, i.e., cells involved in the production of all blood cells.

[0074] In certain embodiments, bone marrow-derived stem cells include erythropoiesis cells. As used herein, the term “erythropoiesis cells” refers to cells involved in erythropoiesis, for example, the production of red blood cells (erythrocytes).

[0075] In certain embodiments, bone marrow-derived stem cells include pluripotent hematopoietic stem cells (HSCs). As used herein, the terms “pluripotent hematopoietic stem cells” or “hematopoietic blasts” refer to stem cells that give rise to all other blood cells through the hematopoietic process.

[0076] In certain embodiments, bone marrow-derived stem cells include myeloid common progenitor cells, lymphoid common progenitor cells, or any combination thereof. In certain embodiments, bone marrow-derived stem cells include mesenchymal stem cells. As used herein, the term "myeloid common progenitor cells" refers to cells that produce myeloid cells. As used herein, the term "lymphoid common progenitor cells" refers to cells that produce lymphocytes.

[0077] In certain embodiments, bone marrow-derived stem cells further include megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. Each possibility represents a distinct embodiment of the present invention.

[0078] In certain embodiments, bone marrow-derived stem cells include mesenchymal stem cells. As used herein, the term “mesenchymal stem cells” refers to pluripotent stromal cells that can differentiate into a variety of cell types, including osteoblasts, chondrocytes, myocytes, and adipocytes.

[0079] In certain embodiments, bone marrow-derived stem cells include bone marrow hematopoietic cells. In certain embodiments, bone marrow-derived stem cells consist of erythropoiesis cells. In certain embodiments, bone marrow-derived stem cells include pluripotent hematopoietic stem cells (HSCs). In certain embodiments, bone marrow-derived stem cells include myeloid common progenitor cells, lymphoid common progenitor cells, or any combination thereof. In certain embodiments, bone marrow-derived stem cells include megakaryocytes, erythrocytes, mast cells, myoblasts, basophils, neutrophils, eosinophils, monocytes, macrophages, natural killer (NK) cells, small lymphocytes, T lymphocytes, B lymphocytes, plasma cells, reticular cells, or any combination thereof. In certain embodiments, bone marrow-derived stem cells consist of mesenchymal stem cells. In certain embodiments, stem cells include a plurality of human bone marrow stem cells obtained from peripheral blood.

[0080] Hematopoietic stem cells (HSCs) are stem cells that give rise to other blood cells. This process is called hematopoiesis. Hematopoietic stem cells give rise to different types of blood cells, which are called the myeloid and lymphoid lineages. Both the myeloid and lymphoid lineages are involved in dendritic cell formation. Myeloid cells include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes, and platelets. Lymphoid cells include T cells, B cells, and natural killer cells.

[0081] Hematopoietic progenitor cell antigen CD34, also known as CD34 antigen, is a protein encoded in humans by the CD34 gene. CD34 is a differentiated cluster of cell surface glycoproteins and functions as a cell-cell adhesion molecule. In certain embodiments, bone marrow stem cells express bone marrow progenitor cell antigen CD34 (are CD34+). In certain embodiments, bone marrow stem cells do not express CD34. In certain embodiments, bone marrow stem cells present bone marrow progenitor cell antigen CD34 on their outer membrane. In certain embodiments, CD34+ cells are derived from umbilical cord blood. As used herein, the term “CD34+ cells” refers to hematopoietic stem cells characterized as CD34-positive, regardless of their origin. In certain embodiments, CD34+ cells are obtained from bone marrow, from bone marrow cells recruited into the bloodstream, or from umbilical cord blood.

[0082] In certain embodiments, stem cells, including hematopoietic stem cells, are obtained from the peripheral blood of a person suffering from a disease or disorder. In certain embodiments, stem cells are obtained from the peripheral blood of a healthy person. As used herein, the term “peripheral blood” refers to blood circulating in the blood system.

[0083] As used herein, the terms “autologous cells” or “cells of autologous origin” refer to cells of the subject itself. The term “autologous mitochondria” refers to mitochondria obtained from the subject's own cells or from cells related to the maternal lineage. The terms “allogeneic cells” or “allogeneic mitochondria” refer to cells or mitochondria from different donor individuals.

[0084] As used herein and in the claims, the term “symbiotic” means sufficient genetic identity or genetic proximity to enable transplantation between individuals without rejection. In relation to mitochondria, the term “symbiotic” is used herein interchangeably with the term “autologous mitochondria,” meaning of the same maternal lineage. Cells enriched with allogeneic mitochondria mean that the exogenous and endogenous mitochondria are substantially genetically distinct, while cells enriched with syngeneic mitochondria mean that the exogenous and endogenous mitochondria are genetically identical or nearly identical, since the exogenous mitochondria originate from a maternally related cell.

[0085] The terms “disease” and “disorder” refer to any distress that is not considered normal or differs from a physiological state. Diseases and disorders can affect virtually any organ, tissue, or function that is part of the body. Non-exclusive examples of diseases and conditions include cancer, muscle diseases and disorders, glycogen storage diseases and disorders, vascular endothelial disorders or diseases, brain disorders or brain diseases, placental disorders or placental diseases, thymic disorders or thymic diseases, autoimmune diseases, kidney diseases or disorders, pancreatic diseases or pancreatic disorders, prostate disorders or prostate diseases, kidney disorders or kidney diseases, blood disorders or blood disorders, heart diseases or heart disorders, skin disorders or skin diseases, immune and inflammatory diseases and disorders, bone diseases or bone disorders, gastrointestinal diseases or gastrointestinal disorders, and eye diseases or eye disorders.

[0086] As used herein, the terms “subject suffering from disease or disorder” or “subject having disease or disorder” refer to a human subject experiencing a debilitating effect caused by a particular condition. Disorders may include cancer, age-related disorders, kidney disease, pancreatic disease, liver disease, muscle disease, brain disease, primary or secondary mitochondrial disease, and other diseases or disorders.

[0087] As used herein, the term “donor” refers to a donor that provides exogenous cells or mitochondria. In some embodiments, the donor is free from disease or disorder, or does not suffer from the same disorder as the subject. In certain embodiments, the donor is the subject, and the cells and / or mitochondria are of autologous origin.

[0088] With respect to mitochondria, the terms “exogenous” or “isolated exogenous” refer to mitochondria originating from a source outside the recipient cell. For example, in some embodiments, exogenous mitochondria are induced or isolated from a donor cell different from the donor of the recipient cell. In some embodiments, exogenous mitochondria are induced or isolated from a donor cell from the same subject as the recipient cell. For example, exogenous mitochondria may be purified, isolated, or obtained from a donor cell and then introduced into recipient cells derived from the same subject or a different donor, resulting in exogenous mitochondria being autologous and allogeneic, respectively. In certain embodiments, exogenous mitochondria are whole mitochondria.

[0089] As used herein, the terms “isolated” and “partially purified” in relation to mitochondria include exogenous mitochondria that have been purified from other cellular components, or at least partially purified. The total amount of mitochondrial proteins in exogenous isolated or partially purified mitochondria is between 10% and 90% of the total amount of cellular proteins in the sample.

[0090] In certain embodiments, exogenous mitochondria constitute at least 1% of the total mitochondrial content in mitochondrial-enriched cells. In certain embodiments, exogenous mitochondria constitute at least 3% of the total mitochondrial content in mitochondrial-enriched cells. In certain embodiments, exogenous mitochondria constitute at least 10% of the total mitochondrial content in mitochondrial-enriched T cells. In some embodiments, exogenous mitochondria constitute at least about 1%, 3%, 5%, 10%, 15%, 20%, 25%, or 30% of the total mitochondrial content in mitochondrial-enriched T cells. In certain embodiments, the total amount of mitochondrial proteins in exogenous mitochondria is 10%–80%, 20–80%, 40–70%, 20–40%, or 20–30% of the total amount of cellular proteins. Each possibility represents a distinct embodiment of the present invention. In certain embodiments, the total amount of mitochondrial proteins in exogenous mitochondria is 10%–80% of the total amount of cellular proteins in the sample. In certain embodiments, the total amount of mitochondrial proteins in exogenous mitochondria is 10% to 80% of the combined weight of mitochondria and other intracellular fractions. In other embodiments, the total amount of mitochondrial proteins in exogenous mitochondria exceeds 80% of the combined weight of mitochondria and other intracellular fractions.

[0091] In certain embodiments, exogenous mitochondria are obtained from human cells or human tissue. In certain embodiments, the cells are selected from the group consisting of placenta, cultured placental cells, or blood cells. In some embodiments, mitochondria are obtained from human stem cells. In some embodiments, the human cells are human somatic cells. In some embodiments, the human cells are cultured cells.

[0092] In certain embodiments, the methods and pharmaceutical compositions provided by the present invention further include the step of administering an active agent that promotes mitochondrial biosynthesis to a mitochondrial donor. As used herein, the term “mitochondrial biosynthesis” refers to the growth and fission of mitochondria. In certain embodiments, the active agent that promotes mitochondrial biosynthesis is erythropoietin (EPO) or a salt thereof. In certain embodiments, the active agent is selected from the group consisting of recombinant human erythropoietin and isolated human erythropoietin.

[0093] In relation to mitochondria, the term "endogenous" refers to mitochondria that are created / expressed / produced by the cell and not introduced into the cell from an external source. In some embodiments, endogenous mitochondria contain proteins and / or other molecules encoded by the cell's genome. In some embodiments, the term "endogenous mitochondria" is equivalent to the term "host mitochondria."

[0094] According to the principles of this invention, exogenous human mitochondria are introduced into cells, and these cells are thus enriched with exogenous mitochondria. It should be understood that such enrichment alters the mitochondrial content of the cell. Naive human cells substantially have one population of host / autologous mitochondria, while cells enriched with exogenous mitochondria that are not autologous mitochondria substantially have two populations of mitochondria: a first population of host / autologous / endogenous mitochondria and another population of introduced mitochondria (i.e., exogenous mitochondria). Thus, the term “enriched” refers to the state of the cell after it has received / integrated exogenous mitochondria. In certain embodiments, exogenous mitochondria are autologous mitochondria. When exogenous mitochondria are autologous mitochondria, there is only one population of mitochondria, and enriched refers to the total mitochondrial content. Determining the number and / or ratio between the two populations of mitochondria is straightforward because the two populations can differ in several aspects, for example, in mitochondrial DNA. For example, a human cell containing at least 1% exogenous mitochondria of its total mitochondrial content is considered to have host / self-derived / endogenous mitochondria and exogenous mitochondria in a ratio of 99:1. For instance, "3% of total mitochondria" means that after enrichment, the original (endogenous) mitochondria account for 97% of the total mitochondrial content, and the introduced (exogenous) mitochondria account for 3% of the total mitochondrial content, which corresponds to an enrichment of 3.1% (3 / 97=). As another example, "33% of total mitochondria" means that after enrichment, the original (endogenous) mitochondria account for 67% of the total mitochondrial content, and the introduced (exogenous) mitochondria account for 33% of the total mitochondrial content, which corresponds to an enrichment of 49.2% (33 / 67=).

[0095] In some embodiments, after exogenous mitochondria have been introduced into target cells, the identification / differentiation of endogenous mitochondria from exogenous mitochondria may be carried out by various means, including, but are not limited to, identifying differences in mtDNA sequences between endogenous and exogenous mitochondria, e.g., different haplotypes; identifying specific mitochondrial proteins derived from the source tissue of the exogenous mitochondria, e.g., cytochrome p450 cholesterol side chain breaks (P450SCCs) from the placenta, UCP1 from brown adipose tissue, etc., or any combination thereof.

[0096] Heteroplasmy is the presence of more than one type of mitochondrial DNA within a cell or organism. Heteroplasmy levels, which are the ratio of mutant mtDNA molecules to wild-type / functional mtDNA molecules, are an important factor in considering the severity of mitochondrial disease. Low levels of heteroplasmy (sufficient mitochondria functioning) are associated with a healthy phenotype, while high levels of heteroplasmy (insufficient mitochondria functioning) are associated with disease. In certain embodiments, the heteroplasmy levels of enriched cells, such as genetically engineered T cells enriched with mitochondria, are at least 1%, 3%, 5%, 15%, 20%, 25%, or 30% lower than the heteroplasmy levels of cells obtained from or derived from the subject or donor.

[0097] As used herein, the term “contact” refers to bringing mitochondria and cells (e.g., T cells and genetically modified T cells) into close proximity to facilitate the entry of exogenous mitochondria into the cells. The terms “introduce” or “insert” mitochondria into cells (e.g., T cells and genetically modified T cells) are interchangeable with the term “contact.”

[0098] As used herein, the phrases “conditions enabling the entry of exogenous mitochondria into T cells” and “conditions enabling the entry of exogenous mitochondria into T cells” generally refer to parameters such as time, temperature, centrifugation, culture medium, and proximity between mitochondria and recipient cells. For example, human cells and human cell lines are routinely incubated in liquid medium and maintained in a sterile environment, such as in a tissue culture incubator, at 37°C and a 5% CO2 atmosphere. According to alternative embodiments disclosed and illustrated herein, cells can be incubated at room temperature in saline supplemented with human serum albumin.

[0099] In certain embodiments, cells are incubated with exogenous mitochondria at a temperature in the range of approximately 16–37°C for a time in the range of 0.5–30 hours. In certain embodiments, cells are incubated with exogenous mitochondria for a time in the range of approximately 1–30 or approximately 5–25 hours. In specific embodiments, incubation is approximately 20–30 hours. In some embodiments, incubation is at least approximately 1, 5, 10, 15, 20, 21, 22, 23, or 24 hours. In other embodiments, incubation is up to 5, 10, 15, 20, or 30 hours. In specific embodiments, incubation is 24 hours. In certain embodiments, incubation is continued until the mitochondrial content in the cells increases by an average of 1%–45% compared to their initial mitochondrial content.

[0100] In some embodiments, incubation is at room temperature (16°C to 30°C). In other embodiments, incubation is at 37°C. In some embodiments, incubation is in a 5% CO2 atmosphere. In other embodiments, incubation does not contain additional CO2 beyond the levels found in air.

[0101] In further embodiments, incubation is performed in culture medium supplemented with human serum albumin (HSA). In additional embodiments, incubation is performed in saline supplemented with HSA. According to a particular exemplary embodiment, conditions that allow human stem cells to be enriched with exogenous mitochondria, thereby enriching the human stem cells with said human exogenous mitochondria, include incubation at room temperature in saline supplemented with 4.5% human serum albumin.

[0102] In certain embodiments, incubation is performed at 37°C. In certain embodiments, incubation is performed for at least 6 hours. In certain embodiments, incubation is performed for at least 12 hours. In certain embodiments, incubation is performed for 12 to 24 hours.

[0103] As used herein, the term “enrich” refers to any action designed to increase the mitochondrial content of a mammalian cell, for example, the number of intact mitochondria, or mitochondrial function. In particular, genetically engineered T cells enriched with exogenous mitochondria will exhibit enhanced function compared to the same T cells before enrichment.

[0104] As used herein, the terms “enrich” and “enrich” refer to any action performed ex vivo that increases the mitochondrial content of human cells, e.g., the number of intact, functional, healthy mitochondria. According to the principles of the present invention, exogenous mitochondria are introduced into human T cells, so that these cells are enriched with exogenous mitochondria. According to some embodiments, exogenous mitochondria constitute more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 10%, more than 15%, or more than 20% of the total mitochondria in mitochondrial-enriched T cells and mitochondrial-enriched genetically engineered T cells.

[0105] Citrate synthase (CS) is localized in the mitochondrial matrix but is encoded by nuclear DNA. Citrate synthase is involved in the first step of the Krebs cycle and is commonly used as a quantitative enzymatic marker for the presence of intact mitochondria (Larsen S. et al., J. Physiol., 2012, Vol. 590(14), pages 3349-3360; Cook GA et al., Biochim. Biophys. Acta., 1983, Vol. 763(4), pages 356-367).

[0106] The mitochondrial dose can be expressed in units of CS activity or mtDNA copy number, which are other quantifiable measures of mitochondrial quantity, as described herein. "Units of CS activity" are defined as the amount that allows for the conversion of 1 micromolar of substrate per minute in a reaction volume of 1 mL.

[0107] In some embodiments, enrichment of cells (e.g., T cells and genetically engineered T cells) with exogenous mitochondria involves introducing mitochondria into cells in doses of at least 0.044 to a maximum of 176 milliunits (mU) of citrate synthase (CS) activity per million cells, at least 0.088 to a maximum of 176 mU of CS activity per million cells, at least 0.2 to a maximum of 150 mU of CS activity per million cells, at least 0.4 to a maximum of 100 mU of CS activity per million cells, at least 0.6 to a maximum of 80 mU of CS activity per million cells, at least 0.7 to a maximum of 50 mU of CS activity per million cells, at least 0.8 to a maximum of 20 mU of CS activity per million cells, at least 0.88 to a maximum of 17.6 mU of CS activity per million cells, and at least 0.44 to a maximum of 17.6 mU of CS activity per million cells.

[0108] As used herein, the term “mitochondrial content” refers to the amount of mitochondria in a cell, or the average amount of mitochondria in multiple cells. As used herein, the term “increased mitochondrial content” refers to a detectable increase in mitochondrial content compared to the mitochondrial content of a cell before mitochondrial enrichment.

[0109] In certain embodiments, the mitochondrial content of cells enriched with exogenous mitochondria is detectably higher than that of naive cells. According to various embodiments, the mitochondrial content of genetically modified T cells enriched with mitochondria is at least 1%, at least 5%, at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, or more higher than that of cells before mitochondrial enrichment. In certain embodiments, fresh T cells or genetically modified T cells are used. In certain embodiments, T cells or genetically modified T cells are frozen and thawed.

[0110] In certain embodiments, the mitochondrial content of cells or genetically engineered T cells enriched with mitochondria is determined by determining the citrate synthase content. In certain embodiments, the mitochondrial content of naive or enriched cells is determined by determining the activity level of citrate synthase. In certain embodiments, the mitochondrial content of naive or enriched cells correlates with the citrate synthase content. In certain embodiments, the mitochondrial content of naive or enriched cells correlates with the activity level of citrate synthase. CS activity can be measured using a commercially available kit, for example, the CS activity kit CS0720 (Sigma).

[0111] Mitochondrial DNA content can be measured by performing quantitative PCR of mitochondrial genes before and after mitochondrial enrichment, normalized against nuclear genes.

[0112] In certain situations, the same cells before mitochondrial enrichment serve as a control to measure CS and ATP activity and determine the level of enrichment.

[0113] In certain embodiments, as used herein, the term “detectably high” refers to a statistically significant increase between a normal value and an increased value. In certain embodiments, as used herein, the term “detectably high” refers to a non-pathological increase, i.e., a level at which no pathological symptoms associated with a significantly high value are apparent. In certain embodiments, as used herein, the term “increased” refers to a value that is 1.05 times, 1.1 times, 1.25 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, or more higher than the corresponding value found in the corresponding cells or mitochondria of one or more healthy subjects, or in cells before mitochondrial enrichment (e.g., T cells and genetically engineered T cells).

[0114] As used herein, the term “increased mitochondrial DNA content” refers to a mitochondrial DNA content that is detectably higher than the mitochondrial DNA content in the cell before mitochondrial enrichment. Mitochondrial content may be determined by measuring the content of SDHA or COX1. In the context of this specification and the claims, “normal mitochondrial DNA” refers to mitochondrial DNA that does not have / possesses mutations or deletions known to be associated with mitochondrial disease. As used herein, the term “normal oxygen (O2) consumption rate” refers to the average O2 consumption of cells from a healthy individual. As used herein, the term “normal citrate synthase activity level” refers to the average citrate synthase activity level in cells from a healthy individual. As used herein, the term “normal adenosine triphosphate (ATP) production rate” refers to the average ATP production rate in cells from a healthy individual.

[0115] The degree of cellular enrichment by exogenous mitochondria can be determined by functional and / or enzymatic assays, including but not limited to oxygen (O2) consumption rate, citrate synthase content or activity level, and adenosine triphosphate (ATP) production rate. Alternatively, cellular enrichment by exogenous mitochondria can be confirmed by detection of donor mitochondrial DNA. According to some embodiments, the degree of cellular enrichment by exogenous mitochondria can be determined by the level of heteroplasmy alteration and / or the copy number of mtDNA per cell.

[0116] TMRM (tetramethylrhodamine methyl ester) or the related TMRE (tetramethylrhodamine ethyl ester) are cell-permeable fluorescent dyes commonly used to assess mitochondrial function in living cells by identifying changes in mitochondrial membrane potential. According to some embodiments, the level of enrichment can be determined by staining with TMRE or TMRM.

[0117] According to some embodiments, the mitochondria include intact mitochondria, ruptured mitochondria, and / or mitochondrial components selected from the group consisting of mitochondrial proteins, mitochondrial nucleic acids, mitochondrial lipids, and mitochondrial sugars.

[0118] According to some embodiments, the integrity of the mitochondrial membrane can be determined by any method known in the art. In non-limiting examples, the integrity of the mitochondrial membrane is measured using a tetramethylrhodamine methyl ester (TMRM) or tetramethylrhodamine ethyl ester (TMRE) fluorescent probe. Each possibility represents a distinct embodiment of the invention. Mitochondria observed under a microscope and showing TMRM or TMRE staining have an intact mitochondrial outer membrane. As used herein, the term “mitochondrial membrane” refers to a mitochondrial membrane selected from the group consisting of the mitochondrial inner membrane, the mitochondrial outer membrane, or both.

[0119] In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched genetically engineered T cells is determined by sequencing at least a statistically representative portion of the total mitochondrial DNA in the cell and determining the relative levels of host / endogenous mitochondrial DNA and exogenous mitochondrial DNA. In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched genetically engineered T cells is determined by single nucleotide polymorphism (SNP) analysis. In certain embodiments, the largest mitochondrial population and / or the largest mitochondrial DNA population is the host / endogenous mitochondrial population and / or the host / endogenous mitochondrial DNA population, and / or the second largest mitochondrial population and / or the second largest mitochondrial DNA population is the exogenous mitochondrial population and / or the exogenous mitochondrial DNA population.

[0120] According to certain embodiments, cell enrichment by exogenous mitochondria can be determined by conventional assays recognized in the art. In certain embodiments, the level of mitochondrial enrichment in mitochondrial-enriched human genetically engineered T cells is determined by (i) the levels of host / endogenous mitochondrial DNA and exogenous mitochondrial DNA, (ii) the levels of mitochondrial proteins selected from the group consisting of citrate synthase (CS), cytochrome C oxidase (COX1), succinate dehydrogenase complex flavin protein subunit A (SDHA), and any combination thereof, (iii) the level of CS activity, or (iv) any combination of (i), (ii), and (iii). Methods for determining these various parameters are well known in the art.

[0121] In certain embodiments, the level of mitochondrial enrichment in genetically engineered T cells enriched with mitochondria is determined by at least one of the following: (i) levels of host mitochondrial DNA and exogenous mitochondrial DNA, (ii) levels of citrate synthase activity, (iii) levels of succinate dehydrogenase complex flavoprotein subunit A (SDHA) or cytochrome C oxidase (COX1), (iv) oxygen (O2) consumption rate, (v) adenosine triphosphate (ATP) production rate, or (vi) any combination thereof. Each possibility represents a distinct embodiment of the present invention. Methods for measuring these various parameters are well known in the art.

[0122] In some embodiments, cell enrichment with exogenous human mitochondria includes incubating cells with exogenous human mitochondria and then washing the mitochondrial-enriched cells (e.g., mitochondrial-enriched genetically engineered T cells). This step provides mitochondrial-enriched cells substantially lacking cell flakes or mitochondrial membrane residues and mitochondria that did not enter the stem cells. In some embodiments, the washing includes centrifuging the mitochondrial-enriched cells after incubating the human cells with the human exogenous mitochondria. According to some embodiments, the method produces mitochondrial-enriched cells isolated from free mitochondria, i.e., mitochondria that did not enter the cells, or other cell flakes, and the pharmaceutical composition contains mitochondrial-enriched cells isolated from free mitochondria. According to some embodiments, mitochondrial-enriched genetically engineered T cells that do not contain a detectable amount of free mitochondria are produced by the method and contained in the pharmaceutical composition.

[0123] In certain embodiments, the method above further comprises enriching T cells or genetically modified T cells with exogenous mitochondria before or during incubation and / or contact. In certain embodiments, the method above further comprises centrifugation of T cells or genetically modified T cells with exogenous mitochondria before, during, or after incubation or contact. In some embodiments, the method above in its various embodiments comprises a single centrifugation step before, during, or after incubation of cells with exogenous mitochondria.

[0124] In certain embodiments, the centrifugal separation speed is 7,000g or 8,000g. According to further embodiments, centrifugal separation is performed at speeds of 300g-8000g, 500g-6000g, 1000g-5000g, 2000g-4000g, 2500g-8500g, 3000g-8000g, 4000g-8000g, 5000-10,000g, 7000g-8000g, or over 2500g. In some embodiments, centrifugal separation is performed over a time range of 2-30 minutes, 3-25 minutes, 5-20 minutes, or 8-15 minutes.

[0125] In some embodiments, centrifugation is performed at temperatures in the range of 2–6°C, 4–37°C, 4–10°C, or 16–30°C. In specific embodiments, centrifugation is performed at 4°C. In some embodiments, the above method in various embodiments includes a single centrifugation before, during, or after incubation of cells with exogenous mitochondria, followed by allowing the cells to stand at a temperature below 30°C. In some embodiments, the conditions that allow human cells to enter with exogenous mitochondria include a single centrifugation before, during, or after incubation of cells with exogenous mitochondria, followed by allowing the cells to stand at a temperature in the range of 16–28°C.

[0126] In some embodiments, at least 10 per kilogram of the subject's body weight5 ~10 10 pieces, 5×10 5 ~1.5×10 7 pieces, or 5 x 10 5 ~4×10 7 Mitochondrial-enriched genetically modified T cells are produced by this method and / or contained in the pharmaceutical composition at a concentration of 10 mitochondrial-enriched genetically modified T cells per kilogram of body weight of the subject. In some embodiments, at least 10 mitochondrial-enriched genetically modified T cells per kilogram of body weight of the subject. 6 ~10 7 Mitochondrial-enriched genetically modified T cells are produced by this method and / or contained in the pharmaceutical composition at a concentration of 10 mitochondrial-enriched genetically modified T cells per kilogram of body weight of the subject. In other embodiments, at least 10 5 one or at least 10 6 Mitochondrial-enriched genetically modified T cells are produced by this method and / or contained in the pharmaceutical composition at a concentration of 5 × 10¹⁵ mitochondrial-enriched genetically modified T cells. In some embodiments, a total of at least 5 × 10¹⁵ mitochondrial-enriched genetically modified T cells are produced by this method and / or contained in the pharmaceutical composition. 5 ~Maximum 5×10 9 Mitochondrial-enriched genetically modified T cells are produced by this method and / or contained in the pharmaceutical composition at a concentration of 10 mitochondrial-enriched genetically modified T cells. In some embodiments, a total of at least 10 6 ~up to 10 9 Mitochondrial-enriched genetically modified T cells are produced by this method and / or included in the pharmaceutical composition at a concentration of 10¹⁵ mitochondrial-enriched genetically modified T cells. In other embodiments, a total of at least 2 × 10¹⁵ mitochondrial-enriched genetically modified T cells are produced by this method and / or included in the pharmaceutical composition. 6 ~Maximum 5×10 8 A number of mitochondrially enriched genetically modified T cells are produced by this method and / or included in this pharmaceutical composition.

[0127] In certain embodiments, the T cells are fresh. In certain embodiments, the T cells are frozen and then thawed before incubation. In certain embodiments, the exogenous mitochondria are fresh. In certain embodiments, the exogenous mitochondria are frozen and then thawed before incubation. In certain embodiments, the mitochondrial-enriched genetically engineered T cells are fresh. In certain embodiments, the mitochondrial-enriched genetically engineered T cells are frozen and then thawed before administration.

[0128] In certain embodiments, T cells are then stored and used after thawing. In further embodiments, exogenous mitochondria are frozen, then stored, and thawed before use. In even further embodiments, mitochondrial-enriched genetically modified T cells are used without freezing and storage. In yet another embodiment, mitochondrial-enriched genetically modified T cells are used after freezing, storage, and thawing. Suitable methods for freezing and thawing cell preparations to maintain viability are well known in the art.

[0129] As used herein, the term “freeze-thaw cycle” refers to freezing exogenous mitochondria at a temperature below 0°C, maintaining the mitochondria at a temperature below 0°C for a predetermined period, and thawing the exogenous mitochondria to room temperature, body temperature, or any temperature above 0°C that allows for the processing of cells by the exogenous mitochondria. As used herein, the term “room temperature” typically refers to a temperature of 18°C ​​to 25°C. As used herein, the term “body temperature” refers to a temperature of 35.5°C to 37.5°C, preferably 37°C.

[0130] In another embodiment, mitochondria subjected to a freeze-thaw cycle were frozen at temperatures below -20°C, below -4°C, or below -70°C. According to another embodiment, mitochondrial freezing is gradual. According to some embodiments, mitochondrial freezing is by flash freezing. As used herein, the term “flash freezing” refers to rapidly freezing mitochondria by subjecting them to a low storage temperature.

[0131] In another embodiment, mitochondria undergoing a freeze-thaw cycle were frozen for at least 30 minutes before thawing. According to another embodiment, the freeze-thaw cycle includes freezing exogenous mitochondria for at least 30, 60, 90, 120, 180, or 210 minutes before thawing. Each possibility represents a distinct embodiment of the present invention. In another embodiment, mitochondria undergoing a freeze-thaw cycle were frozen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 24, 48, 72, 96, or 120 hours before thawing. In another embodiment, mitochondria undergoing a freeze-thaw cycle were frozen for at least 4, 5, 6, 7, 30, 60, 120, or 365 days before thawing. According to another embodiment, the freeze-thaw cycle includes freezing exogenous mitochondria for at least 1, 2, or 3 weeks before thawing. According to another embodiment, the freeze-thaw cycle includes freezing exogenous mitochondria for at least 1, 2, or 3 months before thawing. Each possibility represents a distinct embodiment of the present invention. According to another embodiment, the oxygen consumption of exogenous mitochondria after the freeze-thaw cycle is equal to or higher than the oxygen consumption of exogenous mitochondria before the freeze-thaw cycle.

[0132] According to one particular embodiment, thawing is at room temperature. In another embodiment, thawing is at body temperature. According to yet another embodiment, thawing is at a temperature that allows for the administration of mitochondria by the method of the present invention. According to yet another embodiment, thawing is performed progressively.

[0133] In certain embodiments, the method further includes a prior step of administering to a subject suffering from a disease or disorder, or to a donor, an agent that induces the recruitment of bone marrow cells into the peripheral blood.

[0134] In certain embodiments, the active ingredient that induces the recruitment of bone marrow cells / stem cells produced in the bone marrow into the peripheral blood is selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), 1,1'-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (Plerixafor, CAS No. 155148-31-5), CXCR4 inhibitors, salts thereof, and any combination thereof. Each possibility represents a distinct embodiment of the present invention.

[0135] In certain embodiments, the above method further includes isolating cells from the peripheral blood of a subject suffering from a disease or disorder, and / or from the peripheral blood of a donor. As used herein, the term “isolate from peripheral blood” refers to the isolation of T cells or hematopoietic stem cells from other components of the blood.

[0136] During apheresis, the blood of the subject or donor passes through a device that separates one specific component and returns the rest to circulation. Therefore, it is a medical procedure performed outside the body. In certain embodiments, isolation is performed by apheresis.

[0137] In certain embodiments, the cells are obtained from a subject suffering from a disease or disorder, or from a donor, and the cells have (i) a normal oxygen (O2) consumption rate, (ii) a normal citrate synthase content or activity level, (iii) a normal adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii).

[0138] In certain embodiments, cells that may be T cells are obtained from a subject suffering from a disease or disorder or from a donor, and the T cells have, compared to a subject not suffering from a disease or disorder, (i) a reduced oxygen (O2) consumption rate, (ii) a reduced citrate synthase content or activity level, (iii) a reduced adenosine triphosphate (ATP) production rate, or (iv) any combination of (i), (ii), and (iii).

[0139] In certain embodiments, mitochondrial-enriched genetically engineered T cells have, compared to corresponding T cells that have not undergone mitochondrial enrichment, (i) an increased oxygen (O2) consumption rate, (ii) an increased citrate synthase content or activity level, (iii) an increased adenosine triphosphate (ATP) production rate, (iv) an increased mitochondrial DNA content, or (v) any combination of (i), (ii), (iii), and (iv).

[0140] As used herein, the term “increased oxygen (O2) consumption rate” refers to an oxygen (O2) consumption rate that is detectably higher than the oxygen (O2) consumption rate before mitochondrial enrichment.

[0141] As used herein, the term “increased content of at least one mitochondrial protein” refers to the content of either a nuclear-encoded or mitochondrial-encoded mitochondrial protein, e.g., CS, COX1, and SDHA, that is detectably higher than the content of that mitochondrial protein in the cell before mitochondrial enrichment.

[0142] As used herein, the term “increased citrate synthase content or activity level” means a citrate synthase content or activity level that is detectably higher than the citrate synthase content or activity level in the cell before mitochondrial enrichment.

[0143] As used herein, the term “increased adenosine triphosphate (ATP) production rate” refers to adenosine triphosphate (ATP) production rate that is detectably higher than the adenosine triphosphate (ATP) production rate before mitochondrial enrichment.

[0144] In yet another aspect, the present invention provides a method for treating a disease selected from the group consisting of cancer, infectious diseases, or autoimmune diseases, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a plurality of mitochondrial-enriched genetically modified T cells to a subject in need thereof.

[0145] In some embodiments, the disease is cancer. The term “cancer” refers to a group of diseases characterized by abnormal and uncontrolled cell proliferation (this distinguishes cancer (malignant tumor) from benign tumor) that begins in one site (primary site), invades other sites (secondary sites, metastases), and has the potential to metastasize. Almost every organ can be affected, leading to more than 100 types of cancer that can affect humans. Cancer can result from a number of causes, including genetic predisposition, viral infection, exposure to ionizing radiation, exposure to environmental pollutants, tobacco and / or alcohol use, obesity, poor diet, lack of physical activity, or any combination thereof. As used herein, “neoplasm” or “tumor” includes its grammatical variations and means a new and abnormal proliferation of tissue that may be benign or cancerous. In relevant embodiments, neoplasm leads to a neoplastic disease or disorder, including but not limited to various cancers. For example, such cancers may include cancers of the prostate, pancreas, biliary tract, colon, rectum, liver, kidney, lung, testes, breast, ovaries, brain, and head and neck, as well as melanoma, sarcoma, multiple myeloma, leukemia, and lymphoma.

[0146] Cancers that originate in hematopoietic tissues such as bone marrow, or in cells of the immune system, are called hematologic cancers or blood cancers. Hematologic cancers affect the production and function of blood cells and are classified into three main types: leukemia, lymphoma, and multiple myeloma.

[0147] As used herein, “leukemia” refers to blood caused by the rapid production of abnormal white blood cells. Examples of leukemia include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), and hairy cell leukemia.

[0148] As used herein, “lymphoma” refers to a type of blood cancer that affects the lymphatic system. Examples of lymphomas include AIDS-associated lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, mycosis fungoides, non-Hodgkin lymphoma, primary central nervous system lymphoma, Sézary syndrome, cutaneous T-cell lymphoma, and Waldenström macroglobulinemia.

[0149] As used herein, “myeloma” refers to cancer of plasma cells. Examples of myeloma include chronic myeloproliferative neoplasms, Langerhans cell histiocytosis, multiple myeloma, plasma cell neoplasms, myelodysplastic syndromes, and myelodysplastic / myeloproliferative neoplasms.

[0150] In some embodiments, cancer is hematological cancer. In certain embodiments, cancer includes acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), hairy cell leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0151] In some embodiments, the administration can be combined with one or more additional therapeutic agents. Terms such as “combination therapy” and “combined with” refer to the simultaneous use of two or more drugs or treatments to enhance the response. The compositions of the present invention can be used, for example, in combination with other drugs or treatments being used to treat cancer. Specifically, administration of the compositions of the present invention to a subject can be combined with any anti-cancer therapy. Such therapies can be administered before, concurrently with, or after the administration of the compositions of the present invention.

[0152] As used herein, the terms “cancer therapy” or “cancer treatment” mean any treatment that may be used to treat cancer, such as surgery, radiation therapy, chemotherapy, immunotherapy, and checkpoint inhibitor therapy.

[0153] The following embodiments are provided to further illustrate embodiments of the present invention, but are not intended to limit the scope of the invention. They are typical of those that may be used, but alternatively, other procedures, methodologies, or techniques known to those skilled in the art may be used. [Examples]

[0154] Example 1 In one embodiment, the present invention provides the following method for treating the subjects described herein.

[0155] As described herein, isolated T cells are enriched with mitochondria, where the mitochondria are frozen / thawed using a freeze / thaw cycle.

[0156] Nucleic acid polymers encoding chimeric T cell receptors or chimeric antigen receptors (CARs) are introduced into the cells.

[0157] It is administered to cells enriched with mitochondria.

[0158] Example 2 In one embodiment, the present invention provides the following method for treating the subjects described herein.

[0159] Isolated T cells are introduced with nucleic acid polymers that encode chimeric T cell receptors or chimeric antigen receptors (CARs).

[0160] Cells are enriched with mitochondria.

[0161] It is administered to enriched cells.

[0162] Example 3 Peripheral blood mononuclear cells (PBMCs) from healthy human donors were thawed and cultured with interleukin-2. After a 2-day recovery period, the cells were activated using OKT3 (50 ng / ml) (i.e., day 0 of the protocol).

[0163] For transduction with a retrovirus encoding CD19-CAR with a CD28 co-stimulatory domain, non-tissue culture plates were coated with retronectin overnight at room temperature on day 2. On day 3, the virus was thawed and added to the retronectin-coated plates, and spun at 32°C for 2 hours. The virus was then removed from the plates, and activated donor cells were seeded (0.5 × 10⁶). 6 Cells (1 / ml) were placed in an incubator. As a control, untransduced cells were seeded on virus-free plates. Cells were grown in RPMI medium supplemented with 10% fetal bovine serum (FBS) and IL-2 (100 IU / ml), and the medium was changed every 2-3 days.

[0164] For mitochondrial enrichment, cryopreserved mitochondria (MNV-PLC) isolated from the placenta of healthy individuals were thawed and 1 × 10¹⁶ of them were added on either day 5 or day 10 of cell activation. 6 Cells were supplemented with CS activity at 0.88 mU or 4.4 mU per cell. Mitochondrial-enriched and unenriched cells were centrifuged at 7000 g (A1) or 400 g (A2) for 5 minutes at 4°C. After centrifugation, cells were suspended in the same medium (RPMI-10% FCS supplemented with 100 U / ml IL-2) and incubated at 37°C for 22 hours. After 22 hours, cells were harvested, washed, and resuspended in RPMI. Mitochondrial enrichment was verified by identifying the presence of exogenous placental mitochondria in the cells by sequence analysis. CAR-T cells incubated at 37°C for 22 hours using the same procedure but without exogenous mitochondria were used as a control.

[0165] Mitochondrial enrichment was induced by treating CAR-T cells with 4.4 mU or 0.88 mU of MNV-PLC, as verified by sequence analysis. Mitochondrial enrichment of CAR-T cells was feasible on day 5 or day 10 after cell activation. Mitochondrial enrichment performed on day 5 resulted in a viability of 90.3–95.5%, a 1.05–1.21-fold increase in ATP content compared to control cells, and a 1.7–2.6-fold increase in normalized CS activity compared to control cells. Mitochondrial enrichment performed on day 10 resulted in a viability of 88.5–92.7%, a 1.02–1.53-fold increase in ATP content compared to control cells, and a 1.7–2.13-fold increase in normalized CS activity compared to tp control cells. Control cells were transduced cells subjected to enhancement conditions without the introduction of exogenous mitochondria into the process (Figure 1B). Mitochondrial enrichment performed on day 10 resulted in a twofold or threefold increase in exogenous mitochondrial incorporation compared to mitochondrial enrichment performed on day 5 (8.14% vs. 4.03% in A1, and 6.34% vs. 2.7% in A2) (Figure 1C). T cell proliferation and transduction efficacy were not impaired by mitochondrial enrichment (Figure 2).

[0166] Although the present invention has been described with reference to the above embodiments, modifications and variations will be understood to be included within the spirit and scope of the invention. Accordingly, the present invention is limited only by the appended claims. The present invention may also include the following embodiments. [Aspect 1] A pharmaceutical composition comprising mitochondrial-enriched genetically modified T cells and a pharmaceutically acceptable carrier, wherein the mitochondrial-enriched genetically modified T cells are enriched with exogenous mitochondria. [Aspect 2] The aforementioned mitochondrial-enriched genetically modified T cells a) Obtaining T cells from subjects suffering from a disease or disorder, or from donors, b) Obtaining exogenous mitochondria, c) Producing mitochondrial-enriched T cells by bringing the T cells into contact with the exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the T cells, d) To produce genetically modified T cells with enriched mitochondria by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the aforementioned mitochondrial-enriched T cells. Produced by methods including, The mitochondrial content of the genetically modified T cells enriched with mitochondria is detected to be higher than the mitochondrial content of the T cells. The pharmaceutical composition described in Embodiment 1. [Aspect 3] The aforementioned mitochondrial-enriched genetically modified T cells a) Obtaining T cells from subjects suffering from a disease or disorder, or from donors, b) Producing genetically modified T cells by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the T cells, c) Obtaining exogenous mitochondria, d) Producing mitochondrial-enriched genetically modified T cells by bringing the genetically modified T cells into contact with the exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the genetically modified T cells. Produced by the method described above, The mitochondrial content of the genetically modified T cells enriched with mitochondria is detected to be higher than the mitochondrial content of the genetically modified T cells. The pharmaceutical composition described in Embodiment 1. [Aspect 4] The pharmaceutical composition according to embodiment 2 or 3, wherein the genetically modified T cells and the genetically modified T cells enriched with mitochondria are CAR-T cells. [Aspect 5] The pharmaceutical composition according to embodiment 2 or 3, wherein the genetically modified T cells and the genetically modified T cells enriched with mitochondria are TCR-T cells. [Aspect 6] The pharmaceutical composition according to embodiment 1, wherein the exogenous mitochondria are derived from human cells. [Aspect 7] The pharmaceutical composition according to embodiment 6, wherein the human cells are selected from human placenta, cultured human placental cells, human blood cells, stem cells, and somatic cells. [Aspect 8] The pharmaceutical composition according to embodiment 7, wherein the stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, and pluripotent stem cells. [Aspect 9] The pharmaceutical composition according to embodiment 1, wherein the exogenous mitochondria are of the same or different species. [Aspect 10] The pharmaceutical composition according to embodiment 1, wherein the exogenous mitochondria are of autologous origin. [Aspect 11] At least 5 × 10 per 1 kg of body weight of the subject mentioned above 5 ~at least 5 × 10 10 Individual, mitochondrially enriched genetically modified T cells A pharmaceutical composition according to any one embodiment of embodiments 1 to 10, including the above. [Aspect 12] The genetically engineered T cells enriched with mitochondria showed, compared to the corresponding levels in the T cells before mitochondrial enrichment, (a) Increased mitochondrial DNA content, (b) Increased citrate synthetase (CS) activity levels, (c) Increased content of at least one mitochondrial protein selected from SDHA and COX1, (d) Increased O 2 consumption rate, (e) Increased ATP production rate, or (f) Any combination of them, A pharmaceutical composition according to any one embodiment of embodiments 1 to 11, having at least one of the following. [Aspect 13] The pharmaceutical composition according to any one embodiment of embodiments 1 to 12, wherein the conditions that enable the entry of the exogenous mitochondria into the T cells or genetically modified T cells include incubating the T cells or genetically modified T cells together with the exogenous mitochondria at a temperature in the range of approximately 16 to 37°C for a period of 0.5 to 30 hours. [Aspect 14] The pharmaceutical composition according to any one embodiment of embodiments 1 to 13, wherein the exogenous mitochondria constitute at least 1% of the total mitochondria in the mitochondrial-enriched T cells or the mitochondrial-enriched genetically modified T cells. [Aspect 15] A pharmaceutical composition according to any one embodiment of embodiments 1 to 14, wherein the disease or disorder is cancer. [Aspect 16] The pharmaceutical composition according to embodiment 15, wherein the cancer is a blood cancer. [Aspect 17] The pharmaceutical composition according to any one embodiment of embodiments 1 to 16, wherein the T cells are of autologous origin or allogeneic. [Aspect 18] The conditions that enable the exogenous mitochondria to enter the genetically modified T cells are 10 6 A pharmaceutical composition according to any one of embodiments 1 to 17, comprising a ratio of approximately 0.088 to 176 mU of citrate synthase (CS) activity per T cell. [Aspect 19] A method of treating a disease or disorder in a person who needs it, Administering genetically modified T cells enriched with mitochondria to the subject, wherein the genetically modified T cells enriched with mitochondria are enriched with exogenous mitochondria. The method comprising, and thereby treating the subject. [Aspect 20] The aforementioned mitochondrial-enriched genetically modified T cells a) Obtaining T cells from subjects suffering from a disease or disorder, or from donors, b) Obtaining exogenous mitochondria, c) Producing mitochondrial-enriched T cells by bringing the T cells into contact with the exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the T cells, d) To produce genetically modified T cells with enriched mitochondria by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the aforementioned mitochondrial-enriched T cells. Produced by methods including, The mitochondrial content of the genetically modified T cells enriched with mitochondria is detected to be higher than the mitochondrial content of the T cells. The method described in aspect 19. [Aspect 21] The aforementioned mitochondrial-enriched genetically modified T cells a) Producing genetically modified T cells by introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into T cells obtained from subjects suffering from a disease or disorder, or from donors. b) Obtaining exogenous mitochondria, c) To produce mitochondrial-enriched genetically modified T cells by bringing the genetically modified T cells into contact with the exogenous mitochondria under conditions that allow the exogenous mitochondria to enter the genetically modified T cells. Produced by methods including, The mitochondrial content of the genetically modified T cells enriched with mitochondria is detected to be higher than the mitochondrial content of the T cells. The method according to embodiment 19 or 20. [Aspect 22] The method according to any one embodiment of embodiments 19 to 21, wherein the subject is a human subject. [Aspect 23] The method according to any one embodiment of embodiments 19 to 22, wherein the disease or disorder is cancer. [Aspect 24] The method according to embodiment 23, wherein the cancer is a blood cancer. [Aspect 25] The method according to any one embodiment of embodiments 19 to 24, wherein the administration is by intravenous, intraperitoneal, intraarterial, intrathecal, and intramuscular administration. [Aspect 26] The method according to any one embodiment of embodiments 19 to 25, wherein the administration is by intravenous administration. [Aspect 27] The method according to any one embodiment of embodiments 19 to 26, further comprising administering chemotherapy, radiotherapy, or other anti-cancer therapies to the subject. [Aspect 28] The method according to any one embodiment of embodiments 19 to 27, wherein the pharmaceutical composition is administered simultaneously with, in parallel with, or after the administration of chemotherapy, radiotherapy, or other anticancer therapy. [Aspect 29] The method according to any one embodiment of embodiments 19 to 28, wherein the exogenous mitochondria are derived from human cells or tissues. [Aspect 30] The method according to embodiment 29, wherein the human cells are selected from the group consisting of placenta, cultured placental cells, blood cells, and stem cells. [Aspect 31] The method according to embodiment 30, wherein the stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, and pluripotent stem cells. [Aspect 32] The method according to any one embodiment of embodiments 1 to 31, wherein the exogenous mitochondria are allogeneic or self-derived. [Aspect 33] The method according to any one embodiment of embodiments 19 to 32, wherein, after enrichment with exogenous mitochondria, at least 1% of the total mitochondria in the mitochondrial-enriched T cells or the mitochondrial-enriched genetically modified T cells contain exogenous mitochondria. [Aspect 34] The method according to any one embodiment of aspects 19 to 33, wherein the conditions enabling the entry of the exogenous mitochondria into the T cells or genetically modified T cells include incubating the T cells or genetically modified T cells together with the exogenous mitochondria at a temperature in the range of approximately 16 to 37°C for a period of 0.5 to 30 hours. [Aspect 35] The conditions that enable the exogenous mitochondria to enter the cell are 10 6 The method according to any one embodiment of embodiments 19 to 34, comprising a ratio of approximately 0.088 to 176 mU of citrate synthase (CS) activity per cell.

Claims

1. A pharmaceutical composition comprising mitochondrial-enriched genetically modified T cells and a pharmaceutically acceptable carrier, wherein the mitochondrial-enriched genetically modified T cells are enriched with exogenous mitochondria, a) Introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into the T cells by contacting T cells obtained from the subject with exogenous mitochondria at a ratio of approximately 0.88 mU or approximately 4.4 mU of citrate synthase (CS) activity per 10⁶ T cells; or b) Introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into T cells obtained from the target, and contacting the T cells with exogenous mitochondria at a ratio of approximately 0.88 mU or 4.4 mU of citrate synthase (CS) activity per 10⁶ T cells. It is produced by methods including, The mitochondrial content of the genetically modified T cells enriched with mitochondria is at least 1% higher than that of the T cells before mitochondrial enrichment. The method described above does not involve reducing the amount of mtDNA in the T cells before contacting the exogenous mitochondria with the T cells, and is a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the method further includes activating T cells obtained from the subject before contact with exogenous mitochondria, and contacting the exogenous mitochondria with the T cells 10 days after the activation of the T cells.

3. The pharmaceutical composition according to claim 1 or 2, wherein the mitochondrial content of the genetically modified T cells enriched with mitochondria is at least 5% higher than the mitochondrial content of the T cells before mitochondrial enrichment.

4. The pharmaceutical composition according to claim 3, wherein the mitochondrial content of the genetically modified T cells enriched with mitochondria is at least 10% higher than the mitochondrial content of the T cells before mitochondrial enrichment.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the genetically modified T cells enriched with mitochondria are CAR-T cells.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the genetically modified T cells enriched with mitochondria are TCR-T cells.

7. The pharmaceutical composition according to claim 1, wherein the exogenous mitochondria are derived from human cells.

8. The pharmaceutical composition according to claim 7, wherein the human cells are selected from human placenta, cultured human placental cells, human blood cells, stem cells, and somatic cells.

9. The pharmaceutical composition according to claim 8, wherein the stem cells are selected from the group consisting of induced pluripotent stem cells, embryonic stem cells, and pluripotent stem cells.

10. The pharmaceutical composition according to claim 1, wherein the exogenous mitochondria are homogeneous.

11. The pharmaceutical composition according to claim 1, wherein the exogenous mitochondria are of autologous origin.

12. The genetically engineered T cells enriched with mitochondria showed, compared to the corresponding levels in the T cells before mitochondrial enrichment, (a) Increased mitochondrial DNA content, (b) Increased citrate synthetase (CS) activity levels, (c) Increased content of at least one mitochondrial protein selected from SDHA and COX1, (d) Increased O ２ consumption rate, (e) Increased ATP production rate, or (f) Any combination of them, A pharmaceutical composition according to any one of claims 1 to 11, having at least one of the following.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the exogenous mitochondria constitute at least 1% of the total mitochondria in the mitochondrial-enriched T cells or the genetically modified T cells enriched with mitochondria.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the T cells are of autologous origin or allogeneic.

15. A pharmaceutical composition used in a method for treating a disease or disorder in a subject that requires such treatment, wherein the treatment method comprises administering the pharmaceutical composition to the subject, according to any one of claims 1 to 14.

16. The pharmaceutical composition according to any one of claims 15, wherein the subject is a human subject.

17. The pharmaceutical composition according to claim 15 or 16, wherein the disease or disorder is cancer.

18. The pharmaceutical composition according to claim 17, wherein the cancer is a blood cancer.

19. The pharmaceutical composition according to any one of claims 15 to 18, wherein administration is by intravenous, intraperitoneal, intraarterial, intrathecal, and intramuscular administration.

20. The pharmaceutical composition according to any one of claims 15 to 19, wherein the administration is by intravenous administration.

21. The pharmaceutical composition according to any one of claims 15 to 20, further comprising the subject being subjected to chemotherapy, radiotherapy, or other anti-cancer therapies.

22. The pharmaceutical composition according to claim 21, wherein the pharmaceutical composition is administered simultaneously with, in parallel with, or after the administration of chemotherapy, radiotherapy, or other anticancer therapy.

23. 5 x 10 per 1 kg of body weight of the subject mentioned above ５ ~5 x 10 １０ A pharmaceutical composition according to any one of claims 15 to 22, comprising a number of mitochondrially enriched genetically modified T cells.

24. a) Introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into T cells by contacting T cells obtained from a subject with exogenous mitochondria at a ratio of approximately 0.88 mU or approximately 4.4 mU of citrate synthase (CS) activity per 10⁶ T cells; or b) Introducing nucleic acids encoding chimeric T cell receptors (TCRs) or chimeric antigen receptors (CARs) into T cells obtained from the subject, and contacting the T cells with exogenous mitochondria at a ratio of approximately 0.88 mU or approximately 4.4 mU of citrate synthase (CS) activity per 10⁶ T cells. A method for producing genetically modified T cells enriched with mitochondrial cells using exogenous mitochondria, The mitochondrial content of the genetically modified T cells enriched with mitochondria is at least 1% higher than that of the T cells before mitochondrial enrichment. The above method is a manufacturing method that does not involve reducing the amount of mtDNA in the T cells before contacting the exogenous mitochondria with the T cells.

25. The manufacturing method according to claim 24, wherein the method further includes activating T cells obtained from the subject before contact with exogenous mitochondria, and contacting the exogenous mitochondria with the T cells 10 days after the activation of the T cells.

26. The manufacturing method according to claim 24 or 25, wherein the mitochondrial content of the genetically modified T cells enriched with mitochondria is at least 5% higher than the mitochondrial content of the T cells before mitochondrial enrichment.

27. The manufacturing method according to claim 26, wherein the mitochondrial content of the genetically modified T cells enriched with mitochondria is at least 10% higher than the mitochondrial content of the T cells before mitochondrial enrichment.

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