Methods and compositions for generating mitochondrial-substituted lymphoid cells

Abstract

The present disclosure provides methods and compositions for generating mitochondrial-replaced lymphoid cells, such as T cells, which involve incubating lymphoid cells that have not been subjected to a procedure that reduces or depletes endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor.Furthermore, the present disclosure also provides methods for treating immune disorders associated with heteroplasmic immune cells, as well as methods for reversing symptoms of mitochondrial complex III deficiency, which involve administering mitochondrial-replaced lymphoid cells.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 190,078, filed May 18, 2021, which is incorporated by reference in its entirety.

[0002] 1. Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on May 18, 2022, is named 14595-008-228_SL.txt, and is 3,681 bytes in size.

[0003] 2. Introduction The present disclosure relates in part to the method and composition for generating mitochondrial replacement lymphoid cells.In a specific embodiment, the present disclosure relates to the method for generating mitochondrial replacement lymphoid cells (e.g., T cells) using a composition comprising rapamycin and its equivalent, without prior deletion or depletion of mitochondria, and the therapeutic method for treating disease using such mitochondrial replacement lymphoid cells.The disease that can be treated using mitochondrial replacement lymphoid cells includes the disease caused by inherited and acquired mitochondrial DNA mutation, such as mitochondrial disease, as well as the immune deficiency associated with heteroplasmic immune cells. [Background technology]

[0004] 3.Background Mitochondrial dysfunction can result from a variety of factors, such as genetic disorders. In certain circumstances, mitochondrial disease or disorders can negatively affect lymphocyte function. For example, mitochondrial complex III is involved in the suppressive function of regulatory T cells (Treg), and mitochondrial complex III deficiency can impair Treg function. Mitochondrial dysfunction can also result from genotoxic drugs, aging, oxidative stress inflammation, and / or injury. For example, exposure to therapeutic agents (e.g., nucleoside / nucleotide reverse transcriptase inhibitors) can reduce mitochondrial DNA (mtDNA) copy number. Transfer of mitochondria from healthy cells to damaged or dysfunctional cells may help rescue and restore mitochondrial function.

[0005] Current methods for mitochondria transfer into recipient cells include those that involve partial or complete depletion of endogenous mtDNA.For example, the classical method for removing endogenous mtDNA involves long-term treatment of cells with low concentrations of ethidium bromide (EtBr), which is known to be a carcinogen and teratogen, thereby limiting its therapeutic application.Additional techniques for mitochondria transfer include those that use invasive instruments, which are harmful to recipient cells and inefficient. Summary of the Invention [Means for solving the problem]

[0006] Cell-based therapy involving lymphocytes has emerged as a means to fight disease and alleviate medical conditions.In many cases, the number of lymphocytes available for such therapy is limited.Therefore, any modification to cells must not only be safe for administration to patients, but also be gentle enough not to damage lymphocytes.Therefore, there is a significant unmet need to develop a safe and effective method for transferring mitochondria into lymphocytes, including in the context of cell-based therapy involving lymphocytes.

[0007] 4. Overview In one aspect, provided herein is a method for generating mitochondrial-replaced lymphoid cells, the method comprising incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells in which at least 20% of the endogenous mtDNA has been replaced by exogenous mtDNA. In a specific embodiment, provided herein is a method for generating mitochondrial-replaced lymphoid cells in which at least 20% of endogenous mitochondrial DNA (mtDNA) has been replaced by exogenous mtDNA, comprising incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells in which at least 20% of endogenous mtDNA has been replaced by exogenous mtDNA. In one embodiment, the mTOR inhibitor comprises rapamycin or a derivative thereof. In a specific embodiment, the mTOR inhibitor is rapamycin. In certain embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 100 nM to about 1000 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 200 nM to about 500 nM. In one embodiment, the effective amount of the mTOR inhibitor is at a concentration of about 100 nM. In another embodiment, the effective amount of the mTOR inhibitor is at a concentration of about 200 nM. In another embodiment, the effective amount of the mTOR inhibitor is at a concentration of about 500 nM. In another embodiment, the effective amount of the mTOR inhibitor is at a concentration of about 1000 nM. In a specific embodiment, the mitochondrial replacement lymphoid cells contain at least 20% exogenous mtDNA and 80% or less endogenous mtDNA as measured by TaqMan single nucleotide polymorphism (SNP) assay.

[0008] In specific embodiments, provided herein is a method for generating mitochondrial-replaced lymphoid cells, comprising incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and about 100 nM to about 1000 nM rapamycin for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells.

[0009] In some embodiments, the methods provided herein include culturing 1×10 cells. 6 The exogenous isolated mitochondria contain about 20 μg to 80 μg of protein per cell. In certain embodiments, the methods provided herein further comprise centrifuging the lymphoid cells prior to the incubating step. In one embodiment, the centrifugation is performed at room temperature and 1,500 relative centrifugal force (RCF) for approximately 5 minutes.

[0010] In some embodiments, the sufficient period of time for the methods provided herein is at least about 24 hours. In certain embodiments, the sufficient period of time for the methods provided herein is at least 36 hours. In some embodiments, the sufficient period of time for the methods provided herein is at least 48 hours. In certain embodiments, the sufficient period of time for the methods provided herein is about 2 days or longer. In some embodiments, the sufficient period of time for the methods provided herein is about 7 days or longer. In one embodiment, the sufficient period of time for the methods provided herein is about 2 days to about 7 days.

[0011] In yet another aspect, provided herein is a method for generating mitochondrial-replaced lymphoid cells, comprising: (a) centrifuging lymphoid cells and isolated exogenous mitochondria under conditions sufficient to generate a cell pellet, wherein the lymphoid cells have not been subjected to a procedure that reduces or depletes endogenous mitochondria; and (b) incubating the lymphoid cells with 100 nM-1000 nM rapamycin for approximately 24 hours or longer, thereby generating mitochondrial-replaced lymphoid cells.

[0012] In certain embodiments, the incubating step is for about 7 days or longer. In some embodiments, the incubating step is for about 2 days to about 7 days. In some embodiments, the lymphoid cell of the methods provided herein is a T cell, a B cell, a monocyte, a macrophage, a natural killer (NK) cell, or a granulocyte. In certain embodiments, the lymphoid cell of the methods provided herein is a T cell. In some embodiments, the T cell comprises an exhausted T cell, an aged T cell, or a combination thereof. In certain embodiments, the lymphoid cell of the methods provided herein is a human lymphoid cell.

[0013] In another aspect, provided herein are mitochondrial-replaced lymphoid cells produced by the methods described herein, and compositions comprising such cells.

[0014] In another aspect, provided herein is a composition comprising an effective amount of the mitochondrial-substituted lymphoid cells of the present disclosure and a pharma- ceutically acceptable carrier.

[0015] In another aspect, provided herein is a method for reversing symptoms of mitochondrial complex III deficiency in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of mitochondrial-replaced lymphoid cells of the present disclosure and a pharma- ceutically acceptable carrier. In a specific embodiment, the subject is a human.

[0016] In another aspect, provided herein is a method for treating an immune deficiency associated with heteroplasmic immune cells in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of the mitochondrial-replaced lymphoid cells of the present disclosure and a pharma- ceutically acceptable carrier. In a specific embodiment, the subject is receiving a reverse transcriptase inhibitor. In certain embodiments, the subject has human immunodeficiency virus (HIV). In some embodiments, the subject has hepatitis B virus (HBV). In a specific embodiment, the subject is a human. [Brief description of the drawings]

[0017] 5. Brief description of the drawings [Figure 1] Mitochondrial DNA (mtDNA) sequences. MtDNA sequences isolated from human GJ T cells and EPC100 cells were sequenced and compared. Differences in the mtDNA sequences were detected by sequencing the D-loop, and several differences were observed in the D-loop hypervariable region 1 ("HVR1").

[0018] [Figure 2-1] Primers and probes for SNP assay. Figure 2A shows a set of primers (SEQ ID NOs: 5 and 6) and probes (SEQ ID NOs: 1 and 2) for SNP assay to detect differences in HVR1 of human mtDNA derived from human GJ T cells and EPC100 cells. Figure 2B shows where the primers (SEQ ID NOs: 5 and 6) and probes (SEQ ID NOs: 1 and 2) bind to in HVR1 of human mtDNA derived from human GJ T cells and EPC100 cells. [Figure 2-2] Same as above.

[0019] [Diagram 3] Figure 3A shows the protocol for transferring isolated mitochondria from EPC100 cells into human GJ T cells, and Figure 3B shows the results of a SNP assay to detect replacement of GJ T cell mtDNA with mtDNA from EPC100 cells.

[0020] [Figure 4] Figure 4A shows a protocol for transferring mitochondria from B6 mouse embryonic fibroblast (MEF) cells into mouse NZB T cells, and Figure 4B shows a depiction of the titration of rapamycin in the wells.

[0021] [Figure 5-1] Figure 5A shows the results of the SNP assay on day 2. Figure 5B shows the results of the SNP assay on day 7. Orange indicates the ratio of endogenous mitochondrial genotypes in NZB T cells, and blue indicates the ratio of exogenous mitochondrial genotypes from B6 MEF cells. [Figure 5-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 6. Detailed Description Provided herein is a method for generating mitochondrial-replaced lymphoid cells, which involves incubating lymphoid cells that have not been subjected to a procedure that reduces or depletes endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor.The mitochondrial-replaced lymphoid cells generated according to the method disclosed herein have therapeutic utility, for example, for reversing symptoms of immune deficiency associated with mitochondrial complex III deficiency or heteroplasmic immune cells.

[0023] As used herein, the term "mitochondrial replacement lymphoid cells" is generally intended to mean lymphoid cells in which endogenous mitochondria and / or endogenous mtDNA are replaced by exogenous mitochondria and / or exogenous mtDNA. In certain embodiments, the mitochondrial replacement lymphoid cells are those in which all endogenous mitochondria and / or endogenous mtDNA in the lymphoid cells are replaced by exogenous mitochondria and / or exogenous mtDNA. In specific embodiments, the mitochondrial replacement lymphoid cells have endogenous mitochondria replaced by exogenous mitochondria. In such a situation, the replacement of endogenous mitochondria by exogenous mitochondria is evaluated by evaluating mtDNA markers. In specific embodiments, the mitochondrial replacement lymphoid cells are those in which a certain percentage of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, a mitochondrial-replaced lymphoid cell is one in which about 5% or more, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, or about 95% or more of the endogenous mitochondria and / or endogenous mtDNA in the lymphoid cell have been replaced with exogenous mitochondria and / or exogenous mtDNA. In certain embodiments, the mitochondrial-replaced lymphoid cells are those in which about 5% to about 10%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 20% to about 40%, about 25% to about 50%, about 25% to about 75%, about 50% to about 75%, about 40% to about 50%, about 75% or more to about 85%, or about 75% to about 95% of the endogenous mitochondria and / or endogenous mtDNA in the lymphoid cells have been replaced by exogenous mitochondria and / or exogenous mtDNA.In some embodiments, a mitochondrial-replaced lymphoid cell is one in which at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the endogenous mitochondria and / or endogenous mtDNA in the lymphoid cell have been replaced by exogenous mitochondria and / or exogenous mtDNA.

[0024] As used herein, the term "isolated," when used in reference to mitochondria, generally refers to mitochondria that have been physically separated or removed from other cellular components of their natural biological environment. In specific embodiments, mitochondria are isolated using the techniques described in the Examples below.

[0025] As used herein, the term "isolated" when used in reference to a cell generally means a cell that is substantially free of at least one component in which the referenced cell is found in nature. The term includes a cell that has been removed from some or all of the components in which it is found in its natural environment. The term also includes a cell that has been removed from at least one, some or all of the components in which the cell is found in a non-naturally occurring environment. Thus, an isolated cell is one that is partially or completely separated from other materials in which it is found in nature or in which it is grown, stored or present in a non-naturally occurring environment. Specific examples of isolated cells include partially pure cells (e.g., lymphoid cells) enriched from other cell types (e.g., non-lymphoid cells), and substantially pure cells (e.g., lymphoid cells). Thus, a cell that is referenced as being isolated can be 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% pure, free of other cells and / or materials. In a specific embodiment, the referenced cells are isolated using the techniques described in the Examples below.

[0026] As used herein, the term "exogenous" is understood by those skilled in the art. In general, the term "exogenous" refers to intracellular material (e.g., mitochondria or mtDNA) that is not derived from recipient cells. For example, as described in the following examples, exogenous mitochondria or mtDNA can be isolated from fibroblasts that are introduced into T cells.

[0027] As used herein, the term "endogenous" is commonly understood by those of skill in the art. In general, the term "endogenous" refers to intracellular material (e.g., mitochondria or mtDNA) that is native to the recipient cell.

[0028] As used herein, the term "effective amount" generally refers to the amount of a compound or composition required to realize the desired result(s) under the relevant conditions.

[0029] As used herein, the terms "about" or "approximately" when used in conjunction with a number generally refer to the referenced number as well as any number within 1%, 5%, 10%, 15% or 20% of the referenced number.

[0030] As used herein, the term "sufficient period of time" generally refers to the amount of time over which a desired result or results.

[0031] As used herein, the term "non-invasively," when used in reference to the transfer of exogenous material (e.g., mitochondria and / or mtDNA), is intended to mean generally without the use of invasive instruments (e.g., nanoblades or electroporation) or harmful conditions that damage the structure of the cell.

[0032] As used herein, the term "subject" is intended to generally mean an animal. A subject may be a human or a non-human mammal, such as a dog, cat, bovine, equine, mouse, rat, rabbit, or transgenic species thereof. It is understood that "subject" can also refer to a "patient," e.g., a human patient.

[0033] The terms "heteroplasmy" and "heteroplasmic" are understood by those of skill in the art. In general, the terms "heteroplasmy" and "heteroplasmic" refer to the presence of more than one type of mtDNA genome in an individual or sample.

[0034] The practice of the embodiments provided herein will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, and immunology that are within the skill of those in the art, and such techniques are fully explained in the literature. Examples of particularly useful texts include: Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD (1999); Glover, ed., DNA Cloning, Volumes I and II (1985); Gait, ed., Oligonucleotide Synthesis (1984); Hames & Higgins, eds., Nucleic Acid Hybridization (1984); Hames & Higgins, eds., Transcription and Translation (1984); Freshney, ed., Animal Cell Culture: Immobilized Cells and Enzymes (IRL Press, 1986); Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Scopes, Protein Purification: Principles and Practice (Springer Verlag, NY, 2d ed. 1987); and Weir & Blackwell, eds., Handbook of Experimental Immunology, Volumes I-IV (1986). 6.1 How to generate mitochondrial replacement cells (MirC)

[0035] The present disclosure is based in part on the discovery that the use of mTOR inhibitors can enhance the transfer of donor mitochondria into recipient lymphocytes without the need for any prior reduction or depletion of endogenous mitochondria and / or endogenous mitochondrial DNA (mtDNA) of recipient cells.Therefore, in one aspect, a method is provided herein for generating mitochondrial-replaced lymphoid cells using mTOR inhibitors and without any prior procedure for reducing or depleting endogenous mitochondria and / or endogenous mtDNA.In a specific embodiment, a method is provided herein for generating mitochondrial-replaced lymphoid cells, comprising: incubating lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer exogenous mitochondria into lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells. In another specific embodiment, the present invention provides a method for generating mitochondrial replacement lymphoid cells, comprising incubating lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria with a composition comprising isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer exogenous mitochondria into lymphoid cells, thereby generating mitochondrial replacement lymphoid cells.For example, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria can be those that have not been contacted with a mitochondrial DNA (mtDNA) depleting agent (e.g., ethidium bromide (EtBr), or an enzyme that can degrade mtDNA, such as a restriction enzyme), or that have not been transfected or transduced with a polynucleotide that codes for a mtDNA depleting agent.In some embodiments, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria may not have been contacted with an enzyme capable of degrading mtDNA, nor have they been transfected or transduced with a polynucleotide encoding an enzyme capable of degrading mtDNA. In certain embodiments, the methods provided herein for generating mitochondrial-replaced lymphoid cells are performed in vitro or ex vivo.

[0036] The level of endogenous mtDNA replaced according to the methods provided herein need not result in complete replacement (i.e., 100% replacement) of endogenous mtDNA with exogenous mtDNA. For example, in some embodiments, at least 10% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 15% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 20% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 25% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 30% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 35% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 40% of endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 45% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 50% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 55% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 60% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 65% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 70% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 80% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, at least 90% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, 100% of the endogenous mtDNA is replaced by exogenous mtDNA. Replacement of endogenous mtDNA with exogenous mtDNA can be measured using techniques known to those of skill in the art or described herein (eg, in the Examples).

[0037] In a specific embodiment, provided herein is a method for generating mitochondrial-replaced lymphoid cells in which at least 20% of endogenous mtDNA has been replaced by exogenous mtDNA, comprising incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells in which at least 20% of endogenous mtDNA has been replaced by exogenous mtDNA.

[0038] The replacement of endogenous mtDNA by exogenous mtDNA can be measured using techniques known to those skilled in the art or described herein (e.g., in the examples). For example, various sequencing methods can be used in combination with any of the methods provided herein to evaluate or confirm the transfer of exogenous mitochondria and / or exogenous mtDNA, or to quantify heteroplasmy. In general, mtDNA differences can be detected by sequencing the D-loop of mtDNA. The D-loop contains two regions that accumulate mutations more frequently than anywhere else in the mitochondrial genome. The regions are referred to as hypervariable region (HVR)-1 and HVR2, respectively.

[0039] In some embodiments, in connection with the methods provided herein, endogenous and exogenous mtDNA are sequenced and quantified by sequencing HV1 and / or HV2 of the D-loop of mtDNA. In specific embodiments, the sequencing method comprises a single nucleotide polymorphism (SNP) assay. In other embodiments, the sequencing method comprises digital PCR. In specific embodiments, the digital PCR is droplet digital PCR.

[0040] In general, an effective amount of an mTOR inhibitor is a concentration of about 60 nanomolar (nM) to about 1000 nM. However, factors including cell density, treatment time, type of mTOR inhibitor, and type of lymphocyte can affect the amount of mTOR inhibitor effective to import mitochondria into lymphocytes.

[0041] In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 60 nM to about 1000 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 100 nM to about 1000 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 200 nM to about 500 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 300 nM to about 600 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 400 nM to about 700 nM.

[0042] In certain embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 60 nM. In certain embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 70 nM. In certain embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 80 nM. In certain embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 90 nM. In certain embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 100 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 150 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 200 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 250 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 300 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 350 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 400 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 450 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 500 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 550 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 600 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 650 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 700 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 750 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 800 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 850 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 900 nM. In some embodiments, the effective amount of the mTOR inhibitor is at a concentration of about 1000 nM. In some embodiments, the effective amount of an mTOR inhibitor is a concentration greater than about 1000 nM.

[0043] In order to generate mitochondrial replacement lymphoid cell according to the present disclosure, various mTOR inhibitors can be used, for example, the exemplary mTOR inhibitors described in section 6.2.In certain embodiments, the mTOR inhibitor is rapamycin or its derivatives.In a specific embodiment, the mTOR inhibitor is rapamycin.

[0044] In some embodiments, the mTOR inhibitor is rapamycin or a derivative thereof at a concentration of about 100 nanomolar (nM) to about 1000 nM. In some embodiments, the mTOR inhibitor is rapamycin or a derivative thereof at a concentration of about 200 nanomolar (nM) to about 500 nM. In some embodiments, the mTOR inhibitor is rapamycin or a derivative thereof at a concentration of about 300 nanomolar (nM) to about 600 nM. In some embodiments, the mTOR inhibitor is rapamycin or a derivative thereof at a concentration of about 400 nanomolar (nM) to about 700 nM.

[0045] In certain embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 100 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 150 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 200 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 250 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 300 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 350 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 400 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 450 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 500 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 550 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 600 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 650 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 700 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 750 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 800 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 850 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 900 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration of about 1000 nM. In some embodiments, the effective amount of rapamycin or a derivative thereof is at a concentration greater than 1000 nM.

[0046] In specific embodiments, provided herein is a method for generating mitochondrial-replaced lymphoid cells, comprising incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and about 100 nM to about 1000 nM rapamycin for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells.

[0047] In one embodiment, lymphoid cells that have not been subjected to the procedure of reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100nM of mTOR inhibitor (for example, mTOR inhibitor described in section 6.2, for example, rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to the procedure of reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100nM of mTOR inhibitor (for example, mTOR inhibitor described in section 6.2, for example, rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to the procedure of reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100nM of mTOR inhibitor for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100 nM of an mTOR inhibitor for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 100 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0048] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 200 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0049] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 300 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0050] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 400 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0051] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 500 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0052] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 600 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0053] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 700 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0054] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 800 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0055] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900nM of mTOR inhibitor (for example, mTOR inhibitors described in section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 900 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for greater than 7 days.

[0056] In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 12 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 24 hours.In another embodiment, lymphoid cells that have not been subjected to a procedure for reducing or depleting endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000nM of mTOR inhibitor (for example, mTOR inhibitors described in Section 6.2, such as rapamycin) for about 36 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 48 hours. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for about 2 to 7 days. In another embodiment, lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria are incubated with isolated exogenous mitochondria and about 1000 nM of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) for more than 7 days.

[0057] In some embodiments, mTOR inhibitor (for example, mTOR inhibitor described in section 6.2, for example, rapamycin) is washed out and / or diluted in cell culture after incubation with lymphoid cells and exogenous mitochondria for a certain period of time.For example, in some embodiments, lymphoid cells are incubated with exogenous mitochondria and mTOR inhibitor (for example, mTOR inhibitor described in section 6.2, for example, rapamycin) for about 12 hours or longer, and then culture medium is replaced with fresh medium without mTOR inhibitor, and then cells are cultured for at least another 12 hours.In some embodiments, lymphoid cells are incubated with exogenous mitochondria and mTOR inhibitor (for example, mTOR inhibitor described in section 6.2, for example, rapamycin) for about 24 hours or longer, and then culture medium is replaced. In some embodiments, the lymphoid cells are incubated with exogenous mitochondria and mTOR inhibitors (e.g., mTOR inhibitors described in Section 6.2, e.g., rapamycin) for about 36 hours or longer, and then the culture medium is replaced. In some embodiments, the lymphoid cells are incubated with exogenous mitochondria and mTOR inhibitors (e.g., mTOR inhibitors described in Section 6.2, e.g., rapamycin) for about 48 hours or longer, and then the culture medium is replaced. In some embodiments, the lymphoid cells are incubated with exogenous mitochondria and mTOR inhibitors for about 60 hours or longer, and then the culture medium is replaced. In some embodiments, the lymphoid cells are rinsed one or more times with a suitable buffer (e.g., PBS, Hanks' Balanced Salt Solution (HBSS), Earle's Balanced Salt Solution (EBSS), culture medium, etc., with or without supplements, that maintains the water balance of the lymphoid cells, and then cultured in fresh culture medium that does not contain mTOR inhibitors. In some embodiments, lymphoid cells are incubated with an mTOR inhibitor and exogenous mitochondria and the medium is not changed for the duration of the culture.

[0058] In some embodiments, an mTOR inhibitor (eg, an mTOR inhibitor described in Section 6.2, including, for example, rapamycin) is diluted out of the cell culture after incubation with lymphoid cells and exogenous mitochondria. For example, in some embodiments, after incubating lymphoid cells with exogenous mitochondria and an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, e.g., rapamycin) for about 12 hours or more, a portion of the culture medium is replaced with fresh medium without the mTOR inhibitor (e.g., about 25%, about 50%, or about 75% of the culture medium is replaced with fresh medium), thus diluting the concentration of the mTOR inhibitor, and the cells are then cultured for at least an additional 12 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, 132 hours, about 148 hours, about 172 hours, about 200 hours, about 250 hours, about 275 hours or more). In some embodiments, after incubating lymphoid cells with exogenous mitochondria and an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, e.g., rapamycin) for about 24 hours or more, a portion of the culture medium is replaced with fresh medium without the mTOR inhibitor (e.g., about 25%, about 50%, or about 75% of the culture medium is replaced with fresh medium), thus diluting the concentration of the mTOR inhibitor, and the cells are then cultured for at least an additional 12 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, 132 hours, about 148 hours, about 172 hours, about 200 hours, about 250 hours, about 275 hours or more).In some embodiments, after incubating lymphoid cells with exogenous mitochondria and an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, e.g., rapamycin) for about 36 hours or more, a portion of the culture medium is replaced with fresh medium without the mTOR inhibitor (e.g., about 25%, about 50%, or about 75% of the culture medium is replaced with fresh medium), thus diluting the concentration of the mTOR inhibitor, and the cells are then cultured for at least an additional 12 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, 132 hours, about 148 hours, about 172 hours, about 200 hours, about 250 hours, about 275 hours or more). In some embodiments, after incubating the lymphoid cells with exogenous mitochondria and an mTOR inhibitor for about 48 hours or longer, a portion of the culture medium is replaced with fresh medium without the mTOR inhibitor (e.g., about 25%, about 50%, or about 75% of the culture medium is replaced with fresh medium), thus diluting the concentration of the mTOR inhibitor, and the cells are then cultured for at least an additional 12 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, 132 hours, about 148 hours, about 172 hours, about 200 hours, about 250 hours, about 275 hours or longer). In some embodiments, after incubating lymphoid cells with exogenous mitochondria and an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, e.g., rapamycin) for about 60 hours or more, a portion of the culture medium is replaced with fresh medium without the mTOR inhibitor (e.g., about 25%, about 50%, or about 75% of the culture medium is replaced with fresh medium), thus diluting the concentration of the mTOR inhibitor, and the cells are then cultured for at least an additional 12 hours (e.g., about 12 hours, about 18 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, 132 hours, about 148 hours, about 172 hours, about 200 hours, about 250 hours, about 275 hours or more).In some embodiments, lymphoid cells are incubated with exogenous mitochondria and an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) and the medium is not changed for the duration of the culture.

[0059] Mitochondria-replaced lymphoid cells generated according to the methods provided herein can include various types of lymphoid cells. Non-limiting examples of lymphoid cells suitable for use with the present disclosure include T cells, B cells, monocytes, macrophages, natural killer (NK) cells, or granulocytes.

[0060] In some embodiments, the lymphoid cells are T cells. In certain embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In certain embodiments, the lymphoid cells include a combination of CD4+ T cells and CD8+ T cells. In some embodiments, the lymphoid cells are or include Tregs. In certain embodiments, the lymphoid cells are or include effector T cells. In some embodiments, the lymphoid cells are or include memory T cells, effector T cells, Tregs, or a combination thereof. In specific embodiments, the lymphoid cells are or include T cells that include an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In specific embodiments, the lymphoid cells are or include T cells that have been genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0061] In some embodiments, the T cells are genetically modified T cells to express a chimeric antigen receptor (CAR) or a T cell receptor (TCR). For example, TCR uses a naturally occurring receptor that can also recognize antigens present inside tumor cells. CAR, on the other hand, includes a portion of an antibody that can only recognize a specific antigen on the surface of a cancer cell. CAR-T cells and TCR T cells can become exhausted, despite their use in immunotherapy. Thus, as provided herein, in some embodiments, the mitochondrial-replaced lymphoid cells produced according to the methods described herein, for example, the methods described in section 6.1, can be CAR-T cells or TCR T cells, and can be administered to a subject to treat or ameliorate symptoms of cancer. Non-limiting exemplary cancer types for which the mitochondrial-replaced lymphoid cells described herein that are or include CAR-T cells or TCR Ts can be beneficial include hematological cancers (e.g., acute lymphocytic leukemia, multiple myeloma, B-cell lymphoma, mantle cell lymphoma), as well as solid tumors. In a specific embodiment, the subject is a human subject.

[0062] CAR is generally designed to include an extracellular target binding domain, a hinge region, a transmembrane domain that anchors CAR to the cell membrane, and one or more intracellular domains that transmit activation signals. Depending on the number of costimulatory domains, CAR can be classified as first generation (intracellular domain, e.g., CD3ζ only), second generation (one costimulatory domain and intracellular domain), or third generation CAR (more than one costimulatory domain and intracellular domain). New generation CARs are also under development (see, for example, Guedan S, et al.. Mol Ther Methods Clin Dev. 2018 Dec 31; 12: 145-156). CAR targets for hematological malignancies (e.g., CD19, BCMA) and solid tumors (e.g., HER2, PSCA) are known in the art, and any target is suitable for use with the present disclosure (see, e.g., Dotti G, et al. Immunol Rev. 2014; 257 (1): 107-126). CAR-T cells may be autologous or allogeneic to the subject receiving the CAR-T cells. In some embodiments, the CAR-T cells are allogeneic to the subject. In other embodiments, the CAR-T cells are autologous to the subject.

[0063] In certain embodiments, the CAR comprises a tumor antigen recognition domain, a transmembrane domain, and one or more intracellular signaling domains. In some embodiments, the CAR comprises a tumor antigen recognition domain, a transmembrane domain, one or more costimulatory molecules, and one or more intracellular signaling domains. In a specific embodiment, the CAR comprises a tumor antigen recognition domain, a transmembrane domain, and an intracellular domain. In a specific embodiment, the CAR comprises a tumor antigen recognition domain, a transmembrane domain, an intracellular domain, and at least one costimulatory domain. In another specific embodiment, the CAR comprises a tumor antigen recognition domain, a transmembrane domain, two or more costimulatory domains, and an intracellular domain. In some embodiments, the CAR comprises a constitutively or inducibly expressed chemokine. In certain embodiments, the CAR comprises an intracellular domain of a cytokine receptor (e.g., an IL-2Rβ chain fragment).

[0064] In some embodiments, the T cell is a T cell genetically modified to express a TCR. TCR generally uses a heterodimer consisting of an alpha peptide chain and a beta peptide chain to recognize a polypeptide fragment presented by an MHC molecule, and TCR T cell is a genetically engineered TCR product that can recognize a specific antigen. Generally, an artificially designed high affinity TCR is encoded in the T cell by genetic engineering techniques, thereby enhancing both specific recognition and affinity during the recognition of tumor cells by the T cell. The TCR-T cell may be autologous or allogeneic to the subject receiving the TCR-T cell. In some embodiments, the TCR-T cell is allogeneic to the subject. In other embodiments, the TCR-T cell is autologous to the subject.

[0065] In certain embodiments, the TCR T cells recognize antigens on hematological malignancies (e.g., CMV, WT1, HA-1). In certain embodiments, the TCR T cells recognize antigens on solid tumors (e.g., HBV, p53, mutant KRAS). In certain embodiments, the TCR T cells recognize SL9 associated with HIV.

[0066] In some embodiments, the lymphoid cell that is subjected to the method described herein is dysfunctional T cell.For example, in some embodiments, T cell comprises exhausted T cell, senescent T cell, or a combination thereof.In certain embodiments, lymphoid cell comprises T cell that is isolated from a subject (e.g., human subject) with disease or disorder associated with inherited and acquired mitochondrial DNA mutation, such as mitochondrial disease, or immune deficiency associated with heteroplasmic immune cells.

[0067] The method for generating mitochondrial replacement lymphoid cells may involve human cells and non-human cells. In some embodiments, the lymphoid cells are human lymphoid cells. In other embodiments, the lymphoid cells are non-human lymphoid cells (e.g., mouse lymphoid cells, monkey lymphoid cells, etc.).

[0068] In certain embodiments, the lymphoid cells subjected to the methods described herein are isolated from human subjects with diseases or disorders associated with inherited and acquired mitochondrial DNA mutations, such as mitochondrial diseases, or immunodeficiencies associated with heteroplasmic immune cells. In some embodiments, the lymphoid cells subjected to the methods described herein are isolated from a subject (e.g., a human subject) to which the mitochondrial-replaced lymphoid cells are administered as a treatment. In other words, the mitochondrial-replaced lymphoid cells are derived from autologous lymphoid cells to the subject to which the mitochondrial-replaced lymphoid cells are administered as a treatment. In other embodiments, the lymphoid cells subjected to the methods described herein are isolated from a subject (e.g., a different human subject) different from the subject (e.g., a human subject) to which the mitochondrial-replaced lymphoid cells are administered as a treatment. In other words, in specific embodiments, the mitochondrial-replaced lymphoid cells are derived from allogeneic lymphoid cells to the subject to which the mitochondrial-replaced lymphoid cells are administered. In some embodiments, the lymphoid cells are T cells. In certain embodiments, the lymphoid cells are CD4+ T cells, CD8+ T cells, or a combination thereof. In some embodiments, the lymphoid cell is a T cell genetically modified to express a CAR.

[0069] Lymphoid cells can be isolated from a subject (e.g., a human subject) using techniques known to those skilled in the art or described herein (e.g., those described in the Examples). For example, peripheral blood lymphocytes can be isolated from a subject (e.g., a human subject). In some embodiments, a certain subset of lymphoid cells (e.g., a subset of T cells, e.g., CD4+ T cells, CD8+ T cells, or a combination thereof) can be isolated from peripheral blood lymphocytes using techniques known to those skilled in the art or described herein, such as magnetic separation.

[0070] The amount of isolated exogenous mitochondria incubated with lymphoid cells to generate mitochondrial-replaced lymphoid cells depends on factors such as the amount of lymphoid cells co-incubated with the isolated mitochondria. Generally, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 5 μg to about 100 μg per lymphoid cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 10 μg to about 90 μg per lymphoid cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 20 μg to about 80 μg per lymphoid cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 30 μg to about 70 μg per lymphoid cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 40 μg to about 80 μg per lymphoid cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells / ml. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells / ml. 6In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells / ml. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells / ml. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells / ml. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells / ml. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with lymphoid cells is about 1×10 lymphoid cells. 6 More than 100 μg per piece.

[0071] As provided herein, the isolated exogenous mitochondria of the present disclosure can be obtained from various types of cells that have healthy and functional mitochondria. Assays for determining mitochondrial function are known in the art, including assays such as those described in Section 6.4. Exemplary sources of mitochondria for use in the methods provided herein include fibroblasts, platelet cells, and other lymphoid cells. In certain embodiments, the isolated exogenous mitochondria are obtained from fibroblasts. In some embodiments, the isolated exogenous mitochondria are obtained from platelet cells. In some embodiments, the isolated exogenous mitochondria are obtained from lymphoid cells.

[0072] As provided herein, isolated exogenous mitochondria can be autologous or allogeneic to recipient cells. In some embodiments, isolated exogenous mitochondria are allogeneic to recipient cells. For example, isolated exogenous mitochondria can be obtained from a different subject than recipient cells. In other embodiments, isolated exogenous mitochondria are autologous. For example, exemplary autologous isolated exogenous mitochondria can include mitochondria isolated from the same subject at an earlier time point, for example, from placenta or umbilical cord blood. Another exemplary autologous exogenous mtDNA can include donor mtDNA, for example, isolated from the same subject as recipient cells, modified and then replaced with recipient cells.

[0073] In certain embodiments, mitochondria are obtained from normal peripheral blood collected using leukopak (i.e., a product enriched by leukopheresis). In some embodiments, mitochondria are obtained from CD34+ cells.

[0074] Isolation of mitochondria can be achieved by any of several well-known techniques, including but not limited to those described herein. In certain embodiments, exogenous mitochondria for use in mitochondrial transfer are isolated using a commercially available kit, such as, for example, the Qproteum Mitochondria Isolation Kit (Qiagen, USA) or the MITOISO2 Mitochondria Isolation Kit (Sigma, USA). In other embodiments, exogenous mitochondria for use in mitochondrial transfer are manually isolated (see, for example, Preble et al. J. Vis. Exp. 2014, 91: e51682; Gasnier et al. Anal Biochem 1993; 212 (1): 173-8 and Frezza et al. Nat Protoc 2007; 2 (2): 287-95). For example, as an exemplary manual isolation of mitochondria, donor cells are pelleted and approximately 10 cells grown in culture are isolated. 9 Mitochondria can be isolated from donor cells by washing 1-2 mL of cell pellet from donor cells, swelling the cells in hypotonic buffer, disrupting the cells using a Dounce or Potter-Elvehjem homogenizer with a tight-fitting pestle, and isolating the mitochondria by differential centrifugation. Manual isolation can also include, for example, sucrose density gradient ultracentrifugation, or free-flow electrophoresis. Without wishing to be bound to any particular method, it is understood that the kits and manual methods described herein are exemplary and that any mitochondrial isolation method can be used and is within the skill of one of ordinary skill in the art.

[0075] In some embodiments, isolated donor mitochondria are substantially pure, free from other organelles. In other embodiments, isolated mitochondria may contain impurities and are enriched in mitochondria. For example, in some embodiments, isolated mitochondria are about 90% pure, about 80% pure, about 70% pure, about 60% pure, about 50% pure, or any integer between them. It is generally understood that any impurities contained in isolated donor mitochondria do not affect the viability or function of recipient cells upon mitochondria transfer. In specific embodiments, transfer of exogenous mitochondria, exogenous mtDNA, or combinations thereof is not accompanied by transfer of non-mitochondrial organelles.

[0076] The quantity and quality of isolated mitochondria can be easily determined by several well-known techniques, including but not limited to those described herein and in the cited references.For example, in some embodiments, the quantity of isolated mitochondria is determined by measuring total protein content.To assess total protein content, various methods are available, such as the Biuret and Lowry procedure (see, for example, Hartwig et al., Proteomics, 2009 Jun; 9 (11): 3209-14) and Bradford protein assay (Bradford. Anal Biochem. 1976; 72: 248-54).In other embodiments, the quantity of isolated mitochondria is determined by mtDNA copy number.

[0077] The period of time sufficient for non-invasively transferring exogenous mitochondria into lymphoid cells may be any period of time that allows a greater amount of exogenous mtDNA to be detected than in lymphoid cells that have not been exposed to exogenous mitochondria. In some embodiments, the period of time sufficient for non-invasively transferring exogenous mitochondria into lymphoid cells is any period of time that allows at least 20% of exogenous mtDNA to be transferred into lymphoid cells compared to the amount of exogenous mtDNA transferred into lymphoid cells that have not been incubated with exogenous mtDNA. In certain embodiments, the period of time sufficient for non-invasively transferring exogenous mitochondria into lymphoid cells is any period of time that allows at least 30% of exogenous mtDNA to be transferred into lymphoid cells compared to the amount of exogenous mtDNA transferred into lymphoid cells that have not been incubated with exogenous mtDNA. In some embodiments, the period of time sufficient for non-invasively transferring exogenous mitochondria into lymphoid cells is any period of time during which at least 30% of exogenous mtDNA is transferred into lymphoid cells compared to the amount of exogenous mtDNA transferred into lymphoid cells that have not been incubated with exogenous mtDNA. In certain embodiments, the period of time sufficient for non-invasively transferring exogenous mitochondria into lymphoid cells is any period of time during which at least 40% of exogenous mtDNA is transferred into lymphoid cells compared to the amount of exogenous mtDNA transferred into lymphoid cells that have not been incubated with exogenous mtDNA. In some embodiments, the period of time sufficient for non-invasively transferring exogenous mitochondria into lymphoid cells is any period of time during which at least 50% of exogenous mtDNA is transferred into lymphoid cells compared to the amount of exogenous mtDNA transferred into lymphoid cells that have not been incubated with exogenous mtDNA. In some embodiments, a period of time sufficient to non-invasively transfer exogenous mitochondria into lymphoid cells is any period of time during which at least 60% of the exogenous mtDNA is transferred to lymphoid cells compared to the amount of exogenous mtDNA transferred to lymphoid cells that have not been incubated with exogenous mtDNA.In some embodiments, a sufficient period of time for non-invasively transferring exogenous mitochondria into lymphoid cells is any period during which at least 70% of the exogenous mtDNA is transferred into lymphoid cells compared to the amount of exogenous mtDNA transferred into lymphoid cells that have not been incubated with exogenous mtDNA. Techniques known to those of skill in the art or described herein (e.g., in the Examples) can be used to assess transfer of exogenous mtDNA into lymphoid cells. Generally, a sufficient period of time is at least about 12 hours and less than 2 weeks. In some embodiments, a sufficient period of time is at least 12 hours. In some embodiments, a sufficient period of time is at least 24 hours. In some embodiments, a sufficient period of time is at least 36 hours. In some embodiments, a sufficient period of time is at least 48 hours. In some embodiments, a sufficient period of time is about 2 days or longer. In some embodiments, a sufficient period of time is about 7 days or longer. In some embodiments, a sufficient period of time is about 2 days to about 7 days.

[0078] In some embodiments, the ratio of exogenous mtDNA copy number to endogenous mtDNA copy number in mitochondrial replacement lymphoid cells generated according to the methods provided herein is greater than 4:1. In some embodiments, the ratio is about 4:1. In some embodiments, the ratio is about 3:1. In some embodiments, the ratio is about 2:1. In some embodiments, the ratio is about 1:1. In some embodiments, the ratio is about 0.75:1. In some embodiments, the ratio is about 0.5:1. In some embodiments, the ratio is about 0.25:1. In some embodiments, the ratio is about 0.1:1.

[0079] Generally, the method provided herein is compatible with simple co-incubation of lymphoid cells and isolated exogenous mitochondria. However, it is also possible to promote mitochondrial import by centrifuging lymphoid cells and isolated exogenous mitochondria as needed. In some embodiments, before centrifuging lymphoid cells and isolated exogenous mitochondria, lymphoid cells are treated with mTOR inhibitors (e.g., mTOR inhibitors described in section 6.2, including rapamycin). In some embodiments, mTOR inhibitors, lymphoid cells and isolated exogenous mitochondria are centrifuged together and then incubated together as described herein. In other embodiments, mTOR inhibitors are added after centrifuging lymphoid cells and isolated exogenous mitochondria.

[0080] In another aspect, provided herein is a method for generating mitochondrial-replaced lymphoid cells, the method comprising: (a) centrifuging lymphoid cells and isolated exogenous mitochondria under conditions sufficient to generate a cell pellet, wherein the lymphoid cells have not been subjected to a procedure that reduces or depletes endogenous mitochondria; and (b) incubating lymphoid cells (e.g., T cells or other lymphoid cells described herein) with 100 nM-1000 nM rapamycin for approximately 24 hours or longer, thereby generating mitochondrial-replaced lymphoid cells.

[0081] Incubation of lymphoid cells with rapamycin after centrifugation may be for any time sufficient to generate mitochondrial-replaced lymphoid cells. In one embodiment, the incubating step is for approximately 7 days or longer. In one embodiment, the incubating step is for approximately 2 days to approximately 7 days. In one embodiment, the medium is changed to 50% fresh medium during the incubation period.

[0082] Centrifugation conditions can be easily determined by those skilled in the art, and can vary in speed and time as long as cells and mitochondria are not damaged and mitochondrial import is promoted.For example, centrifugation conditions can include centrifugation at room temperature and approximately 1,500 relative centrifugal force (also referred to as "g") for approximately 5 minutes.In a specific embodiment, centrifugation is as described in the following examples.

[0083] In one embodiment, the centrifugation is at approximately 500 RCF at room temperature for approximately 5 minutes. In another embodiment, the centrifugation is at approximately 750 RCF at room temperature for approximately 5 minutes. In another embodiment, the centrifugation is at approximately 1,000 RCF at room temperature for approximately 5 minutes. In another embodiment, the centrifugation is at approximately 1,500 RCF at room temperature for approximately 5 minutes. In another embodiment, the centrifugation is at approximately 2,000 RCF at room temperature for approximately 5 minutes. In another embodiment, the centrifugation is at approximately 2,500 RCF at room temperature for approximately 5 minutes. In another embodiment, the centrifugation is at approximately 3,000 RCF at room temperature for approximately 5 minutes.

[0084] In one embodiment, the centrifugation is at approximately 500 RCF at room temperature for approximately 10 minutes. In another embodiment, the centrifugation is at approximately 750 RCF at room temperature for approximately 10 minutes. In another embodiment, the centrifugation is at approximately 1,000 RCF at room temperature for approximately 10 minutes. In another embodiment, the centrifugation is at approximately 1,500 RCF at room temperature for approximately 10 minutes. In another embodiment, the centrifugation is at approximately 2,000 RCF at room temperature for approximately 10 minutes. In another embodiment, the centrifugation is at approximately 2,500 RCF at room temperature for approximately 10 minutes. In another embodiment, the centrifugation is at approximately 3,000 RCF at room temperature for approximately 10 minutes.

[0085] In another embodiment, the centrifugation is at approximately 500 RCF at room temperature for approximately 15 minutes. In another embodiment, the centrifugation is at approximately 750 RCF at room temperature for approximately 15 minutes. In another embodiment, the centrifugation is at approximately 1,000 RCF at room temperature for approximately 15 minutes. In another embodiment, the centrifugation is at approximately 1,500 RCF at room temperature for approximately 15 minutes. In another embodiment, the centrifugation is at approximately 2,000 RCF at room temperature for approximately 15 minutes. In another embodiment, the centrifugation is at approximately 2,500 RCF at room temperature for approximately 15 minutes. In another embodiment, the centrifugation is at approximately 3,000 RCF at room temperature for approximately 15 minutes.

[0086] In another embodiment, the centrifugation is at room temperature and approximately 500 RCF for less than 1 hour. In another embodiment, the centrifugation is at room temperature and approximately 750 RCF for less than 1 hour. In another embodiment, the centrifugation is at room temperature and approximately 1,000 RCF for less than 1 hour. In another embodiment, the centrifugation is at room temperature and approximately 1,500 RCF for less than 1 hour. In another embodiment, the centrifugation is at room temperature and approximately 2,000 RCF for less than 1 hour. In another embodiment, the centrifugation is at room temperature and approximately 2,500 RCF for less than 1 hour. In another embodiment, the centrifugation is at room temperature and approximately 3,000 RCF for less than 1 hour.

[0087] In one embodiment, the centrifugation is at about 4° C. and about 500 RCF for about 5 minutes. In another embodiment, the centrifugation is at about 4° C. and about 750 RCF for about 5 minutes. In another embodiment, the centrifugation is at about 4° C. and about 1,000 RCF for about 5 minutes. In another embodiment, the centrifugation is at about 4° C. and about 1,500 RCF for about 5 minutes. In another embodiment, the centrifugation is at about 4° C. and about 2,000 RCF for about 5 minutes. In another embodiment, the centrifugation is at about 4° C. and about 2,500 RCF for about 5 minutes. In another embodiment, the centrifugation is at about 4° C. and about 3,000 RCF for about 5 minutes.

[0088] In another embodiment, the centrifugation is at about 4° C. and about 500 RCF for about 10 minutes. In another embodiment, the centrifugation is at about 4° C. and about 750 RCF for about 10 minutes. In another embodiment, the centrifugation is at about 4° C. and about 1,000 RCF for about 10 minutes. In another embodiment, the centrifugation is at about 4° C. and about 1,500 RCF for about 10 minutes. In another embodiment, the centrifugation is at about 4° C. and about 2,000 RCF for about 10 minutes. In another embodiment, the centrifugation is at about 4° C. and about 2,500 RCF for about 10 minutes. In another embodiment, the centrifugation is at about 4° C. and about 3,000 RCF for about 10 minutes.

[0089] In another embodiment, the centrifugation is at about 4° C. and about 500 RCF for about 15 minutes. In another embodiment, the centrifugation is at about 4° C. and about 750 RCF for about 15 minutes. In another embodiment, the centrifugation is at about 4° C. and about 1,000 RCF for about 15 minutes. In another embodiment, the centrifugation is at about 4° C. and about 1,500 RCF for about 15 minutes. In another embodiment, the centrifugation is at about 4° C. and about 2,000 RCF for about 15 minutes. In another embodiment, the centrifugation is at about 4° C. and about 2,500 RCF for about 15 minutes. In another embodiment, the centrifugation is at about 4° C. and about 3,000 RCF for about 15 minutes.

[0090] In another embodiment, the centrifugation is at about 4° C. and approximately 500 RCF for less than 1 hour. In another embodiment, the centrifugation is at about 4° C. and approximately 750 RCF for less than 1 hour. In another embodiment, the centrifugation is at about 4° C. and approximately 1,000 RCF for less than 1 hour. In another embodiment, the centrifugation is at about 4° C. and approximately 1,500 RCF for less than 1 hour. In another embodiment, the centrifugation is at about 4° C. and approximately 2,000 RCF for less than 1 hour. In another embodiment, the centrifugation is at about 4° C. and approximately 2,500 RCF for less than 1 hour. In another embodiment, the centrifugation is at about 4° C. and approximately 3,000 RCF for less than 1 hour. In some embodiments, the methods provided herein do not involve centrifugation.

[0091] In certain embodiments, lymphoid cells are starved for a certain period of time, and then incubated with an mTOR inhibitor (e.g., an mTOR inhibitor as described in Section 6.2, e.g., rapamycin) and isolated exogenous mitochondria. For example, in some embodiments, lymphoid cells are starved for 3-6 hours, 3-9 hours, 6-9 hours, 6-12 hours, 6-18 hours, 12-24 hours, or 24-48 hours, and then incubated with an mTOR inhibitor (e.g., an mTOR inhibitor as described in Section 6.2, e.g., rapamycin) and isolated exogenous mitochondria. In certain embodiments, lymphoid cells are starved by depriving them of glucose, essential amino acids (e.g., glutamine), and / or serum. For example, in some embodiments, lymphoid cells are cultured in a cell culture medium lacking one or more nutrients (e.g., glucose-free, serum-free, and / or glutamine-free).

[0092] In certain embodiments, lymphoid cells are starved for a certain period of time, and then centrifuged with isolated exogenous mitochondria. For example, in some embodiments, lymphoid cells are starved for 3-6 hours, 3-9 hours, 6-9 hours, 6-12 hours, 6-18 hours, 12-24 hours, or 24-48 hours, and then centrifuged with isolated exogenous mitochondria. In certain embodiments, lymphoid cells are starved by depriving the lymphoid cells of glucose, essential amino acids (e.g., glutamine), and / or serum. After centrifugation, lymphoid cells (e.g., T cells or other lymphoid cells described herein) can be incubated with an effective amount of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, e.g., rapamycin) for a period of time sufficient to non-invasively transfer exogenous mitochondria into lymphoid cells as described above. In some embodiments, the lymphoid cells are cultured in a cell culture medium lacking one or more nutrients (e.g., glucose-free, serum-free, and / or glutamine-free) and then the lymphoid cells are centrifuged with the isolated exogenous mitochondria.

[0093] In certain embodiments, the step of incubating lymphoid cells with an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) and isolated exogenous mitochondria is performed under starvation conditions. In certain embodiments, lymphoid cells are starved by depriving them of glucose, essential amino acids (e.g., glutamine), and / or serum. For example, in some embodiments, lymphoid cells are incubated in a cell culture that (1) lacks one or more nutrients (e.g., glucose-free, serum-free, and / or glutamine-free) and (2) contains an effective amount of an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin). In some embodiments, the step of incubating lymphoid cells with an mTOR inhibitor (e.g., an mTOR inhibitor described in Section 6.2, such as rapamycin) and isolated exogenous mitochondria is performed under conditions that do not involve starvation.

[0094] In a specific embodiment, provided herein is a method for generating mitochondrial-substituted lymphoid cells using the methods described in Example 8.1.

[0095] In a specific embodiment, the mitochondrial-replaced lymphoid cells exhibit one, two or more improved functions compared to the lymphoid cells from which they were generated. For lymphoid cell functions that can be improved in mitochondrial-replaced lymphoid cells, see, e.g., Section 6.4. 6.2 Rapamycin analogues and other mTOR inhibitors

[0096] A variety of compounds have been found to inhibit mTOR, including rapamycin, also known as sirolimus (CAS number 53123-88-9; C51H79NO13), and rapamycin derivatives (e.g., rapamycin analogs, also known as "rapalogs"). Non-limiting examples of rapamycin derivatives include, for example, temsirolimus (CAS number 162635-04-3; C56H87NO16), everolimus (CAS number 159351-69-6; C53H83NO14), ridaforolimus (CAS number 572924-54-0; C53H84NO14P), WYE-125132 (WYE-132), and zotarolimus (ABT-578). In a specific embodiment, the mTOR inhibitor is rapamycin.

[0097] In some embodiments, mTOR inhibitors suitable for use in any of the methods described herein are those that inhibit both mTORC1 and mTORC2, such as, for example, AZD8055, Torin1, Torkinib, or Omipalisib.

[0098] In certain embodiments, the mTOR inhibitor disclosed herein also inhibits one or more substrates other than mTOR, such as dual kinase inhibitors. Inhibitors with specificity for mTOR and one or more substrates are known in the art. By way of example, dual PI3K / mTOR inhibitors are one type of mTOR inhibitor suitable for use with the present disclosure, which inhibits mTOR and another substrate. Non-limiting examples of dual PI3K / mTOR inhibitors include, for example, Dactolisib (also known as BEZ235), PI-103, Bimiralisib (also known as PQR309), GDC-0084, and Gedatolisib. 6.3 Treatment Method

[0099] As provided herein, the mitochondrial-replaced lymphoid cells generated according to the methods of the present disclosure are suitable for use as cell-based therapy, for example, in the methods described in Sections 6.3.1 and 6.3.2. For example, in some embodiments, an effective amount of the mitochondrial-replaced lymphoid cells generated according to the methods described in Section 6.1 can be combined with a pharma- ceutically acceptable carrier to provide a pharmaceutical composition. In some embodiments, a composition (e.g., a pharmaceutical composition) is provided herein that includes the mitochondrial-replaced lymphoid cells generated according to the methods described herein, for example, in the methods described in Section 6.1, and a pharma- ceutically acceptable carrier. In some embodiments, a composition (e.g., a pharmaceutical composition) is provided herein that includes an effective amount of the mitochondrial-replaced lymphoid cells generated according to the methods described herein, for example, in the methods described in Section 6.1, and a pharma- ceutically acceptable carrier.

[0100] As used herein, the term "pharmaceutical acceptable" when used in reference to a carrier shall mean that the carrier, diluent or excipient is not toxic or otherwise undesirable (i.e., the material can be administered to a subject without causing any undesired biological effects) and is compatible with the other ingredients of the formulation. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic is administered. Such pharmaceutical carriers may be sterile liquids, such as saline solution. Saline solution may be a carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions may also be used as liquid carriers, particularly injectable solutions. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired.

[0101] In certain embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 1×10 cells. 6 ~Approx. 1×10 7 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 10×10 cells. 6 ~About 900×106 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 50×10 cells. 6 ~About 800×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 100×10 cells. 6 ~Approx. 700×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 200×10 cells. 6 ~About 900×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 250×10 cells. 6 ~Approx. 750×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 50×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 150×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 300×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 450×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 600×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 850×10 cells. 6 There are 10 pieces.

[0102] In certain embodiments, the effective amount of mitochondrial-replaced lymphoid cells is empirically determined, such as based on the subject's body weight or disease or disorder burden. In some embodiments, the effective amount of mitochondrial-replaced lymphoid cells is about 1.0×10 cells. 6 cells / kg~cells approx. 1.0×10 7 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 1.0×10 cells / kg. 6 cells / kg~cells approx. 500×10 6In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 1.0×10 cells / kg. 6 pcs / kg~cells approx. 50×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 1.0×10 cells / kg. 6 cells / kg~cells approx. 10×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 1.0×10 cells / kg. 6 cells / kg~cells approx. 5.0×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 10×10 cells / kg. 6 cells / kg~cells approx. 600×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 50×10 cells / kg. 6 cells / kg~cells approx. 750×10 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 1.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 2.5×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 5.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 10.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 50.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 250.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced lymphoid cells is about 500×10 cells / kg. 6 Pieces / kg.

[0103] In certain embodiments, treatment with mitochondrial-replaced lymphoid cells results in one, two, or more, or all of the following: (1) a reduction in the severity, progression, spread, and / or frequency of occurrence of one or more symptoms, (2) elimination of one or more symptoms and / or underlying causes, (3) prevention of the occurrence of one or more symptoms and / or their underlying causes, and (4) amelioration or correction of damage. In specific embodiments, treatment includes therapeutic treatment as well as prophylactic or suppressive measures against a condition, disease, or disorder.

[0104] In some embodiments, the mitochondrial-replaced lymphoid cells for use as cell-based therapy, for example in the methods described in Sections 6.3.1 and 6.3.2, are autologous or allogeneic to the subject receiving the mitochondrial-replaced lymphoid cells. In some embodiments, the mitochondrial-replaced lymphoid cells for use as cell-based therapy, for example in the methods described in Sections 6.3.1 and 6.3.2, are autologous to the subject receiving the mitochondrial-replaced lymphoid cells. In some embodiments, the mitochondrial-replaced lymphoid cells for use as cell-based therapy, for example in the methods described in Sections 6.3.1 and 6.3.2, are allogeneic to the subject receiving the mitochondrial-replaced lymphoid cells. 6.3.1 Methods of Treating Mitochondrial Diseases or Disorders

[0105] In one aspect, provided herein is a method for ameliorating symptoms of mitochondrial complex III deficiency in a subject in need thereof, comprising administering to the subject an effective amount of mitochondrial-replaced lymphoid cells produced according to the methods described in Section 6.1, and a pharmaceutically acceptable carrier.

[0106] Mitochondrial complex III is essential for the suppressive function of regulatory T cells (Tregs). For example, it has been shown that Treg cells require mitochondrial complex III to maintain immunoregulatory gene expression and suppressive function (see Weinberg, S. et al. Nature vol. 565,7740 (2019) : 495-499). Mitochondrial complex III deficiency is a genetic disease. Mitochondrial complex III deficiency is generally caused by mutations in the nuclear DNA of the BCS1L, UQCRB and UQCRQ genes and is inherited in an autosomal recessive manner. However, it can also be caused by mutations in the mitochondrial DNA of the MTCYB gene, which can be maternally transmitted or occur sporadically, resulting in a milder condition.

[0107] Thus, in some embodiments, the mitochondrial replacement lymphoid cells are Treg cells, and the mitochondrial replacement lymphoid cells are administered to a subject with mitochondrial complex III deficiency. In a specific embodiment, the subject is a human subject.

[0108] In certain embodiments, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the severity, progression, spread, and / or frequency of occurrence of symptoms, eliminating symptoms and / or underlying causes, preventing the occurrence of symptoms and / or their underlying causes, and improving or correcting damage. In one embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the severity of symptoms. In one embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the progression of symptoms. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the spread of symptoms. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency reduces the frequency of occurrence of symptoms. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes eliminating the symptoms. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes preventing the occurrence of symptoms of mitochondrial complex III deficiency. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes improving damage caused by mitochondrial complex III deficiency. In another embodiment, the method for ameliorating symptoms of mitochondrial complex III deficiency comprises correcting the damage caused by mitochondrial complex III deficiency. 6.3.2 Methods of Treating Immune Disorders Associated with Heteroplasmic Immune Cells.

[0109] Also provided herein is a method for treating an immune deficiency associated with heteroplasmic immune cells in a subject in need thereof, comprising administering to the subject an effective amount of mitochondrial-replaced lymphoid cells produced according to the methods described in Section 6.1, and a pharmaceutically acceptable carrier.

[0110] In certain embodiments, the heteroplasmic immune cells are the result of undesirable pharmacological side effects. For example, nucleoside reverse transcriptase inhibitors (NRTIs) inhibit the replication of human immunodeficiency virus (HIV)-1 and are useful for treating HIV. However, NRTIs also exhibit side effects in human tissues that may be the result of the inhibition of human mitochondrial polymerase gamma (poly-gamma) by NRTIs. In one embodiment, the subject is receiving a reverse transcriptase inhibitor. In one embodiment, the subject has human immunodeficiency virus (HIV). In a specific embodiment, the subject is a human.

[0111] Similarly, many of the drugs approved by FDA for the treatment of Hepatitis B virus (HBV) are nucleoside RT inhibitors (NRTIs) that target reverse transcriptase (RT or P gene product) and suppress viral replication. Thus, in some embodiments, the subject has Hepatitis B virus (HBV). In a specific embodiment, the subject is a human.

[0112] Heteroplasmy can also result from various types of mutations. Thus, the methods provided herein are not limited to treating heteroplasmic immune cells caused by NRTI inhibitors.

[0113] In some embodiments, treating an immune deficiency associated with heteroplasmic immune cells includes reducing the severity, progression, spread, and / or frequency of occurrence of symptoms, eliminating symptoms and / or underlying causes, preventing the onset of symptoms and / or their underlying causes, and improving or correcting damage. In one embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes reducing the severity of the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes reducing the progression of the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes reducing the spread of the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes reducing the frequency of occurrence of symptoms of the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes eliminating the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes preventing the onset of symptoms of the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes improving damage due to the immune deficiency. In another embodiment, treating an immune deficiency associated with heteroplasmic immune cells includes correcting damage due to the immune deficiency. 6.4 Biological Assays for Lymphatic Function

[0114] Successful generation of mitochondrial-replaced lymphoid cells results in lymphoid cells with improved function compared to lymphoid cells without mitochondrial replacement. A variety of functional assays can be used to measure and evaluate the phenotype of mitochondrial-replaced lymphoid cells.

[0115] In some embodiments, mitochondria-replaced lymphoid cells have improved mitochondrial function compared to lymphoid cells without mitochondrial replacement. Those skilled in the art will understand how to assess mitochondrial function. For example, cell-based assays such as Seahorse Bioscience XF Extracellular Flux Analyzer can be used to determine basal oxygen consumption, glycolysis rate, ATP production, and respiratory capacity to assess mitochondrial dysfunction. Similarly, Oroboros 02K respirometer can be used to establish quantitative functional mitochondrial diagnostics. It is understood that the above assay examples are illustrative and are not all-inclusive for assessing mitochondrial function.

[0116] Increased cell proliferation can also be an indicator of improved lymphoid cell function.An exemplary assay for measuring cell proliferation of lymphoid cells is mixed lymphocyte reaction (MLR) assay.MLR assay generally involves combining a population of mitochondrial-replaced lymphoid cells, such as CD4+ T cells, with a different population of lymphocytes and measuring proliferation.In some embodiments, the mitochondrial-replaced lymphoid cells produced according to the methods provided herein have increased cell proliferation compared to lymphoid cells that are not incubated with isolated exogenous mitochondria and mTOR inhibitors.

[0117] Another exemplary assay that can be used to evaluate the function of mitochondrial replacement cells, for example in cytotoxic T cells, is cytotoxic T cell (CTL) assay.CTL assay shows the presence and cytotoxic activity of T cells against a specific antigen, and allows the influence of test items on this immune function to be investigated.Thus, in some embodiments, mitochondrial replacement lymphoid cells produced according to the method provided herein have increased CTL response compared to lymphoid cells that are not incubated with isolated exogenous mitochondria and mTOR inhibitors.

[0118] DNA stability is important for the function of T cells. Therefore, another non-limiting exemplary assay that can be used to evaluate the function of mitochondrial replacement cells is to measure DNA damage response. Double-strand breaks (DSBs) are critical damage to genome stability, and therefore are repaired quickly and precisely to maintain cell homeostasis. The initial response to DSBs is phosphorylation of minor histone H2A variants at Ser-139, which forms γH2AX. Thus, as an example, DNA damage response can be assayed by detecting phosphorylation of histone 2A X (H2AX), and phosphorylation can be measured using any assay known in the art, such as by flow cytometry or by immunoblotting.

[0119] For example, the following assay can be carried out to detect DNA damage response: Lymphoid cells (e.g., T cells) are washed twice with PBS, suspended with 70% ethanol, cooled at -20°C for 60 min, then washed twice with PBS and stained with PE anti-H2A.X phospho antibody (Biolegend) and APC mouse anti-CD3 antibody (Biolegend) for 60 min at 37°C in a humidified 5% CO2 incubator. Cells are then washed and resuspended in autoMACSTM Running Buffer (Miltenyi Biotec, Bergisch Gladbach, Germany) and immediately subjected to flow cytometry analysis. Data can be analyzed using FlowJo software (BD Bioscience, Franklin Lakes, NJ, USA).

[0120] Ca2+ signaling is crucial for lymphoid cell (e.g., T cell) activation as a means to rapidly activate and integrate multiple signaling pathways to produce widespread changes in gene expression and function. A variety of assays for measuring Ca2+ signaling are known in the art (see Samakai E, et al., Signaling Mechanisms Regulating T Cell Diversity and Function. Boca Raton (FL): CRC Press / Taylor & Francis; 2018. Chapter 10.). Thus, in some embodiments, the mitochondrial-replaced lymphoid cells produced according to the methods provided herein have increased Ca2+ signaling compared to lymphoid cells that have not been incubated with isolated exogenous mitochondria and mTOR inhibitors.

[0121] Telomere length can also serve as an indicator of the function of mitochondrial replacement cells.Telomere length can be measured using any method known in the art.One exemplary technique is by measuring absolute telomere length by qPCR.Thus, in some embodiments, the mitochondrial replacement lymphoid cell produced according to the method provided herein has reduced telomere shortening compared with lymphoid cell that is not incubated with exogenous mitochondria and mTOR inhibitors that are isolated.

[0122] As provided herein, in some embodiments, the lymphoid cells used to generate the mitochondrial replacement cells are senescent, and the mitochondrial replacement cells exhibit reduced senescence. Thus, measurement of the senescence-associated secretory phenotype (SASP) can serve as a functional assay. SASP includes increased secretion of inflammatory cytokines (e.g., interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNFα), growth factors, and proteases, as well as reduced and / or slower rate of cell population doubling, shortening of telomeres, increased DNA damage response (DDR), or combinations thereof. FACS analysis for senescence markers (e.g., CD57 / KIR / KLRG1) can also be used. Thus, in some embodiments, the mitochondrial replacement lymphoid cells generated according to the methods provided herein have reduced senescence compared to lymphoid cells that have not been incubated with isolated exogenous mitochondria and an mTOR inhibitor.

[0123] In some embodiments, the lymphoid cells used to generate mitochondrial replacement cells are exhausted T cells, and the mitochondrial replacement cells show reduced T cell exhaustion. FACS analysis for exhaustion markers (e.g., PD-1 / TIM3 / LAG3) can be used to measure T cell exhaustion. Thus, in some embodiments, the mitochondrial replacement lymphoid cells generated according to the methods provided herein have reduced exhaustion compared to lymphoid cells that are not incubated with isolated exogenous mitochondria and mTOR inhibitors. 7. Embodiment

[0124] The present invention provides the following non-limiting embodiments.

[0125] A1 1. A method for generating a mitochondrial-replaced lymphoid cell in which at least 20% of the endogenous mitochondrial DNA (mtDNA) is replaced by exogenous mtDNA, comprising: incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and an effective amount of a mammalian target of rapamycin (mTOR) inhibitor for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrially-replaced lymphoid cells in which at least 20% of the endogenous mtDNA has been replaced by exogenous mtDNA. A method comprising:

[0126] A2 The method of embodiment A1, wherein the mTOR inhibitor comprises rapamycin or a derivative thereof.

[0127] A3 The method of embodiment A1 or embodiment A2, wherein the mTOR inhibitor is rapamycin.

[0128] A4 The method of any one of embodiments A1 to A3, wherein the effective amount of the mTOR inhibitor is at a concentration of about 100 nM to about 1000 nM.

[0129] A5 The method of any one of embodiments A1 to A3, wherein the effective amount of the mTOR inhibitor is at a concentration of about 200 nM to about 500 nM.

[0130] A6 The method of any one of embodiments A1 to A3, wherein the effective amount of the mTOR inhibitor is at a concentration of about 100 nM.

[0131] A7 The method of any one of embodiments A1 to A3, wherein the effective amount of the mTOR inhibitor is at a concentration of about 200 nM.

[0132] A8 The method of any one of embodiments A1 to A3, wherein the effective amount of the mTOR inhibitor is at a concentration of about 500 nM.

[0133] A9 The method of any one of embodiments A1 to A3, wherein the effective amount of the mTOR inhibitor is at a concentration of about 1000 nM.

[0134] A10 The method of any one of embodiments A1 to A9, wherein the mitochondrial replaced lymphoid cells comprise at least 20% exogenous mtDNA and 80% or less endogenous mtDNA as measured by TaqMan single nucleotide polymorphism (SNP) assay.

[0135] A11 1. A method for generating mitochondrial-replaced lymphoid cells, the method comprising: incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria with isolated exogenous mitochondria and about 100 nM to about 1000 nM rapamycin for a sufficient period of time to non-invasively transfer the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrial-replaced lymphoid cells.

[0136] A12 The isolated exogenous mitochondria are cultured at 1×10 cells. 6 The method of any one of embodiments A1 to A11, wherein the amount of protein is about 20 μg to 80 μg per unit.

[0137] A13 The method of any one of embodiments A1 to A12, further comprising centrifuging the lymphoid cells prior to the incubating step.

[0138] A14 The method of embodiment A13, wherein the centrifugation step is carried out at room temperature and 1,500 relative centrifugal force (RCF) for approximately 5 minutes.

[0139] A15 The method of any one of embodiments A1 to A14, wherein the sufficient period of time is at least about 24 hours.

[0140] A16 The method of any one of embodiments A1 to A14, wherein the sufficient period of time is at least 36 hours.

[0141] A17 The method of any one of embodiments A1 to A14, wherein the sufficient period of time is at least 48 hours.

[0142] A18 The method of any one of embodiments A1 to A14, wherein the sufficient period of time is about 2 days or longer.

[0143] A19 The method of any one of embodiments A1 to A14, wherein the sufficient period of time is about 7 days or longer.

[0144] A20 The method of any one of embodiments A1 to A14, wherein the sufficient period of time is from about 2 days to about 7 days.

[0145] A21 1. A method for generating mitochondrial-substituted lymphoid cells, comprising: (a) centrifuging lymphoid cells and isolated exogenous mitochondria under conditions sufficient to produce a cell pellet, wherein the lymphoid cells have not been subjected to a procedure that reduces or depletes endogenous mitochondria; (b) incubating the lymphoid cells with 100 nM to 1000 nM rapamycin for approximately 24 hours or longer, thereby generating mitochondrial-replaced lymphoid cells. A method comprising:

[0146] A22 The method of embodiment A21, wherein the incubating step is for approximately 7 days or longer.

[0147] A23 The method of embodiment A21, wherein the incubating step is for about 2 days to about 7 days.

[0148] A24 The method of any one of embodiments A1 to A23, wherein the lymphoid cell is a T cell, a B cell, a monocyte, a macrophage, a natural killer (NK) cell, or a granulocyte.

[0149] A25 The method of embodiment A24, wherein the lymphoid cell is a T cell.

[0150] A26 The method of embodiment A25, wherein the T cells comprise exhausted T cells, senescent T cells, or a combination thereof.

[0151] A27 The method of any one of embodiments A1 to A26, wherein the lymphoid cells are human lymphoid cells.

[0152] A28 A composition comprising an effective amount of mitochondrial-replaced lymphoid cells produced by the method of any one of embodiments A1 to A27, and a pharma- ceutically acceptable carrier.

[0153] A29 A method for ameliorating a symptom of mitochondrial complex III deficiency in a subject in need thereof, comprising administering to the subject a composition of embodiment A28.

[0154] A30 A method for treating an immune deficiency associated with heteroplasmic immune cells in a subject in need thereof, comprising administering to the subject the composition of embodiment A28.

[0155] A31 The method of embodiment A30, wherein the subject is receiving a reverse transcriptase inhibitor.

[0156] A32 The method of embodiment A31, wherein the subject has human immunodeficiency virus (HIV).

[0157] A33 The method of embodiment A31, wherein the subject has Hepatitis B virus (HBV).

[0158] A34 The method of any one of embodiments A29 to A33, wherein the subject is a human. EXAMPLES

[0159] 8. Working Example The examples in this section are offered by way of illustration and not by way of limitation. The following examples are presented as exemplary embodiments of the invention. The following examples should not be construed as limiting the broad scope of the invention. Example 1 8.1 Example 1: Methods and Compositions for Treating Mitochondrial Diseases and Other Diseases Associated with Heteroplasmy Using Lymphoid Cells

[0160] This example demonstrates that endogenous mitochondria and / or mtDNA in lymphocytes (e.g., T lymphocytes) can be replaced by exogenous mitochondria using rapamycin without prior depletion of endogenous mtDNA. 8.1.1 Materials and Methods

[0161] Isolation and cell culture of primary mouse T lymphocytes: All animal experiments were performed in accordance with the animal experiment guidelines issued by our institution's Animal Care and Use Committee (M2019-536). For isolation of mouse T lymphocytes, spleens were removed from mice after anesthetization and sacrifice. The spleens were washed with PBS (FUJIFILM Wako Pure Chemical Corp.), then ground and filtered to extract splenocytes. Mouse T cells were highly purified from splenocytes by immunomagnetic negative selection using the EasySep Mouse T Cell Isolation Kit (Veritas, Santa Clara, CA, USA) according to the manufacturer's recommendations. Isolated mouse T cells were cultured in Advanced RPMI1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, 20 mM L-glutamine (Thermo Fisher Scientific incorporated), and 20 μM recombinant human IL-2 (PeproTech, Rocky Hill, NJ, USA) and activated with Dynabeads mouse T-activator CD3 / CD28 (Thermo Fisher Scientific incorporated). Cells were incubated at 37°C in a humidified 5% CO2 incubator.

[0162] Isolation of mitochondria and transfer into mouse T cells: Mitochondria were isolated from B6 mouse embryonic fibroblasts (MEFs). Briefly, cells were harvested from culture dishes using homogenization buffer [HB; 20 mM HEPES-KOH (pH 7.4), 220 mM mannitol and 70 mM sucrose] containing a protease inhibitor mixture (Sigma-Aldrich, St. Louis, Missouri, USA). The cell pellet was resuspended in HB and incubated on ice for 5 min. Cells were disrupted by stabbing 10 times with a 27-gauge needle on ice. The homogenate was centrifuged twice (400×g, 4° C.; 5 min) to remove unbroken cells. Mitochondria were harvested by centrifugation (6000×g, 4° C.; 5 min) and resuspended in HB. The amount of isolated mitochondria was expressed as protein concentration using a Bio-Rad protein assay kit (Bio-Rad Laboratories, incorporated, Richmond, CA, USA). Isolated mitochondria were mixed with mouse T cells in standard medium and centrifuged at 1,500 g for 5 min at room temperature. The pellet was gently resuspended, and various concentrations of rapamycin were added and incubated at 37°C under 5% CO2 for 24 h.

[0163] Isolation and cell culture of human T cells: Heparinized venous blood was obtained from the median cubital vein according to standard procedures. Human peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood using density gradient centrifugation with 1.077g / ml Percoll (GE Healthcare Life Sciences, Buckinghamshire, England). Cells were cultured in TexMACS medium (Miltenyi Biotec) supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin (Thermo Fisher Scientific incorporated), 20μM IL-7, and 10μM IL-15 on cell culture plates coated with anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec). Cells were incubated at 37°C in a humidified 5% CO2 incubator.

[0164] Mitochondrial isolation and transfer into human T cells: An immortalized human endometrial gland-derived mesenchymal cell line, EPC100 (Japanese Collection of Research Bioresources Cell Bank, JCRB1538), was used as an exemplary donor of exogenous mitochondria for transfer into human primary T cells using the same protocol as in the mouse.

[0165] MtDNA sequencing: To purify mtDNA from genomic DNA (Jayaprakash AD. Nucleic Acids Res. 2015), exonuclease V (ExoV, New England Biolabs Ltd, Ipswich, MA, USA) was used to remove only nuclear DNA. Briefly, total DNA was extracted from cells using NucleoSpin Tissue (MACHEREY-NAGEL) and heated at 70 °C for 30 min to inactivate any remaining proteinase K. In the first digestion, the total DNA sample (4–8 μg in 35 μl) was added in the following steps: 10× New England Biolabs (NEB) 4 Buffer (6 μl), 10 mM ATP (12 μl), ExoV from NEB-M0345S (4 μl) and HO (3 μl). The digests were left at 37°C for 48 hours, heat inactivated at 70°C for 30 minutes, and purified using N Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA). In the second digestion, the following was added to the ExoV-treated DNA (35 μl): NEB4 10× Buffer (6 μl), 10 mM ATP (12 μl), ExoV from NEB-M0345S (4 μl), and H2O (3 μl). The digests were left at 37°C for 16 hours, heat inactivated at 70°C for 30 minutes, and purified using Wizard SV Gel and PCR Clean-Up System (Promega Corporation, Madison, WI, USA). These ExoV-treated mtDNAs were processed for deep sequencing. Sequencing data were generated as fastq format files. Unmapped reads were filtered for quality (sequences with more than 10 consecutive nucleotides with Q<20 were excluded), mapped to the reference mitochondrial genome (GRCh38), and converted to BAM files. These files were analyzed for heteroplasmy using mtDNA-server (mtdna-server.uibk.ac.at / index.html).

[0166] TaqMan Single Nucleotide Polymorphism (SNP) Assay: Based on the differences in mitochondrial HVR1 sequences between human GJ T cells and EPC100 cells, and the differences in mitochondrial ND1 between B6 and NZB mice, the probes and primer sets listed in Tables 1 and 2 were designed. These provide sensitivity and specificity in the TaqMan SNP assay. To determine the ratio of mutations, wild-type allele-specific TaqMan probes and mutant allele-specific TaqMan probes were designed for the TaqMan SNP assay. The extracted DNA (1 ng) was used for quantitative PCR using TaqMan Universal PCR Master Mix kit (Thermo Fisher Scientific Incorporated) on a CFX connect real-time system (Bio-Rad Laboratories, Incorporated) under the following conditions: initial denaturation (95°C for 10 min), followed by 40 cycles of PCR (95°C for 15 s and 60°C for 1 min). A calibration curve was made using the known CN of the plasmid containing the amplified mtDNA fragment for the target. The mtDNA copy number was estimated from the ratio of 12S rRNA content on mtDNA to ACTB (or Actb) content on nuclear DNA by relative quantification based on delta cycle threshold.

[0167] Table 1 [Table 1]

[0168] Table 2 [Table 2] 8.1.2 Results

[0169] As shown in Figure 1, differences between the mtDNA sequences derived from human GJ T cells and EPC100 cells were detected by sequencing the D-loop, and some differences were observed in the D-loop hypervariable region ("HVR"). Based on the differences in the D-loop HVR, a set of probes and primers was designed for use in an SNP assay to detect mitochondria replacement (Figure 2A). The assay was validated by using a cell population of human GJ T cells and EPC100 cells at a given mixing ratio before the experimental sample as a test sample. The assay demonstrated an accurate ratio of genotypes according to the mixing ratio of the cell populations (data not shown). Thus, the TaqMan probe allows us to easily detect and distinguish between mtDNA derived from GJ T cells and mtDNA derived from EPC100 cells. Thus, the SNP assay is a useful tool to identify mtDNA from two different sources and to be able to distinguish between them.

[0170] Human GJ T cells were cultured in vitro for 2 days in TexMacs with 5% FBS and IL-7 and IL-15. Mitochondrial transfer was performed by culturing human GJ T cells in the absence of rapamycin or in the presence of various concentrations of rapamycin (50 nM, 100 nM, 200 nM, or 500 nM) with or without mitochondria from EPC100 cells ("Day 0"). 24 hours after culture in the presence of rapamycin, 50% of the medium was replenished with TexMacs with 5% FBS and IL-7 and IL-15, and no further rapamycin was added. After changing the medium, cells were cultured for an additional 1 or 6 days, for a total of 2 or 7 days after transfer of all mitochondria.

[0171] Two and seven days after culturing human GJ T cells with mitochondria derived from EPC100 cells, SNP assays were performed to detect the transfer of mitochondria from EPC100 cells to human GJ T cells. See Figure 3A for an outline of the protocol. As shown in Figure 3B, mtDNA derived from EPC100 cells was detected in human GJ T cells cultured for two and seven days in the presence of 50-500 nM rapamycin, whereas mtDNA derived from EPC100 cells was not detected in human GJ T cells cultured without EPC100 mitochondria. The mtDNA content in T cells contacted with donor mitochondria in the presence of rapamycin consisted of 40-50% mtDNA derived from EPC100 cells, and the amount of donor mtDNA increased with increasing amounts of rapamycin. For example, the portion of donor mtDNA detected in T cells after contact with donor mitochondria in the presence of 50 nM rapamycin was less than 40% after 7 days, whereas the portion of donor mtDNA detected in T cells after contact with donor mitochondria in the presence of 500 nM rapamycin was approximately 50% after 7 days. Thus, when T cells were cultured under rapamycin, mitochondrial depletion was not required to achieve successful transfer of mtDNA into T cells.

[0172] 3 x 10 per well 6Mouse NZB T cells were cultured in Advanced RPMI with 10% FBS and IL-2 / CD3 / CD28 with 40 μg mitochondria from B6 MEFs in the absence or presence of various concentrations of rapamycin (100 nM, 200 nM, 500 nM or 1000 nM) without depletion of NZB T cell mtDNA. B6 T cells used as mitochondrial donors have 91 polymorphisms in their mtDNA compared to mtDNA from current inbred laboratory mice, including B6 T cells. After 24 hours of incubation with rapamycin, 50% of the medium was replenished with Advanced RPMI with 10% FBS and IL-2 / CD3 / CD28, without further rapamycin. Cells were cultured for a total of 2 or 7 days before assessing mitochondrial replacement.

[0173] Two and seven days after cells were cultured with B6 MEF mitochondria in the presence or absence of rapamycin, SNP assays were performed to detect replacement of NZB T cell mtDNA with B6 MEF mtDNA. See Figure 4A for a scheme showing the protocol and Figure 4B for a depiction of the titration of rapamycin in the wells. As shown in Figures 5A and 5B, increased replacement of NZB T cell mtDNA was detected after culture with rapamycin, whereas no replacement of NZB T cell mtDNA was detected without rapamycin. For example, after centrifugation and 2 days of rapamycin treatment, approximately 45-80% donor mtDNA was present in the recipient T cells (Figure 5A). By day 7, the amount of donor mtDNA in the recipient T cells increased to approximately 70-90% (Figure 5B). The amount of replacement of NZB T cell mtDNA with B6 MEF mtDNA increased in a concentration-dependent manner. Furthermore, the amount of B6 MEF mtDNA in NZB T cells at day 7 was higher than at day 2.

[0174] Taken together, the results demonstrate that mitochondrial transfer can be facilitated by simultaneous rapamycin treatment during contact of T cells with donor mitochondria. Thus, this example demonstrates the successful provision of mitochondrial replacement in T cells using a protocol involving rapamycin, without endonucleolytic reduction of endogenous mtDNA, and without genetic manipulation.

[0175] The embodiments described above are intended to be merely illustrative; those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific compounds, materials, and procedures. All such equivalents are considered to be within the scope of the invention and are encompassed by the appended claims.

Claims

**Claim 1** A method for generating mitochondrially replaced lymphoid cells in which at least 20% of the endogenous mitochondrial DNA (mtDNA) is replaced by exogenous mtDNA, comprising: incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria for a sufficient period of time with isolated exogenous mitochondria and an effective amount of a mammalian mammalian target of rapamycin (mTOR) inhibitor, thereby non-invasively transferring the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrially replaced lymphoid cells in which at least 20% of the endogenous mtDNA is replaced by exogenous mtDNA A method comprising: **Claim 2** The method according to claim 1, wherein the mTOR inhibitor comprises rapamycin or a derivative thereof. **Claim 3** The method according to claim 1, wherein the mTOR inhibitor is rapamycin. **Claim 4** The method according to claim 1, wherein the effective amount of the mTOR inhibitor is at a concentration of about 100 nM to about 1000 nM. **Claim 5** The method according to claim 1, wherein the effective amount of the mTOR inhibitor is at a concentration of about 200 nM to about 500 nM. **Claim 6** The method according to claim 1, wherein the effective amount of the mTOR inhibitor is at a concentration of about 100 nM. **Claim 7** The method according to claim 1, wherein the effective amount of the mTOR inhibitor is at a concentration of about 200 nM. **Claim 8** The method according to claim 1, wherein the effective amount of the mTOR inhibitor is at a concentration of about 500 nM. **Claim 9** The method according to claim 1, wherein the effective amount of the mTOR inhibitor is at a concentration of about 1000 nM. **Claim 10** The method according to claim 1, wherein the mitochondrially replaced lymphoid cells contain at least 20% exogenous mtDNA and 80% or less endogenous mtDNA as measured by TaqMan single nucleotide polymorphism (SNP) assay. **Claim 11** A method for generating mitochondrially replaced lymphoid cells, comprising: incubating lymphoid cells that have not been subjected to a procedure to reduce or deplete endogenous mitochondria for a sufficient period of time with isolated exogenous mitochondria and about 100 nM to about 1000 nM rapamycin, thereby non-invasively transferring the exogenous mitochondria into the lymphoid cells, thereby generating mitochondrially replaced lymphoid cells.

12. The isolated exogenous mitochondria have about 20 μg to 80 μg of protein per 1 × 10 ６ cells, and the method according to claim 1 or claim 11.

13. The method according to claim 1 or claim 11, further comprising centrifuging the lymphoid cells before the incubating step.

14. The method according to claim 13, wherein the centrifuging step is carried out at room temperature at 1,500 relative centrifugal force (RCF) for approximately 5 minutes.

15. The method according to claim 1 or claim 11, wherein the sufficient period is at least approximately 24 hours.

16. The method according to claim 1 or claim 11, wherein the sufficient period is at least 36 hours.

17. The method according to claim 1 or claim 11, wherein the sufficient period is at least 48 hours.

18. The method according to claim 1 or claim 11, wherein the sufficient period is approximately 2 days or longer.

19. The method according to claim 1 or claim 11, wherein the sufficient period is approximately 7 days or longer.

20. The method according to claim 1 or claim 11, wherein the sufficient period is from approximately 2 days to approximately 7 days.

21. A method for generating mitochondria-replaced lymphoid cells, comprising: (a) centrifuging lymphoid cells and isolated exogenous mitochondria under conditions sufficient to produce a cell pellet, wherein the lymphoid cells have not been subjected to a procedure to reduce or deplete endogenous mitochondria; and (b) incubating the lymphoid cells with rapamycin at 100 nM to 1000 nM for approximately 24 hours or longer, thereby generating mitochondria-replaced lymphoid cells. The method comprising the steps.

22. The method according to claim 21, wherein the incubating step is approximately 7 days or longer.

23. The method according to claim 21, wherein the incubating step is from approximately 2 days to approximately 7 days.

24. The method according to claim 1, claim 11 or claim 21, wherein the lymphoid cells are T cells, B cells, monocytes, macrophages, natural killer (NK) cells, or granulocytes.

25. The method according to claim 24, wherein the lymphoid cells are T cells.

26. The method according to claim 25, wherein the T cells include exhausted T cells, senescent T cells, or a combination thereof. Claim 27 The method according to claim 1, claim 11 or claim 21, wherein the lymphocytic cells are human lymphocytic cells. Claim 28 A composition comprising an effective amount of mitochondrial replacement lymphocytic cells generated by the method according to claim 1, claim 11 or claim 21 and a pharmaceutically acceptable carrier. Claim 29. The composition according to claim 28, for ameliorating the symptoms of mitochondrial complex III deficiency in a subject in need thereof. Claim 30. The composition according to claim 28, for treating immunodeficiency associated with heteroplasmic immune cells in a subject in need thereof. Claim 31 The composition according to claim 30, wherein the subject is receiving a reverse transcriptase inhibitor. Claim 32 The composition according to claim 31, wherein the subject has human immunodeficiency virus (HIV). Claim 33 The composition according to claim 31, wherein the subject has hepatitis B virus (HBV). Claim 34 The composition according to claim 29, wherein the subject is human. Claim 35. The composition according to claim 30, wherein the subject is human.

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