Methods and compositions for reducing immune cell exhaustion using mitochondrial replacement

Abstract

The present disclosure provides methods and compositions for generating mitochondrial-replaced T cells from exhausted T cells, comprising reducing exhausted T cell mitochondrial DNA (mtDNA) and incubating with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells in which expression of at least one exhaustion marker is altered by at least 5%, at least 10%, 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more, and the mitochondrial-replaced T cells have improved effector function compared to the exhausted T cells. Additionally, the present disclosure also provides methods for treating or reversing age-related diseases (e.g., cancer or autoimmune diseases) and reversing symptoms of chronic infections (e.g., chronic viral infections) involving administration of mitochondrial-replaced T cells.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 190,115, 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 14595-009-228_SL.txt, and is 2,410 bytes in size.

[0003] 2. Field The present disclosure relates, in part, to methods and compositions for reducing immune cell exhaustion using mitochondrial replacement. In a specific aspect, the present disclosure relates to a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (e.g., a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the exhausted T cells with reduced mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells, such that expression of exhaustion markers (e.g., programmed cell death-1 (PD-1)) in the mitochondrial-replaced T cells is reduced by at least 5%, at least 10%, at least 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more, compared to exhausted T cells. The disclosure also relates to therapeutic methods for treating diseases (e.g., cancer) and chronic viral infections using such mitochondrial-replaced T cells. [Background technology]

[0004] 3.Background T cell exhaustion commonly accompanies a state of T cell dysfunction resulting from chronic antigen stimulation, for example in the context of chronic viral infections and cancer (see Wherry, E. (2011) Nat Immunol 12, 492-499). Hallmarks of T cell exhaustion include insufficient effector function, persistent expression of inhibitory receptors, and / or a transcriptional state distinct from functional effector or memory T cells. Exhaustion prevents optimal control of infections and tumors.

[0005] Cancer immunotherapy has recently emerged as a promising strategy to support and strengthen the ability of a patient's immune system to attack tumors. One approach to immunotherapy involves harvesting and engineering a patient's own T cells to treat the patient's cancer. Two exemplary approaches to engineering T cells include engineering the patient's T cells to express a specific T cell receptor (TCR) or engineering the T cells to express a chimeric antigen receptor (CAR). TCRs use naturally occurring receptors that can also recognize antigens present inside tumor cells. These pieces of antigen are shuttled to the cell surface and "presented" to the immune system as part of a collection of proteins called the MHC complex. CARs, on the other hand, contain portions of antibodies that can recognize specific antigens on the surface of cancer cells. Despite their potential as a treatment, CAR-T cells can also become exhausted and require immune checkpoint blockade so that their functionality can be restored. T cell exhaustion has also been reported in various human chronic viral infections, such as human immunodeficiency virus (HIV), hepatitis B (HBV), and hepatitis C (HCV). T cell exhaustion also occurs under conditions of antigen persistence, more commonly occurring during certain non-viral infections, such as malaria and Mycobacterium tuberculosis. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Wherry, E. (2011) Nat Immunol 12, 492-499 Summary of the Invention [Means for solving the problem]

[0007] Thus, there is a significant unmet need to reduce and / or reverse T cell exhaustion in settings of chronic antigen stimulation.

[0008] 4. Overview In one aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells with reduced mitochondrial DNA (mtDNA) with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having reduced expression of one, two or more markers of T cell exhaustion (e.g., PD-1 expression) relative to expression of one, two or more markers of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In a specific embodiment, reduced expression of one, two or more markers of T cell exhaustion by the mitochondrial-replaced T cells is indicative of one, two or more improved effector functions of the T cells. In specific embodiments, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising incubating exhausted T cells having a reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having at least 5%, at least 10%, 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more reduced expression of programmed cell death-1 (PD-1) relative to expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived.In another embodiment, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising: (a) electroporating the exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having programmed cell death-1 (PD-1) expression that is at least 5%, at least 10%, at least 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In another embodiment, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with at least 5%, at least 10%, at least 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more reduced expression of programmed cell death-1 (PD-1) compared to expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In some embodiments, the nucleic acid sequence is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence is DNA (e.g., cDNA).

[0009] In a specific aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells having a reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with improved effector function relative to the exhausted T cells. In a specific embodiment, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells having a reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with at least 5%, at least 10%, at least 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more reduced expression of programmed cell death-1 (PD-1) relative to expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived, wherein the mitochondrial-replaced T cells have improved effector function relative to the exhausted T cells. In some embodiments, the nucleic acid sequence is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence is DNA (e.g., cDNA).

[0010] In another embodiment, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the exhausted T cells having reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with improved effector function relative to the exhausted T cells. In specific embodiments, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in specific embodiments, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the exhausted T cells having reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having expression of PD-1 reduced by at least 5%, at least 10%, at least 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more compared to expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived, wherein the mitochondrial-replaced T cells have improved effector function compared to the exhausted T cells. In some embodiments, the nucleic acid sequence is RNA (e.g., mRNA). In some embodiments, the nucleic acid sequence is DNA (eg, cDNA).

[0011] In some embodiments, PD-1 expression is reduced by about 5% to about 60%, about 10% to about 50%, about 20% to about 50%, about 10% to about 40%, about 10% to about 30%, about 10% to about 20%, about 20% to about 40%, or about 20% to about 30% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived.

[0012] In certain embodiments, incubation of exhausted T cells with isolated exogenous mitochondria is performed in the presence of rapamycin. In specific embodiments, rapamycin is present at a concentration of 100 nM to 1000 nM.

[0013] In certain embodiments, the expression of PD-1 by the mitochondria-replaced T cells is at most 1.1-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the expression of PD-1 by the mitochondria-replaced T cells is at most 1.15-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the expression of PD-1 by the mitochondria-replaced T cells is at most 1.20-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the expression of PD-1 by the mitochondria-replaced T cells is at most 1.25-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the expression of PD-1 by the mitochondria-replaced T cells is at most 1.3-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is at most 1.4-fold reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is at most 1.5-fold reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is at most 2-fold reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is at most 5-fold reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is at least 5% reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is at most 10% reduced compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells were derived.In certain embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by at most 15% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by at most 20% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by at most 25% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by at most 30% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by at most 35% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by at most 40% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by at most 45% compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by at most 50% compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by at most 60% compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 5% to about 60% compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 10% to about 50% compared to PD-1 expression by the exhausted T cells from which the mitochondrial-replaced T cells are derived.In some embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by about 20% to about 50% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by about 10% to about 40% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by about 10% to about 30% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by about 10% to about 20% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, the expression of PD-1 by the mitochondrial-replaced T cells is reduced by about 10% to about 30% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 20% to about 40% compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 10% to about 30% compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 20% to about 30% compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 1.1-fold to about 2.0-fold compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 1.1-fold to about 1.75-fold compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 1.1-fold to about 1.5-fold compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 1.1-fold to about 1.25-fold compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 1.25-fold to about 2.0-fold compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondrial-replaced T cells is reduced by about 1.25-fold to about 5.0-fold compared to PD-1 expression by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression by the mitochondria-replaced T cells is approximately 1.25- to approximately 1.5-fold reduced compared to PD-1 expression by the exhausted T cells from which the mitochondria-replaced T cells are derived.

[0014] In some embodiments, the method reduces expression of PD-1, T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), lymphocyte activation gene 3 (LAG3), T-cell immunoglobulin and ITIM domain (TIGIT), TOX, or a combination thereof.

[0015] In certain embodiments, isolated exogenous mitochondria are administered to exhausted T cells at a concentration of 1×10 6 Each individual contains approximately 20 μg to 80 μg of protein.

[0016] In some embodiments, the mitochondrial-replaced T cells contain at least 20% exogenous mtDNA. In certain embodiments, the mitochondrial-replaced T cells contain at least 20% exogenous mtDNA and no more than 80% exogenous mtDNA as measured by TaqMan single nucleotide polymorphism (SNP) assay.

[0017] In a specific embodiment, the period of time sufficient for the generation of mitochondrial-replaced T cells is at least about 24 hours. In a specific embodiment, the period of time sufficient for the generation of mitochondrial-replaced T cells is at least 36 hours. In some embodiments, the period of time sufficient for the generation of mitochondrial-replaced T cells is at least 48 hours. In a specific embodiment, the period of time sufficient for the generation of mitochondrial-replaced T cells is about 24 hours to about 48 hours, about 36 hours to 48 hours, about 24 hours to about 72 hours, or about 48 hours to about 72 hours.

[0018] In some embodiments, improved effector function includes one, two or more, or all of the following: increased proliferation, increased cytotoxicity, or increased secretion of cytokines.

[0019] In certain embodiments, the exhausted T cells comprise an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In a specific embodiment, the exhausted T cells are genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0020] In another aspect, provided herein are mitochondrial-replaced T cells produced by the methods described herein, and compositions comprising such cells.

[0021] In another aspect, provided herein is a composition comprising an effective amount of the mitochondrial-replaced T cells of the present disclosure and a pharma- ceutically acceptable carrier.

[0022] In another aspect, provided herein is a method for reversing symptoms of a chronic viral infection in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of the mitochondrial-replaced T cells of the present disclosure and a pharma- ceutically acceptable carrier. In certain embodiments, the chronic viral infection is a human immunodeficiency virus (HIV) infection, a hepatitis B virus (HBV) infection, a cytomegalovirus infection (CMV), or a severe acute respiratory syndrome (SARS)-coronavirus (CoV)-2 infection. In a specific embodiment, the subject is a human.

[0023] In another aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising an effective amount of the mitochondrial-replaced T cells of the present disclosure and a pharma- ceutically acceptable carrier. In a specific embodiment, the subject is a human.

[0024] In another aspect, provided herein is a composition comprising an effective amount of mitochondrial-replaced T cells comprising an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR) and a pharma- ceutically acceptable carrier. In a specific embodiment, the mitochondrial-replaced T cells are genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0025] In another aspect, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject a composition comprising mitochondrial-replaced T cells as described herein, comprising an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and a pharma- ceutically acceptable carrier. In another aspect, provided herein is a method for improving cancer symptoms in a subject in need thereof, comprising administering to the subject a composition comprising mitochondrial-replaced T cells as described herein, comprising an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and a pharma-ceutically acceptable carrier. In a specific embodiment, the subject is a human.

[0026] In another aspect, provided herein is a method for treating a disease or condition associated with, accompanied by, or caused by T cell exhaustion in a subject in need thereof, comprising administering to the subject a composition comprising mitochondrial-replaced T cells as described herein, comprising an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR), wherein the disease or condition is (a) cancer; (b) viral infection (e.g., a chronic viral infection); (c) bacterial infection; (d) obesity or a metabolic disorder; (e) alcoholism; (f) hyperactivity; (g) excessive mental stress; (h) hypoxia; (i) injury; (j) aging; (k) age-related immunological dysfunction; (l) fibrotic disease; (m) macular disease; (n) muscular degenerative disease; or (o) neurodegenerative disease. In another aspect, provided herein is a method for reversing symptoms of a disease or condition associated with, accompanied by, or caused by T cell exhaustion in a subject in need thereof, comprising administering to the subject a composition comprising mitochondrial-replaced T cells as described herein, comprising an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR), wherein the disease or condition is (a) cancer; (b) viral infection (e.g., chronic viral infection); (c) bacterial infection; (d) obesity or metabolic disorder; (e) alcoholism; (f) hyperactivity; (g) excessive mental stress; (h) hypoxia; (i) injury; (j) aging; (k) age-related immunological dysfunction; (l) fibrotic disease; (m) macular disease; (n) muscular degenerative disease; or (o) neurodegenerative disease. In a specific embodiment, the subject is a human.

[0027] In another aspect, provided herein is a method for treating a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, comprising administering to the subject a composition comprising mitochondrial-replaced T cells as described herein, wherein the disease or condition is (a) CD8+ T cell dysfunction; (b) CD4+ T cell dysfunction; (c) T cell priming dysfunction; (d) memory T cell dysfunction; (e) effector B cell dysfunction; (f) B cell priming dysfunction; (g) memory B cell dysfunction; (h) congenital lymphoid cell dysfunction; (i) congenital T cell dysfunction; (j) congenital B cell dysfunction, or (k) a combination of (a)-(j). In another aspect, provided herein is a method for ameliorating a symptom of a disease or condition related to or involving: (a) CD8+ T cell dysfunction; (b) CD4+ T cell dysfunction; (c) impaired T cell priming; (d) impaired memory T cell dysfunction; (e) impaired effector B cell dysfunction; (f) impaired B cell priming; (g) impaired memory B cell dysfunction; (h) impaired lymphoid cell function; (i) impaired congenital T cell function; (j) impaired congenital B cell function; or (k) a combination of (a)-(j). In a specific embodiment, the subject is a human. 5. Brief description of the drawings [Brief description of the drawings]

[0028] [Figure 1] Scheme of an experimental system illustrating the modulation of human T cell exhaustion. Co-culture of ephrin type B receptor 4 (EPHB4)-specific chimeric antigen receptor (CAR) T cells with Rh30, a human rhabdomyosarcoma cell that constitutively expresses EPHB4, for 3 days increased the exhaustion marker PD-1. On day 3, endogenous mitochondrial DNA was depleted in CAR-T cells using XbaIR mRNA. On day 5, normal human derived fibroblast (NHDF)-derived donor mitochondria were co-cultured with CAR-T cells to generate mitochondrial replacement (Mir) CAR-T cells (Mir CAR-T cells). On day 7, FACS and TaqMan SNP assays were performed.

[0029] [Diagram 2] 12S rRNA, a surrogate marker for mtDNA content, was halved on day 3, confirming that XbaIR caused effective mtDNA reduction (Figure 2A), and TaqMan SNP assays were performed on day 7, showing that Mir CAR-T cells contained approximately 40% NHDF-derived donor mtDNA, while exhausted and parental CAR-T cells (referred to as YG cells) were indistinguishable and contained negligible amounts of NHDF-derived donor mtDNA (Figure 2B). Abbreviations: electroporation (EP); normal human dermal fibroblasts (NHDF).

[0030] [Figure 3-1] FACS analysis of CD3 / PD-1 expression in unstained CAR-T cells (Figure 3A), control CAR-T cells (Figure 3B), and Mir CAR-T cells (Figure 3C) on day 7. The x-axis is PD-1 and the y-axis is CD3. [Figure 3-2] Same as above. [Figure 3-3] Same as above.

[0031] [Figure 4-1] PD-1 antigen expression analysis on day 2 (Figure 4A) and mean fluorescence intensity (MFI) bar graph illustrating reduced PD-1 antigen expression in Mir CAR-T cells compared to parental exhausted CAR-T cells (Figure 4B). [Figure 4-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] 6. Detailed Description Provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising incubating exhausted T cells having a reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having at least 5%, at least 10%, at least 20% (e.g., at most 1.25-fold), at least 30%, at least 40%, at least 50%, at least 60%, or more reduced expression of a marker of T cell exhaustion (e.g., programmed cell death-1 (PD-1)) compared to expression of a marker of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells were derived. The mitochondrial-replaced T cells disclosed herein have utility as a therapy, for example, to ameliorate symptoms of chronic viral infection or symptoms of cancer.

[0033] As used herein, the term "mitochondrial replacement T cells" is generally intended to mean T cells in which endogenous mitochondria and / or endogenous mtDNA have been replaced by exogenous mitochondria and / or exogenous mtDNA. In certain embodiments, mitochondrial replacement T cells are those in which all endogenous mitochondria and / or endogenous mtDNA in T cells have been replaced by exogenous mitochondria and / or exogenous mtDNA. In specific embodiments, mitochondrial replacement T 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. In specific embodiments, mitochondrial replacement T cells are those in which a certain percentage of endogenous mtDNA has been replaced by exogenous mtDNA. In some embodiments, a mitochondrial-replaced T 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 T cell have been replaced with exogenous mitochondria and / or exogenous mtDNA.In certain embodiments, mitochondrial-replaced T cells have between about 5% and about 10%, between about 10% and about 20%, between about 10% and about 30%, between about 10% and about 40%, between about 20% and about 40%, between about 25% and about 50%, between about 25% and about 75%, between about 50% and about 75%, between about 40% and about 50%, between about 75% or more and about 85%, between about 75% and about 95%, between about 30% and about 90%, between about 40% and about 50%, between about 50% and about 60%, between about 60% and about 75%, between about 75% and about 95%, between about 80% and about 90%, between about 90% and about 100%, between about 100% and about 150%, between about 150% and about 200%, between about 200% and about 300%, between about 300% and about 400%, between about 400% and about 500%, between about 50% and about 600%, between about 50% and about 600%, between about 50% and about 75%. 0% to about 90%, about 50% to about 90%, about 30% to about 85%, about 40% to about 85%, about 50% to about 85%, about 30% to about 80%, about 40% to about 80%, about 50% to about 80%, about 30% to about 75%, about 40% to about 75%, about 50% to about 75%, about 60% to about 90%, about 60% to about 85%, about 60% to about 80%, about 60% to about 75% is replaced by exogenous mitochondria and / or exogenous mtDNA. In some embodiments, a mitochondrial-replaced T 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 T cell have been replaced with exogenous mitochondria and / or exogenous mtDNA.

[0034] 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.

[0035] 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., T cells) enriched from other cell types (e.g., non-T cells), and substantially pure cells (e.g., T 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 cell is isolated using the techniques described in the Examples below.

[0036] As used herein, the term "exogenous" is generally 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] As used herein, the term "sufficient period of time" generally refers to the amount of time over which a desired result or results.

[0041] 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.

[0042] As used herein, the term "XbaIR" has the typical meaning of being the restriction endonuclease XbaI that recognizes and cleaves the following sequence of DNA: [ka]

[0043] As used herein, the term "substantially free" is generally intended to mean an amount at or near the threshold of detection in an appropriate assay (e.g., PCR for nucleotides, or immunoassay for proteins). When the term is used in reference to XbaIR or nucleotides encoding XbaI (e.g., a fusion protein comprising MTS and XbaIR), it is generally intended to mean an amount that does not interfere with replacement of mtDNA.

[0044] 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 Methods for generating mitochondrial-replaced T cells from exhausted T cells

[0045] The present disclosure is based in part on the discovery that the introduction of exogenous mitochondria into exhausted T cells can reduce T cell exhaustion phenotype. Furthermore, the present disclosure is based in part on the discovery that the introduction of exogenous mitochondria into exhausted T cells can improve the effector function of exhausted T cells. Thus, in one aspect, a method is provided herein for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with altered (e.g., reduced or increased) levels of at least one marker of T cell exhaustion (e.g., PD-1 expression or IL-2 secretion) compared to the levels of at least one marker of T cell exhaustion by exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one marker of T cell exhaustion is increased by at least 0.1-fold or reduced by 0.1-fold compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.2-fold or reduced by 0.2-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.3-fold or reduced by 0.3-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.4-fold or reduced by 0.4-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.5-fold or reduced by 0.5-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.6-fold or reduced by 0.6-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased by at least 0.7-fold or reduced by 0.7-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.8-fold or reduced by 0.8-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.9-fold or reduced by 0.9-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.0-fold or reduced by 1.0-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.1-fold or reduced by 1.1-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.15-fold or reduced by 1.15-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.20-fold or reduced by 1.20-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.25-fold or reduced by 1.25-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.3-fold or reduced by 1.3-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.4-fold or reduced by 1.4-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.5-fold or decreased by 1.5-fold compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased or decreased by at least 1.75-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased or decreased by at least 2-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 2.5-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased or decreased by at least 3-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 4-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased or decreased by at least 5-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.2-fold to about 1.25-fold, or an increase of about 0.2-fold to about 1.25-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.2-fold to about 1.0-fold, or an increase of about 0.2-fold to about 1.0-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.2-fold to about 0.8-fold, or an increase of about 0.2-fold to about 0.8-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.2-fold to about 0.6-fold, or an increase of about 0.2-fold to about 0.6-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.8-fold to about 1.25-fold, or an increase of about 0.8-fold to about 1.25-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.5-fold to about 1.25-fold, or an increase of about 0.5-fold to about 1.25-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an increase of about 0.8-fold to about 1.0-fold, or an increase of about 0.8-fold to about 1.0-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold, or decreased by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, the levels of two, three, or more markers of T cell exhaustion in the mitochondria-replaced T cells are altered compared to the levels of two, three, or more markers by the exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, the levels of one, two, three, or more markers of T cell exhaustion by the mitochondria-replaced T cells are altered compared to the exhausted T cells from which the mitochondria-replaced T cells are derived, indicating one, two, or more improved effector functions of the T cells. In certain embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the RNA level compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the protein level compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the RNA and protein levels compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, the methods provided herein for generating mitochondrial-replaced T cells are performed in vitro or ex vivo.

[0046] Thus, in one aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with altered (e.g., reduced or increased) levels of at least one marker of T cell exhaustion (e.g., PD-1 expression or IL-2 secretion) compared to the level of at least one marker of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 5% compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 10% compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 15% compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 20% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 25% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 30% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 35% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 40% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by approximately 45% compared to exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased or decreased by at least 50% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 60% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 70% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 80% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 90% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 95% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 100% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 200% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 300% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 400% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 500% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased or decreased by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In a specific embodiment, the levels of two, three or more markers of T cell exhaustion are altered in the mitochondria-replaced T cells compared to the levels of two, three or more markers by the exhausted T cells from which the mitochondria-replaced T cells are derived. In a specific embodiment, the levels of one, two, three or more markers of T cell exhaustion are altered by the mitochondria-replaced T cells compared to the exhausted T cells from which the mitochondria-replaced T cells are derived, thereby indicating one, two or more improved effector functions of T cells. In a specific embodiment, the levels of one, two, three or more markers of T cell exhaustion are altered at the RNA level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the protein level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In a specific embodiment, the levels of one, two, three or more markers of T cell exhaustion are altered at the RNA level and the protein level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the methods provided herein for generating mitochondrial-replaced T cells are performed in vitro or ex vivo.

[0047] In another aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising: (a) transfecting or transforming exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the mitochondrial DNA (mtDNA)-reduced exhausted T cells with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having altered (e.g., reduced or increased) levels of at least one marker of T cell exhaustion (e.g., PD-1 expression or IL-2 secretion) compared to the level of at least one marker of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 0.1-fold compared to the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.2-fold or reduced by 0.2-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.3-fold or reduced by 0.3-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.4-fold or reduced by 0.4-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.5-fold or reduced by 0.5-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.6-fold or reduced by 0.6-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased by at least 0.7-fold or reduced by 0.7-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.8-fold or reduced by 0.8-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.9-fold or reduced by 0.9-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.0-fold or reduced by 1.0-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.1-fold or reduced by 1.1-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.15-fold or reduced by 1.15-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.20-fold or reduced by 1.20-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.25-fold or reduced by 1.25-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.3-fold or reduced by 1.3-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.4-fold or reduced by 1.4-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.5-fold or decreased by 1.5-fold compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In some embodiments, at least one exhaustion marker is increased at least 2-fold or reduced at most 2-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased at least 2.5-fold or reduced at most 2.5-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased at least 3-fold or reduced at most 3-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased at least 5-fold or reduced at most 5-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased about 0.2-fold to about 1.25-fold or reduced about 0.2-fold to about 1.25-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by about 0.2-fold to about 1.0-fold or reduced by about 0.2-fold to about 1.0-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by about 0.2-fold to about 0.8-fold or reduced by about 0.2-fold to about 0.8-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by about 0.2-fold to about 0.6-fold or reduced by about 0.2-fold to about 0.6-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by about 0.8-fold to about 1.25-fold or reduced by about 0.8-fold to about 1.25-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by about 0.5-fold to about 1.25-fold or decreased by about 0.5-fold to about 1.25-fold compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased by about 0.8-fold to about 1.0-fold, or decreased by about 0.8-fold to about 1.0-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold, or decreased by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived.

[0048] In another aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising: (a) transfecting or transforming exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the mitochondrial DNA (mtDNA)-reduced exhausted T cells with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having an altered (e.g., reduced or increased) level of at least one marker of T cell exhaustion (e.g., PD-1 expression or IL-2 secretion) compared to the level of at least one marker of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 5% compared to the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 10% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 15% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 20% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 25% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 30% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 35% compared to exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased or decreased by at least 40% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by approximately 45% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 50% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 60% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 70% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 80% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 90% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 95% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 100% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 200% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 300% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 400% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 500% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In some embodiments, at least one exhaustion marker is increased or decreased by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, the level of two, three or more markers of T cell exhaustion in the mitochondria-replaced T cells is altered compared to the level of two, three or more markers by the exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, the level of one, two, three or more markers of T cell exhaustion by the mitochondria-replaced T cells is altered compared to the exhausted T cells from which the mitochondria-replaced T cells are derived, indicating one, two or more improved effector functions of the T cells. In certain embodiments, the level of one, two, three or more markers of T cell exhaustion is altered at the RNA level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, the level of one, two, three or more markers of T cell exhaustion is altered at the protein level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the level of one, two, three or more markers of T cell exhaustion is altered at the RNA level and protein level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the method provided herein for generating mitochondria-replaced T cells is carried out in vitro or ex vivo.

[0049] In a specific aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the mitochondrial DNA (mtDNA)-reduced exhausted T cells with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells in which the level of at least one marker of T cell exhaustion (e.g., PD-1 expression or IL-2 secretion) is altered (e.g., reduced or increased) compared to the level of at least one marker of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 0.1-fold compared to the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.2-fold or reduced by 0.2-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.3-fold or reduced by 0.3-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.4-fold or reduced by 0.4-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.5-fold or reduced by 0.5-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.6-fold or reduced by 0.6-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased by at least 0.7-fold or reduced by 0.7-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.8-fold or reduced by 0.8-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 0.9-fold or reduced by 0.9-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.0-fold or reduced by 1.0-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.1-fold or reduced by 1.1-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.15-fold or reduced by 1.15-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.2-fold or reduced by 1.2-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.25-fold or reduced by 1.25-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.3-fold or reduced by 1.3-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 1.4-fold or reduced by 1.4-fold compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one marker of exhaustion is increased or decreased by at least 1.5-fold relative to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased by at least 1.75-fold or reduced by 1.75-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 2.5-fold or reduced by 2.5-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased by at least 3-fold or reduced by at most 3-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 4-fold or reduced by at most 4-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased by at least 2-fold or reduced by at most 2-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased by at least 5-fold or reduced by at most 5-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker exhibits an increase of about 0.2-fold to about 1.25-fold, or an increase of about 0.2-fold to about 1.25-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker exhibits an increase of about 0.2-fold to about 1.0-fold, or an increase of about 0.2-fold to about 1.0-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker exhibits an increase of about 0.2-fold to about 0.8-fold, or an increase of about 0.2-fold to about 0.8-fold compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker exhibits between about a 0.2-fold increase and about a 0.6-fold increase, or between about a 0.2-fold decrease and about a 0.6-fold decrease, relative to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, the at least one exhaustion marker exhibits an approximately 0.8-fold increase to approximately 1.25-fold increase, or an approximately 0.8-fold decrease to approximately 1.25-fold decrease, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an approximately 0.5-fold increase to approximately 1.25-fold increase, or an approximately 0.5-fold decrease to approximately 1.25-fold decrease, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the at least one exhaustion marker exhibits an approximately 0.8-fold increase to approximately 1.0-fold increase, or an approximately 0.8-fold decrease to approximately 1.0-fold decrease, compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, at least one exhaustion marker is increased by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold, or decreased by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold, compared to the exhausted T cells from which the mitochondria-replaced T cells were derived. In specific embodiments, the levels of two, three, or more markers of T cell exhaustion in the mitochondria-replaced T cells are altered compared to the levels of two, three, or more markers by the exhausted T cells from which the mitochondria-replaced T cells were derived. In specific embodiments, the levels of one, two, three, or more markers of T cell exhaustion by the mitochondria-replaced T cells are altered compared to the exhausted T cells from which the mitochondria-replaced T cells were derived, indicating one, two, or more improved effector functions of the T cells. In certain embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the RNA level compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the protein level compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, the levels of one, two, three or more markers of T cell exhaustion are altered at the RNA and protein levels compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, the methods provided herein for generating mitochondrial-replaced T cells are performed in vitro or ex vivo.

[0050] In a specific aspect, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, the method comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the mitochondrial DNA (mtDNA)-reduced exhausted T cells with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells in which the level of at least one marker of T cell exhaustion (e.g., PD-1 expression or IL-2 secretion) is altered (e.g., reduced or increased) compared to the level of at least one marker of T cell exhaustion by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, the at least one marker of exhaustion is increased or reduced by at least 5% compared to the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 10% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 15% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 20% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 25% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 30% compared to exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 35% compared to exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, at least one exhaustion marker is increased or decreased by at least 40% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by approximately 45% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 50% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 60% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 70% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 80% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 90% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased or decreased by at least 95% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 100% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 200% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 300% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 400% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, at least one exhaustion marker is increased by at least 500% compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In some embodiments, at least one exhaustion marker is increased or decreased by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, the level of two, three or more markers of T cell exhaustion in the mitochondria-replaced T cells is altered compared to the level of two, three or more markers by the exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, the level of one, two, three or more markers of T cell exhaustion by the mitochondria-replaced T cells is altered compared to the exhausted T cells from which the mitochondria-replaced T cells are derived, indicating one, two or more improved effector functions of the T cells. In certain embodiments, the level of one, two, three or more markers of T cell exhaustion is altered at the RNA level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, the level of one, two, three or more markers of T cell exhaustion is altered at the protein level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the level of one, two, three or more markers of T cell exhaustion is altered at the RNA level and protein level compared to the exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, the method provided herein for generating mitochondria-replaced T cells is carried out in vitro or ex vivo.

[0051] Various markers of T cell exhaustion are known in the art. Non-limiting examples of T cell exhaustion markers include increased expression of cell surface molecules that inhibit immune response, called "immune checkpoint receptors" (e.g., programmed cell death-1 (PD-1), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte-associated protein 4 (CTLA-4), 2B4 / CD244 / SLAMF4) compared to functional T cells, reduced production of chemokines (e.g., TNF-alpha, interferon-gamma, cc(beta)), and reduced cytokine production (e.g., IL-2). Assays for determining T cell function are known in the art, and include assays such as those described in Section 6.3.

[0052] In some embodiments, the exhausted T cells comprise an increased level of a T cell exhaustion marker compared to functional T cells. In certain embodiments, the exhausted T cells comprise an immune checkpoint receptor selected from the group consisting of PD-1, TIM3, LAG3, TIGIT, CD150, CTLA-4, or 2B4 / CD244 / SLAMF4 that has increased expression compared to functional T cells. In some embodiments, the exhausted T cells comprise an increased expression of PD-1 compared to functional T cells. In some embodiments, the exhausted T cells comprise an increased expression of TIM3 compared to functional T cells. In some embodiments, the exhausted T cells comprise an increased expression of LAG3 compared to functional T cells. In some embodiments, the exhausted T cells comprise an increased expression of TIGIT compared to functional T cells. In some embodiments, the exhausted T cells comprise an increased expression of CD150 compared to functional T cells. In some embodiments, the exhausted T cells comprise an increased expression of CTLA-4 compared to functional T cells. In one embodiment, exhausted T cells have increased expression of 2B4 / CD244 / SLAMF4 compared to functional T cells.

[0053] In some embodiments, the exhausted T cells comprise a transcription factor selected from the group consisting of TOX, TOX2, and NR4A that has increased expression relative to functional T cells. In some embodiments, the exhausted T cells comprise a TOX that has increased expression relative to functional T cells. In some embodiments, the exhausted T cells comprise a TOX2 that has increased expression relative to functional T cells. In some embodiments, the exhausted T cells comprise NR4A that has increased expression relative to functional T cells.

[0054] In some embodiments, exhausted T cells comprise reduced levels of T cell exhaustion markers compared to functional T cells. In certain embodiments, exhausted T cells comprise reduced chemokine production (e.g., TNF-alpha, interferon-gamma, CC (β-chemokines)) and / or reduced cytokine production (e.g., IL-2) compared to functional T cells. In some embodiments, exhausted T cells comprise reduced production of TNF-α compared to functional T cells. In some embodiments, exhausted T cells comprise reduced production of IFN-γ compared to functional T cells. In some embodiments, exhausted T cells comprise reduced production of CC chemokines compared to functional T cells. In some embodiments, exhausted T cells comprise reduced production of IL-2 compared to functional T cells.

[0055] Thus, in one embodiment, the exhausted T cells of the present disclosure have increased expression of immune checkpoint receptors, and the mitochondria-replaced T cells generated from the exhausted T cells according to the methods provided herein have reduced expression of immune checkpoint receptors compared to the exhausted T cells. In certain embodiments, the mitochondria-replaced T cells have reduced expression of at least PD-1 compared to the exhausted T cells. In a specific embodiment, the mitochondria-replaced T cells have reduced PD-1 expression by at least 10% compared to the exhausted T cells. In a specific embodiment, the mitochondria-replaced T cells have reduced PD-1 expression by at least 20% compared to the exhausted T cells. In a specific embodiment, the mitochondria-replaced T cells have reduced PD-1 expression by at least 30% compared to the exhausted T cells. In a specific embodiment, the mitochondria-replaced T cells have reduced PD-1 expression by at least 40% compared to the exhausted T cells. In a specific embodiment, the mitochondria-replaced T cells have reduced PD-1 expression by at least 50% compared to the exhausted T cells. In a specific embodiment, the mitochondria-replaced T cells have reduced PD-1 expression by at least 60% compared to the exhausted T cells. In specific embodiments, PD-1 expression is reduced by at least 70% in the mitochondria-replaced T cells compared to exhausted T cells. In specific embodiments, PD-1 expression is reduced by at least 80% in the mitochondria-replaced T cells compared to exhausted T cells. In specific embodiments, PD-1 expression is reduced by at least 90% in the mitochondria-replaced T cells compared to exhausted T cells. In specific embodiments, PD-1 expression is reduced by at least 95% in the mitochondria-replaced T cells compared to exhausted T cells. In some embodiments, PD-1 expression is reduced by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% in the mitochondria-replaced T cells compared to the exhausted T cells from which the mitochondria-replaced T cells are derived.

[0056] In a specific embodiment, mitochondrial-replaced T cells have at most 1.1-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, mitochondrial-replaced T cells have at most 1.2-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, mitochondrial-replaced T cells have at most 1.25-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, mitochondrial-replaced T cells have at most 1.3-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, mitochondrial-replaced T cells have at most 1.4-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, mitochondrial-replaced T cells have at most 1.5-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, mitochondrial-replaced T cells have at most 1.6-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 1.7-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 1.8-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 1.9-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 2-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 3-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 4-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have at most 5-fold reduced PD-1 expression compared to exhausted T cells. In a specific embodiment, the mitochondrial-replaced T cells have more than 5-fold reduced PD-1 expression compared to exhausted T cells. In certain embodiments, mitochondrial-replaced T cells exhibit approximately 0.2- to approximately 1.25-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.In certain embodiments, mitochondrial-replaced T cells have about 0.2- to about 1.0-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, mitochondrial-replaced T cells have about 0.2- to about 0.8-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, mitochondrial-replaced T cells have about 0.2- to about 0.6-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, mitochondrial-replaced T cells have about 0.8- to about 1.25-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, mitochondrial-replaced T cells have about 0.5- to about 1.25-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, mitochondrial-replaced T cells have about 0.8- to about 1.0-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived. In some embodiments, mitochondrial-replaced T cells have about 1.25- to about 1.50-fold, about 1.50- to about 2-fold, about 1.75- to about 2-fold, or about 2- to about 4-fold reduced PD-1 expression compared to exhausted T cells from which the mitochondrial-replaced T cells are derived.

[0057] In a specific embodiment, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells with at least 1.1-fold reduced expression of programmed cell death-1 (PD-1) compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression is at most 1.2-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression is at most 1.25-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In some embodiments, PD-1 expression is at most 1.50-fold reduced compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were derived. In certain embodiments, PD-1 expression is at most 1.75-fold reduced compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, PD-1 expression is at most 2-fold reduced compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is at most 2.5-fold reduced compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, PD-1 expression is at most 3-fold reduced compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is at most 4-fold reduced. In certain embodiments, PD-1 expression is approximately 1.1- to approximately 1.25-fold reduced compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, PD-1 expression is reduced by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived.In some embodiments, PD-1 expression is reduced by about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold compared to expression of PD-1 by exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, expression of PD-1 and one, two, three or more other markers of T cell exhaustion is reduced in the mitochondria-replaced T cells compared to expression of PD-1 and one, two, three or more other markers of T cell exhaustion by exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, reduced expression of one, two, three or more markers of T cell exhaustion by the mitochondria-replaced T cells compared to expression of the same one, two, three or more markers by exhausted T cells from which the mitochondria-replaced T cells are derived indicates one, two or more improved effector functions of the T cells. In certain embodiments, expression of PD-1, and in some embodiments, expression of one, two, three or more other markers of T cell exhaustion, is reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, expression of PD-1, and in some embodiments, expression of one, two, three or more other markers of T cell exhaustion, is reduced at the protein level compared to expression of the same markers by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, expression of PD-1, and in some embodiments, expression of one, two, three or more other markers of T cell exhaustion, is reduced at the RNA level and protein level compared to expression of the corresponding markers by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, the nucleic acid sequence is RNA (e.g., mRNA). In some embodiments, the RNA is unmodified RNA. In some embodiments, the RNA is modified RNA. In some embodiments, the nucleic acid sequence is DNA (e.g., cDNA). In some embodiments, the DNA is unmodified DNA. In some embodiments, the DNA is modified DNA.Non-limiting examples of modified RNA or DNA include tritylated bases and unusual bases such as inosine.

[0058] In a specific embodiment, provided herein is a method for generating mitochondrial-replaced T cells from exhausted T cells, comprising incubating exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having at least 5% reduced expression of programmed cell death-1 (PD-1) compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 10% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 15% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 20% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 25% compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 30% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 35% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 40% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 45% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 50% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 60% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived.In certain embodiments, PD-1 expression is reduced by at least 70% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 80% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 90% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, PD-1 expression is reduced by at least 95% compared to PD-1 expression by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, PD-1 expression is reduced by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% compared to expression of PD-1 by exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, expression of PD-1 and one, two, three or more other markers of T cell exhaustion is reduced in the mitochondria-replaced T cells compared to expression of PD-1 and one, two, three or more other markers of T cell exhaustion by exhausted T cells from which the mitochondria-replaced T cells are derived. In specific embodiments, expression of one, two, three or more markers of T cell exhaustion by the mitochondrial-replaced T cells is reduced relative to expression of the same one, two, three or more markers by the exhausted T cells from which the mitochondrial-replaced T cells are derived, indicating one, two or more improved effector function of T cells. In certain embodiments, expression of PD-1, and in some embodiments one, two, three or more other markers of T cell exhaustion, is reduced at the RNA level relative to expression of the corresponding markers by the exhausted T cells from which the mitochondrial-replaced T cells are derived.In some embodiments, expression of PD-1, and in some embodiments, expression of one, two, three or more other markers of T cell exhaustion, is reduced at the protein level compared to expression of the same markers by exhausted T cells from which the mitochondria-replaced T cells are derived. In certain embodiments, expression of PD-1, and in some embodiments, expression of one, two, three or more other markers of T cell exhaustion, is reduced at the RNA level and protein level compared to expression of the corresponding markers by exhausted T cells from which the mitochondria-replaced T cells are derived. In some embodiments, the nucleic acid sequence is RNA (e.g., mRNA). In some embodiments, the RNA is unmodified RNA. In some embodiments, the RNA is modified RNA. In some embodiments, the nucleic acid sequence is DNA (e.g., cDNA). In some embodiments, the DNA is unmodified DNA. In some embodiments, the DNA is modified DNA. Non-limiting examples of modified RNA or modified DNA include tritylated bases and unusual bases such as inosine.

[0059] In a specific embodiment, the expression of PD-1 and one, two, three or more other markers of T cell exhaustion is reduced in the mitochondria-replaced T cells compared to the expression of the corresponding markers in the exhausted T cells from which the mitochondria-replaced T cells are derived. In a specific embodiment, the expression of one, two, three or more markers of T cell exhaustion by the mitochondria-replaced T cells is reduced compared to the expression of the corresponding markers in the exhausted T cells from which the mitochondria-replaced T cells are derived, thereby indicating one, two or more improved effector functions of the T cells. In certain embodiments, the expression of PD-1, and in some embodiments, the expression of one, two, three or more other markers of T cell exhaustion is reduced at the RNA level compared to the expression of the corresponding markers in the exhausted T cells from which they are derived. In some embodiments, the expression of PD-1, and in some embodiments, the expression of one, two, three or more other markers of T cell exhaustion is reduced at the protein level compared to the expression of the corresponding markers in the exhausted T cells from which they are derived. In certain embodiments, expression of one PD-1, and in some embodiments one, two, three or more other markers of T cell exhaustion, is reduced at the RNA and protein levels relative to the expression of the corresponding markers in the exhausted T cells from which they are derived.

[0060] In certain embodiments, the mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 compared to the expression of the corresponding marker by exhausted T cells, and at least one additional immune checkpoint receptor is also reduced compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, the mitochondrial-replaced T cells have at least 1.1-fold to about 1.25-fold, at most 1.25-fold to about 1.50-fold, at most 1.50-fold to about 2-fold, at most 1.75-fold to about 2-fold, or at most 2-fold to about 4-fold reduced expression of PD-1 compared to the expression of the corresponding marker by exhausted T cells, and at least one additional immune checkpoint receptor is also reduced compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, expression of PD-1 and at least one additional immune checkpoint receptor is reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and at least one additional immune checkpoint receptor is reduced at the protein level compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and at least one additional immune checkpoint receptor is reduced at the RNA and protein levels compared to expression of the corresponding markers by exhausted T cells.

[0061] In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding marker by exhausted T cells, and expression of at least one additional immune checkpoint receptor is also reduced compared to expression of the corresponding marker by exhausted T cells. In certain embodiments, the expression of PD-1 is reduced by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% in mitochondria-replaced T cells compared to the expression of the corresponding marker by exhausted T cells, and the expression of at least one additional immune checkpoint receptor is also reduced compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of the at least one additional immune checkpoint receptor are reduced at the RNA level compared to the expression of the corresponding marker by exhausted T cells. In some embodiments, the expression of PD-1 and the expression of the at least one additional immune checkpoint receptor are reduced at the protein level compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least one additional immune checkpoint receptor is reduced at the RNA and protein levels relative to expression of corresponding markers by exhausted T cells.

[0062] In certain embodiments, mitochondrial-replaced T cells exhibit reduced expression of PD-1 by at least 1.1 fold, at most 1.25 fold, at most 1.5 fold, at most 2.0 fold, at most 2.5 fold, at most 3.0 fold, or at most 4.0 fold compared to expression of the corresponding marker by exhausted T cells, and reduced expression of at least one immune checkpoint receptor selected from the group consisting of TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to expression of the corresponding marker by exhausted T cells. In certain embodiments, the mitochondria-replaced T cells have reduced expression of PD-1 by about 1.1-fold to about 1.25-fold, about 1.25-fold to about 1.50-fold, about 1.50-fold to about 2-fold, about 1.75-fold to about 2-fold, or about 2-fold to about 4-fold compared to the expression of the corresponding marker by exhausted T cells, and also have reduced expression of at least one immune checkpoint receptor selected from the group consisting of TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of the at least one additional immune checkpoint receptor are reduced at the RNA level compared to the expression of the corresponding marker by exhausted T cells. In some embodiments, the expression of PD-1 and the expression of the at least one additional immune checkpoint receptor are reduced at the protein level compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least one additional immune checkpoint receptor is reduced at the RNA and protein levels relative to expression of corresponding markers by exhausted T cells.

[0063] In certain embodiments, the mitochondrial-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding marker by exhausted T cells, and reduced expression of at least one immune checkpoint receptor selected from the group consisting of TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to expression of the corresponding marker by exhausted T cells. In certain embodiments, the mitochondria-replaced T cells have reduced expression of PD-1 by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% compared to the expression of the corresponding marker by the exhausted T cells, and the expression of at least one immune checkpoint receptor selected from the group consisting of TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 is also reduced compared to the expression of the corresponding marker by the exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of the at least one additional immune checkpoint receptor are reduced at the RNA level compared to the expression of the corresponding marker by the exhausted T cells. In some embodiments, expression of PD-1 and expression of at least one additional immune checkpoint receptor are reduced at the protein level relative to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least one additional immune checkpoint receptor are reduced at the RNA and protein levels relative to expression of the corresponding markers by exhausted T cells.

[0064] In certain embodiments, the mitochondrial-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding marker by exhausted T cells, and reduced expression of at least one immune checkpoint receptor selected from the group consisting of TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to expression of the corresponding marker by exhausted T cells. In certain embodiments, the mitochondria-replaced T cells have reduced expression of PD-1 by about 5% to about 50%, about 10% to about 50%, about 10% to about 40%, about 10% to about 30%, about 20% to about 50%, about 20% to about 40%, about 10% to about 20%, about 20% to about 30%, about 25% to about 50%, or about 40% to about 60% compared to the expression of the corresponding marker by the exhausted T cells, and the expression of at least one immune checkpoint receptor selected from the group consisting of TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 is also reduced compared to the expression of the corresponding marker by the exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of the at least one additional immune checkpoint receptor are reduced at the RNA level compared to the expression of the corresponding marker by the exhausted T cells. In some embodiments, expression of PD-1 and expression of at least one additional immune checkpoint receptor are reduced at the protein level relative to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least one additional immune checkpoint receptor are reduced at the RNA and protein levels relative to expression of the corresponding markers by exhausted T cells.

[0065] In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 and LAG3 compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 and TIGIT compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 and CD160 compared to exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 and CTLA-4 compared to exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, expression is reduced at the RNA level compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, expression is reduced at the protein level compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, expression is reduced at both the RNA and protein levels compared to the expression of the corresponding markers by exhausted T cells.

[0066] In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 and LAG3 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 and TIGIT by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 and CD160 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 and CTLA-4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of the corresponding markers by exhausted T cells.In certain embodiments, the expression of PD-1 and 2B4 / CD244 / SLAMF4 is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% in mitochondria-replaced T cells compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression is reduced at the RNA level compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the expression is reduced at the protein level compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression is reduced at the RNA level and the protein level compared to the expression of the corresponding markers by exhausted T cells.

[0067] In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 compared to the expression of the corresponding markers by exhausted T cells, and at least two additional immune checkpoint receptors are also reduced compared to the expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, LAG3, and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, TIGIT, and CD160 compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, CTLA-4, and LAG3 compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of at least two additional immune checkpoint receptors are reduced at the RNA level compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of the at least two additional immune checkpoint receptors are reduced at the protein level relative to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of the at least two additional immune checkpoint receptors are reduced at the RNA and protein levels relative to expression of the corresponding markers by exhausted T cells.

[0068] In certain embodiments, the mitochondria-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of the corresponding markers by exhausted T cells, and the expression of at least two additional immune checkpoint receptors is also reduced compared to the expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, the mitochondria-replaced T cells have reduced expression of PD-1, LAG3, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have reduced expression of PD-1, TIGIT, and CD160 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have reduced expression of PD-1, CTLA-4, and LAG3 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least two additional immune checkpoint receptors are reduced at the RNA level relative to expression of corresponding markers by exhausted T cells.In some embodiments, expression of PD-1 and expression of the at least two additional immune checkpoint receptors are reduced at the protein level relative to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of the at least two additional immune checkpoint receptors are reduced at the RNA and protein levels relative to expression of the corresponding markers by exhausted T cells.

[0069] In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 compared to the expression of the corresponding markers by exhausted T cells, and at least three additional immune checkpoint receptors are also reduced compared to the expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, LAG3, TIGIT, and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, CTLA-4, TIGIT, and CD160 compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, CTLA-4, TIGIT, and LAG3 compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression of PD-1 and at least three additional immune checkpoint receptors are reduced at the RNA level compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of the at least three additional immune checkpoint receptors are reduced at the protein level relative to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of the at least three additional immune checkpoint receptors are reduced at the RNA and protein levels relative to expression of the corresponding markers by exhausted T cells.

[0070] In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells, and expression of at least three additional immune checkpoint receptors is also reduced compared to expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, the mitochondria-replaced T cells have reduced expression of PD-1, LAG3, TIGIT, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the mitochondria-replaced T cells have reduced expression of PD-1, CTLA-4, TIGIT, and CD160 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, CTLA-4, TIGIT, and LAG3 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells.In certain embodiments, expression of PD-1 and expression of at least three additional immune checkpoint receptors are reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of at least three additional immune checkpoint receptors are reduced at the protein level compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least three additional immune checkpoint receptors are reduced at the RNA and protein levels compared to expression of the corresponding markers by exhausted T cells.

[0071] In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 compared to the expression of the corresponding markers by exhausted T cells, and at least four additional immune checkpoint receptors are also reduced compared to the expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, LAG3, TIGIT, CD160, and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the expression of PD-1, CTLA-4, TIGIT, LAG3 and CD160 is reduced by at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold in mitochondria-replaced T cells compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the expression of PD-1, CTLA-4, TIGIT, TIM3 and LAG3 is reduced by at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold in mitochondria-replaced T cells compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of at least four additional immune checkpoint receptors are reduced at the RNA level compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of the at least four additional immune checkpoint receptors are reduced at the protein level relative to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of the at least four additional immune checkpoint receptors are reduced at the RNA and protein levels relative to expression of the corresponding markers by exhausted T cells.

[0072] In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells, and expression of at least four additional immune checkpoint receptors is also reduced compared to expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, LAG3, TIGIT, CD160, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells. In some embodiments, the expression of PD-1, CTLA-4, TIGIT, LAG3 and CD160 is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% in the mitochondria-replaced T cells compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the expression of PD-1, CTLA-4, TIGIT, TIM3 and LAG3 is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% in the mitochondria-replaced T cells compared to the expression of the corresponding markers by exhausted T cells.In certain embodiments, expression of PD-1 and expression of at least four additional immune checkpoint receptors are reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of at least four additional immune checkpoint receptors are reduced at the protein level compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least four additional immune checkpoint receptors are reduced at the RNA and protein levels compared to expression of the corresponding markers by exhausted T cells.

[0073] In certain embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1 compared to the expression of the corresponding markers by exhausted T cells, and at least five additional immune checkpoint receptors are also reduced compared to the expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, TIM3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to exhausted T cells. In some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, TIM3, LAG3, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells exhibit at most 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 relative to expression of the corresponding markers by exhausted T cells.By way of example, in some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, TIM3, LAG3, TIGIT, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have at least 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold reduced expression of PD-1, TIM3, LAG3, TIGIT, CD160, and 2B4 / CD244 / SLAMF4 compared to exhausted T cells. By way of example, in some embodiments, the expression of PD-1, TIM3, LAG3, TIGIT, CD160, and CTLA-4 is reduced at most 1.1-fold, at most 1.25-fold, at most 1.5-fold, at most 2.0-fold, at most 2.5-fold, at most 3.0-fold, or at most 4.0-fold in mitochondria-replaced T cells compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of at least five additional immune checkpoint receptors are reduced at the RNA level compared to the expression of the corresponding markers by exhausted T cells. In some embodiments, the expression of PD-1 and the expression of at least five additional immune checkpoint receptors are reduced at the protein level compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, the expression of PD-1 and the expression of at least five additional immune checkpoint receptors are reduced at the RNA level and protein level compared to the expression of the corresponding markers by exhausted T cells.

[0074] In certain embodiments, mitochondrial-replaced T cells have reduced expression of PD-1 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% relative to expression of the corresponding markers by exhausted T cells, and expression of at least five additional immune checkpoint receptors is also reduced relative to expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells. In some embodiments, mitochondrial-replaced T cells exhibit reduced expression of PD-1, TIM3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells. In some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, TIM3, LAG3, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells.By way of example, in some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, TIM3, LAG3, TIGIT, CTLA-4, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, TIM3, LAG3, TIGIT, CD160, and 2B4 / CD244 / SLAMF4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells. By way of example, in some embodiments, mitochondrial-replaced T cells have reduced expression of PD-1, TIM3, LAG3, TIGIT, CD160, and CTLA-4 by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of the corresponding markers by exhausted T cells.In certain embodiments, expression of PD-1 and expression of at least five additional immune checkpoint receptors are reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of at least five additional immune checkpoint receptors are reduced at the protein level compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least five additional immune checkpoint receptors are reduced at the RNA and protein levels compared to expression of the corresponding markers by exhausted T cells.

[0075] In certain embodiments, mitochondrial-replaced T cells have at least a 1.1-fold, at most a 1.25-fold, at most a 1.5-fold, at most a 2.0-fold, at most a 2.5-fold, at most a 3.0-fold, or at most a 4.0-fold reduction in expression of PD-1, and at least six additional immune checkpoint receptors. By way of example, in some embodiments, mitochondrial-replaced T cells have at least a 1.1-fold, at most a 1.25-fold, at most a 1.5-fold, at most a 2.0-fold, at most a 2.5-fold, at most a 3.0-fold, or at most a 4.0-fold reduction in expression of PD-1, TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least six additional immune checkpoint receptors are reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of at least six additional immune checkpoint receptors are reduced at the protein level compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least six additional immune checkpoint receptors are reduced at the RNA and protein levels compared to expression of the corresponding markers by exhausted T cells.

[0076] In certain embodiments, the mitochondrial-replaced T cells have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% reduced expression of PD-1 and at least six additional immune checkpoint receptors. By way of example, in some embodiments, the mitochondrial-replaced T cells have at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% reduced expression of PD-1, TIM3, LAG3, TIGIT, CD160, CTLA-4, and 2B4 / CD244 / SLAMF4 compared to the expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least six additional immune checkpoint receptors are reduced at the RNA level compared to expression of the corresponding markers by exhausted T cells. In some embodiments, expression of PD-1 and expression of at least six additional immune checkpoint receptors are reduced at the protein level compared to expression of the corresponding markers by exhausted T cells. In certain embodiments, expression of PD-1 and expression of at least six additional immune checkpoint receptors are reduced at the RNA and protein levels compared to expression of the corresponding markers by exhausted T cells.

[0077] Mitochondrially-replaced T cells in which expression of programmed cell death-1 (PD-1), and in some embodiments one, two, three or more other markers of T cell exhaustion, is at most 1.1 fold, at most 1.25 fold, at most 1.5 fold, at most 2.0 fold, at most 2.5 fold, at most 3.0 fold, or at most 4.0 fold reduced, relative to PD-1 expression by the exhausted T cells from which they are derived, is understood to encompass both expression on T cells as well as expression within the T cells. It is understood that mitochondrially-replaced T cells in which expression of programmed cell death-1 (PD-1), and in some embodiments one, two, three or more other markers of T cell exhaustion, is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to expression of PD-1 by exhausted T cells from which the mitochondrially-replaced T cells are derived, encompasses both expression on T cells as well as expression within T cells. In some embodiments, expression of one, two, three or more other markers of T cell exhaustion on the mitochondrially-replaced T cells is reduced compared to expression of PD-1, and in some embodiments the corresponding one, two, three or more other markers of T cell exhaustion, on exhausted T cells from which the mitochondrially-replaced T cells are derived. In certain embodiments, the mitochondrial-replaced T cells have reduced expression of PD-1, and in some embodiments, one, two, three or more other markers of T cell exhaustion, compared to expression of the corresponding markers by exhausted T cells, compared to expression of PD-1 in exhausted T cells from which the mitochondrial-replaced T cells are derived, and in some embodiments, compared to expression of the corresponding one, two, three or more other markers of T cell exhaustion.

[0078] In some embodiments, the marker of T cell exhaustion is an intracellular protein. Non-limiting examples of intracellular proteins suitable as markers of T cell exhaustion include transcription factors TOX, TOX2, and NR4A. Thus, in some embodiments, the expression of TOX, TOX2, and / or NR4A is reduced at most 2-fold in mitochondria-replaced T cells compared to the expression of the corresponding marker in exhausted T cells. In some embodiments, the expression of TOX, TOX2, and / or NR4A is reduced at most 5-fold in mitochondria-replaced T cells compared to the expression of the corresponding marker in exhausted T cells. In some embodiments, the expression of TOX, TOX2, and / or NR4A is reduced at most 10-fold in mitochondria-replaced T cells compared to the expression of the corresponding marker in exhausted T cells. In some embodiments, the expression of TOX, TOX2, and / or NR4A is reduced at most 15-fold in mitochondria-replaced T cells compared to the expression of the corresponding marker in exhausted T cells. In some embodiments, mitochondrial-replaced T cells exhibit at least a 20-fold reduction in expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells. In some embodiments, mitochondrial-replaced T cells exhibit at least a 20-fold reduction in expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells.

[0079] In some embodiments, the mitochondria-replaced T cells have at least 20% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 30% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 40% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 50% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have at least 60% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding markers in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have greater than 70% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding marker in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have greater than 80% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding marker in exhausted T cells. In some embodiments, the mitochondria-replaced T cells have greater than 90% reduced expression of TOX, TOX2, and / or NR4A compared to the expression of the corresponding marker in exhausted T cells. In certain embodiments, expression is reduced at the RNA level compared to the expression of the corresponding marker by exhausted T cells. In some embodiments, expression is reduced at the protein level compared to the expression of the corresponding marker by exhausted T cells. In certain embodiments, expression is reduced at the RNA level and the protein level compared to the expression of the corresponding marker by exhausted T cells.

[0080] In some embodiments, exhausted T cells show one or more markers of senescence. Examples of markers of senescence include reduced expression of costimulatory molecules (e.g., CD27, and / or CD28); increased expression of KLRG-1 and / or CD57; upregulation of G1 regulatory proteins (e.g., p15, p16, and p21); downregulation of Cdk2 and cyclin D3 expression; reduced Cdk2 and Cdk6 kinase activity; loss of human telomerase RNA component (hTERC) expression; reduced telomerase activity; increased expression of TIGIT; or combinations thereof. Senescent cells also generally show one or more phenotypic markers, such as 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 cell population doubling rate, shortened telomeres, increased DNA damage response (DDR), or combinations thereof.

[0081] As provided herein, mitochondrial-replaced T cells generated according to the methods described herein, e.g., the methods described in Section 6.1, exhibit one, two or more improved effector functions compared to the exhausted T cells from which the mitochondrial-replaced T cells are derived. See, e.g., Section 6.3 for T cell effector functions that can be improved in mitochondrial-replaced T cells.

[0082] In certain embodiments, the improved effector function comprises increased proliferation, increased cytotoxicity, increased secretion of cytokines, or a combination thereof. In specific embodiments, the improved effector function comprises increased proliferation. In certain embodiments, the improved effector function comprises increased cytotoxicity. In some embodiments, the improved effector function comprises increased secretion of cytokines.

[0083] As provided herein, in one aspect, a method for generating mitochondrial-replaced T cells from exhausted T cells involves electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number. In some embodiments, the nucleotide sequence encoding XbaIR comprises DNA. In some embodiments, the nucleotide sequence encoding XbaIR comprises RNA. As provided herein, in one aspect, a method for generating mitochondrial-replaced T cells from exhausted T cells involves electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number. In some embodiments, the nucleic acid sequence is RNA (e.g., mRNA). In some embodiments, the RNA is unmodified RNA. In some embodiments, the RNA is modified RNA. In some embodiments, the nucleic acid sequence is DNA (e.g., cDNA). In some embodiments, the DNA is unmodified DNA. In some embodiments, the DNA is modified DNA. Non-limiting examples of modified RNA or DNA include tritylated bases and unusual bases such as inosine.

[0084] Various methods are known in the art for introducing nucleotides (e.g., present in a plasmid DNA expression vector cassette or as mRNA). In some embodiments, the nucleotides are electroporated. In specific embodiments, the electroporation method is flow electroporation, such as MaxCyte Flow Electroporation. In other specific embodiments, the electroporation method includes nucleofection technology, such as Lonza's Nucleofector™ technology. However, it is understood that the above methods for introducing nucleotides are intended to be non-limiting and merely exemplary methods, and that any method known in the art can be used to introduce nucleotides. For example, in some embodiments, the nucleotides are introduced by cationic lipid transfection, or any other means of introducing nucleotides into cells. In some embodiments, the nucleotides are introduced by viral transduction.

[0085] In certain embodiments, particularly in clinical settings, a MaxCyte electroporator that meets the standards of the Pharmaceutical and Quasi-drug Industry (PHI) and the standards for conducting clinical trials of pharmaceuticals can be used for mRNA transfection. Transfection can be performed using the MaxCyte electroporator according to the manufacturer's protocol. Furthermore, it is understood that the above methods are merely exemplary and that any means of introducing mRNA can be used.

[0086] Specific targeting of endonucleases to mitochondria can be achieved by incorporating mitochondrial targeting sequences (MTS) adjacent to endonuclease coding sequences, thereby generating fusion proteins that target mitochondria. Powerful MTSs have been identified and shown to be capable of targeting proteins fused to their N-terminus to specific compartments, and they are called mitochondrial targeting sequences. MTSs suitable for the method of the present invention are well known to those skilled in the art (see, for example, U.S. Patent No. 8,039,587 B2, the entirety of which is incorporated herein by reference). For example, MTSs for mitochondrial matrix can be used, such as MTSs that are targeting peptides derived from cytochrome c oxidase subunit IV (COX4), subunit VIII (COX8), or subunit X (COX10). In principle, any target sequence derived from any nuclear-encoded mitochondrial matrix or inner membrane enzyme, or an artificial sequence capable of rendering the fusion protein a mitochondrially imported protein (hydrophobic moment greater than 5.5, at least two basic residues, amphipathic alpha-helical conformation; see, e.g., Bedwell et al., Mol Cell Biol. 9(3) (1989), 1014-1025) is useful for the purposes of the present invention.

[0087] In certain embodiments, the MTS is human MTS. In other embodiments, the MTS is from another species. Non-limiting examples of such sequences are cytochrome c oxidase subunit X (COX10) MTS (MAASPHTLSSRLLTGCVGGSVWYLERRT) (SEQ ID NO: 1) and cytochrome c oxidase subunit VIII (COX8) MTS (MSVLTPLLLRSLTGSARRLMVPRA) (SEQ ID NO: 2). Additional non-limiting examples of MTS sequences are the native MTS of each individual mitochondrial protein that is encoded by nuclear DNA, translated (produced) in the cytoplasm, and transported to mitochondria, as well as citrate synthase (cs), lipoamide dehydrogenase (LAD), and C6ORF66 (ORF). The various MTSs may be interchangeable between each mitochondrial enzyme. Thus, in some embodiments, the MTS targets a mitochondrial matrix protein. In a specific embodiment, the mitochondrial matrix protein is human cytochrome C oxidase subunit VIII.

[0088] In certain aspects, the methods provided herein involve culturing exhausted T cells transfected or transformed with or otherwise comprising a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR (in a specific embodiment, the nucleic acid sequence comprises a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR) to reduce endogenous mitochondrial DNA (mtDNA) copy number. In a specific aspect, the methods provided herein involve culturing exhausted T cells transfected or transformed with or otherwise comprising a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number with minimal cytotoxicity until the cells are at least substantially free of nucleotide sequences encoding XbaIR. In a specific aspect, the methods provided herein involve culturing exhausted T cells transfected or transformed with or otherwise comprising a nucleic acid sequence comprising a nucleotide sequence encoding XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number with minimal cytotoxicity until the cells are at least substantially free of XbaIR. Cytotoxicity can be measured using techniques known to those skilled in the art or described herein. Techniques known to those skilled in the art or described herein (e.g., in the Examples) can be used to assess the amount of nucleotides encoding XbaIR (e.g., PCR or qPCR), or the amount of XbaIR or fusion protein (e.g., immunoblot).

[0089] In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 1 day to about 10 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 2 days to about 7 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 1 day to about 5 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 2 days to about 4 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 2 days to about 3 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 1 day to about 4 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 1 day to about 3 days. In some embodiments, the period of time sufficient to reduce the endogenous mitochondrial DNA (mtDNA) copy number is about 2 days to about 7 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 1 day. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 2 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 3 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 4 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 5 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 6 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 7 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 8 days.In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 9 days. In some embodiments, the period of time sufficient to reduce endogenous mitochondrial DNA (mtDNA) copy number is about 10 days.

[0090] In certain aspects, the methods provided herein involve incubation of exhausted T cells with isolated exogenous mitochondria in the presence of an effective amount of rapamycin or a derivative thereof. In some embodiments, the rapamycin or a derivative thereof is at a concentration of about 100 nanomolar (nM) to about 1000 nM. In some embodiments, the rapamycin or a derivative thereof is at a concentration of about 200 nanomolar (nM) to about 500 nM. In some embodiments, the rapamycin or a derivative thereof is at a concentration of about 300 nanomolar (nM) to about 600 nM. In some embodiments, the rapamycin or a derivative thereof is at a concentration of about 400 nanomolar (nM) to about 700 nM.

[0091] 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.

[0092] Non-limiting examples of rapamycin derivatives (e.g., rapamycin analogs, also known as "rapalogs") include, for example, temsirolimus (CAS No. 162635-04-3; C56H87NO16), everolimus (CAS No. 159351-69-6; C53H83NO14), ridaforolimus (CAS No. 572924-54-0; C53H84NO14P), WYE-125132 (WYE-132), and zotarolimus (ABT-578).

[0093] In specific embodiments, the methods provided herein involve incubation of exhausted T cells with isolated exogenous mitochondria in the presence of an effective amount of rapamycin. In some embodiments, the rapamycin is at a concentration of about 100 nanomolar (nM) to about 1000 nM. In some embodiments, the rapamycin is at a concentration of about 200 nM to about 500 nM. In some embodiments, the rapamycin is at a concentration of about 300 nM to about 600 nM. In some embodiments, the rapamycin is at a concentration of about 400 nM to about 700 nM. In certain embodiments, the effective amount of rapamycin is at a concentration of about 100 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 150 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 200 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 250 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 300 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 350 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 400 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 450 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 500 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 550 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 600 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 650 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 700 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 750 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 800 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 850 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 900 nM. In some embodiments, the effective amount of rapamycin is at a concentration of about 1000 nM. In some embodiments, the effective amount of rapamycin is at a concentration of greater than 1000 nM.

[0094] The amount of isolated exogenous mitochondria that are incubated with exhausted T cells to generate mitochondrial-replaced T cells depends on factors such as the amount of exhausted T cells that are co-incubated with the isolated mitochondria. Generally, the amount of isolated exogenous mitochondria that are incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 5 μg to about 100 μg per T cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 10 μg to about 90 μg per T cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 20 μg to about 80 μg per T cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 30 μg to about 70 μg per T cell. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 In some embodiments, the amount of isolated exogenous mitochondria incubated with exhausted T cells is about 1×10 T cells. 6 More than 100 μg per piece.

[0095] 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 6.3. 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.

[0096] 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.

[0097] 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. 9Mitochondria are isolated from donor cells by washing 1-2 mL of cell pellet from each donor cell, 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. In a specific embodiment, mitochondria are isolated and transferred using the method scheme described in Section 8.

[0098] 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.

[0099] 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 evaluating total protein content.To evaluate 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.

[0100] The period of time sufficient to incubate exhausted T cells with reduced endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells may be any period of time that allows a greater amount of exogenous mtDNA to be detected than in exhausted T cells that have not been exposed to exogenous mitochondria. In some embodiments, the period of time sufficient to incubate exhausted T cells with reduced endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time that allows at least 20% of the exogenous mtDNA to be transferred to exhausted T cells compared to the amount of exogenous mtDNA transferred to exhausted T cells that have not been incubated with exogenous mtDNA. In some embodiments, the period of time sufficient to incubate exhausted T cells with reduced endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time that allows at least 30% of the exogenous mtDNA to be transferred to exhausted T cells compared to the amount of exogenous mtDNA transferred to exhausted T cells that have not been incubated with exogenous mtDNA. In some embodiments, a period of time sufficient to incubate exhausted T cells with depleted endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time during which at least 40% of the exogenous mtDNA is transferred into the exhausted T cells compared to the amount of exogenous mtDNA transferred into exhausted T cells not incubated with exogenous mtDNA. In some embodiments, a period of time sufficient to incubate exhausted T cells with depleted endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time during which at least 50% of the exogenous mtDNA is transferred into the exhausted T cells compared to the amount of exogenous mtDNA transferred into exhausted T cells not incubated with exogenous mtDNA. In some embodiments, a period of time sufficient to incubate exhausted T cells with reduced endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time during which at least 60% of the exogenous mtDNA is transferred to the exhausted T cells compared to the amount of exogenous mtDNA transferred to exhausted T cells that have not been incubated with exogenous mtDNA.In some embodiments, a period of time sufficient to incubate exhausted T cells with reduced endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time during which at least 70% of the exogenous mtDNA is transferred to the exhausted T cells compared to the amount of exogenous mtDNA transferred to exhausted T cells that have not been incubated with exogenous mtDNA. In some embodiments, a period of time sufficient to incubate exhausted T cells with reduced endogenous mtDNA with isolated exogenous mitochondria to generate mitochondrial-replaced T cells is any period of time during which a majority (i.e., greater than 50%) of the exogenous mtDNA, about 20% to about 95%, about 25% to about 90%, about 30% to about 85%, about 35% to about 80%, about 40% to about 75%, about 30% to about 60%, 40% to about 70%, about 40% to about 85%, about 40% to about 80%, about 50% to about 80%, about 60% to about 90%, about 65% to about 95%, is transferred into exhausted T cells relative to the amount of exogenous mtDNA transferred into exhausted T cells that have not been incubated with exogenous mtDNA. Transfer of exogenous mtDNA into exhausted T cells can be assessed using techniques known to those of skill in the art or described herein (e.g., in the Examples).

[0101] 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. In some embodiments, a sufficient period of time is about 1 day to about 7 days.

[0102] As an example, mitochondrial transfer can be performed using simple co-incubation of exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) and isolated exogenous mitochondria. However, it is also possible to facilitate mitochondrial transfer by centrifuging the T cells and isolated exogenous mitochondria as needed. Other means of mitochondrial transfer include heat shock, injection, and / or the use of nanoblades.

[0103] 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 (RCF, also referred to as "g") for approximately 5 minutes.In a specific embodiment, centrifugation is as described in the following examples.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] In some embodiments, the ratio of exogenous mtDNA copy number to endogenous mtDNA copy number in mitochondrial replacement T cells generated by 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.

[0113] 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. In some embodiments, the majority (i.e., greater than 50%) of the endogenous mtDNA is replaced by the exogenous mtDNA, in some embodiments, about 20% to about 95% of the endogenous mtDNA is replaced by the exogenous mtDNA.In some embodiments, about 25% to about 90% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 30% to about 85% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 35% to about 80% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 40% to about 75% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 20% to about 95% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 30% to about 60% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 40% to about 70% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 40% to about 85% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 40% to about 80% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 30% to about 85% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 50% to about 80% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 60% to about 90% of the endogenous mtDNA is replaced by exogenous mtDNA. In some embodiments, about 65% to about 95% of the endogenous mtDNA is replaced by exogenous mtDNA. Replacement of endogenous mtDNA by exogenous mtDNA can be assessed using techniques known to those of skill in the art or described herein (e.g., in the Examples).

[0114] As provided herein, the level of endogenous mtDNA copy number that is reduced according to the methods provided herein need not be a complete depletion. In some embodiments, the endogenous mtDNA copy number is reduced by a majority (i.e., more than 50%). In some embodiments, the endogenous mtDNA copy number is reduced by about 20% to about 95%. In some embodiments, the endogenous mtDNA copy number is reduced by about 25% to about 90%. In some embodiments, the endogenous mtDNA copy number is reduced by about 30% to about 90%. In some embodiments, the endogenous mtDNA copy number is reduced by about 30% to about 85%. In some embodiments, the endogenous mtDNA copy number is reduced by about 35% to about 80%. In some embodiments, the endogenous mtDNA copy number is reduced by about 40% to about 75%. In some embodiments, the endogenous mtDNA copy number is reduced by about 20% to about 95%. In some embodiments, the endogenous mtDNA copy number is reduced by about 30% to about 60%. In some embodiments, the endogenous mtDNA copy number is reduced by about 40% to about 70%. In some embodiments, the endogenous mtDNA copy number is reduced by about 40% to about 85%. In some embodiments, the endogenous mtDNA copy number is reduced by about 40% to about 80%. In some embodiments, the endogenous mtDNA copy number is reduced by about 30% to about 85%. In some embodiments, the endogenous mtDNA copy number is reduced by about 50% to about 80%. In some embodiments, the endogenous mtDNA copy number is reduced by about 60% to about 80%. In some embodiments, the endogenous mtDNA copy number is reduced by about 60% to about 90%. In some embodiments, the endogenous mtDNA copy number is reduced by about 65% to about 95%. In some embodiments, the endogenous mtDNA copy number is reduced by about 10%. In some embodiments, the endogenous mtDNA copy number is reduced by about 10%. In some embodiments, the endogenous mtDNA copy number is reduced by about 20%. In some embodiments, the endogenous mtDNA copy number is reduced by about 30%. In some embodiments, the endogenous mtDNA copy number is reduced by about 40%. In some embodiments, the endogenous mtDNA copy number is reduced by about 50%. In some embodiments, the endogenous mtDNA copy number is reduced by about 60%. In some embodiments, the endogenous mtDNA copy number is reduced by about 70%.In some embodiments, the endogenous mtDNA copy number is reduced by about 80%. In some embodiments, the endogenous mtDNA copy number is reduced by about 85%. In some embodiments, the endogenous mtDNA copy number is reduced by about 90%. In some embodiments, the endogenous mtDNA copy number is reduced by about 95%. Techniques known to those skilled in the art or described herein (e.g., in the Examples) can be used to assess the amount of endogenous mtDNA copy number before and after reduction.

[0115] In certain embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. In certain embodiments, the T cells are double positive CD4+CD8+ T cells. In some embodiments, the T cells are or comprise Tregs. In certain embodiments, the T cells are or comprise effector T cells. In some embodiments, the T cells are or comprise memory T cells, effector T cells, Tregs, or a combination thereof.

[0116] 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 cancer cells. CAR-T cells and TCR T cells can be exhausted, despite their use in immunotherapy. Thus, as provided herein, in some embodiments, the mitochondrial-replaced T cells made from exhausted T cells 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 T cells described herein 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.

[0117] CARs are generally designed to include an extracellular target binding domain, a hinge region, a transmembrane domain that anchors the CAR to the cell membrane, and one or more intracellular domains that transmit activation signals. CARs can be classified into first generation (intracellular domain, e.g., CD3ζ only), second generation (one costimulatory domain and intracellular domain), or third generation CARs (more than one costimulatory domain and intracellular domain) according to the number of costimulatory domains. 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.

[0118] 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).

[0119] 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. In general, 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. TCR-T cell can 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. CAR-T cell and TCR T cell can be generated using techniques known to those skilled in the art, such as the example of one method of generating CAR T cell.

[0120] 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.

[0121] As provided herein, the methods for generating mitochondrial-replaced T cells from exhausted T cells described in this disclosure are also applicable to generating mitochondrial-replaced T cells from senescent T cells. Senescent cells generally exhibit one or more phenotypic markers, such as 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, shortened telomeres, increased DNA damage response (DDR), or a combination thereof.

[0122] In some embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from senescent cells, and the mitochondrial-replaced T cells have reduced senescence compared to senescent T cells that have not been incubated with isolated exogenous mitochondria. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from senescent cells and have reduced secretion of inflammatory cytokines (e.g., interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNFα)). In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from senescent cells and have reduced secretion of growth factors. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from senescent cells and have reduced secretion of proteases. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from senescent cells and have increased proliferation. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from senescent cells and have reduced telomere shortening. In certain embodiments, mitochondrial-replaced T cells generated according to the methods provided herein are generated from senescent cells and have a reduced DNA damage response (DDR).

[0123] In some embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from exhausted T cells, and the mitochondrial-replaced T cells have reduced senescence compared to exhausted T cells that have not been incubated with isolated exogenous mitochondria. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from exhausted T cells and have reduced secretion of inflammatory cytokines (e.g., interferon gamma (IFNγ) and / or tumor necrosis factor alpha (TNFα)). In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from exhausted T cells and have reduced secretion of growth factors. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from exhausted T cells and have reduced secretion of proteases. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from exhausted T cells and have increased proliferation. In certain embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein are made from exhausted T cells and have reduced telomere shortening. In certain embodiments, mitochondrial-replaced T cells generated according to the methods provided herein are generated from exhausted T cells and have a reduced DNA damage response (DDR).

[0124] In a specific embodiment, provided herein is a method for generating mitochondrial-replaced T cells using the methods described in Example 8.1. 6.2 Treatment method

[0125] As provided herein, mitochondrial-replaced T cells generated according to the methods of the present disclosure are suitable for use as a cell-based therapy or therapies, e.g., in the methods described in Sections 6.2.1, 6.2.2, and 6.2.3. For example, in some embodiments, an effective amount of mitochondrial-replaced T 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.

[0126] In some embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising mitochondrial-replaced T cells generated according to the methods described herein. In some embodiments, provided herein are compositions (e.g., pharmaceutical compositions) comprising an effective amount of mitochondrial-replaced T cells generated according to the methods described herein and a pharma- ceutically acceptable carrier.

[0127] 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.

[0128] In certain embodiments, an effective amount of mitochondrial-replaced T cells is about 1×10 cells. 6 ~Approx. 1×10 7 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 10×10 cells. 6 ~About 900×10 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 50×10 cells. 6 ~About 800×10 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 100×10 cells. 6 ~Approx. 700×10 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 200×10 cells. 6 ~About 900×10 6In some embodiments, an effective amount of mitochondrial-replaced T cells is about 250×10 cells. 6 ~Approx. 750×10 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 50×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 150×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 300×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 450×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 600×10 cells. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 850×10 cells. 6 There are 10 pieces.

[0129] In certain embodiments, the effective amount of mitochondrial-replaced T 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 T 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 T cells is about 1.0×10 cells / kg. 6 cells / kg~cells approx. 500×10 6 In some embodiments, an effective amount of mitochondrial-replaced T 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 T 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 T 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 T cells is about 10×10 cells / kg. 6cells / kg~cells approx. 600×10 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 50×10 cells / kg. 6 cells / kg~cells approx. 750×10 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 1.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 2.5×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 5.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 10.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 50.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 250.0×10 cells / kg. 6 In some embodiments, an effective amount of mitochondrial-replaced T cells is about 500×10 cells / kg. 6 Pieces / kg.

[0130] In certain embodiments, mitochondrial replacement may be beneficial for any T cell therapy applied in treatment that is prone to T cell exhaustion.For example, in certain embodiments, the mitochondrial replacement T cells provided herein, for example, the mitochondrial replacement T cells generated according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by cancer.In certain embodiments, the mitochondrial replacement T cells provided herein, for example, the mitochondrial replacement T cells generated according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by viral infection.In certain embodiments, the mitochondrial replacement T cells provided herein, for example, the mitochondrial replacement T cells generated according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by bacterial infection.In certain embodiments, the mitochondrial replacement T cells provided herein, for example, the mitochondrial replacement T cells generated according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by fungal infection. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells produced according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by obesity or metabolic disorders.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells produced according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by alcoholism.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells produced according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by hyperactivity.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells produced according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by excessive mental stress.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells produced according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by hypoxia.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells produced according to the method described in section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell exhaustion is caused by injury.

[0131] In certain embodiments, the mitochondrial-replaced T cells provided herein, e.g., generated according to the methods described in Section 6.1 or in the Examples below, are suitable for use in treating conditions in which age-related T cell senescence occurs.

[0132] In certain embodiments, the mitochondrial-replaced T cells provided herein, e.g., generated according to the methods described in Section 6.1 or in the Examples below, are suitable for use in treating age-related immunological dysfunction.

[0133] In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of disease conditions in which CD8+ T cell dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which CD4+ T cell dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which T cell priming dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which memory T cell dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which B cell dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in treating conditions in which B cell priming dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in treating conditions in which memory B cell dysfunction is observed. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in treating conditions in which congenital lymphoid cell dysfunction is observed.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which congenital T cell dysfunction is observed.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which congenital B cell dysfunction is observed.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of conditions in which CD8+ T cell dysfunction is observed.

[0134] In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in treating disease conditions in cancer patients involving T cell exhaustion. For example, the expression of PD-1 ligands PD-L1 and / or PD-L2 correlates with the prognosis of several human malignancies, such as esophageal cancer, hepatocellular carcinoma, soft tissue sarcoma, non-small cell lung cancer, breast cancer, ovarian cancer, melanoma, pancreatic cancer, cervical cancer, and colon cancer. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in treating patients with solid cancers that exhibit a lack or reduction in T cell infiltration into tumors. In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in treating cancer patients with T cells that exhibit either a T cell exhaustion phenotype or a senescence phenotype.

[0135] Anti-cancer therapy can promote tissue dysfunction and the premature onset of various aging symptoms in treated cancer patients (see Wang B, et al., Trends Cancer. 2020 Oct; 6 (10): 838-857). Thus, in some embodiments, the mitochondrial-replaced T cells provided herein, e.g., generated according to the methods described in Section 6.1 or the Examples below, are suitable for use in treating the accumulation and persistence of senescent cells induced by anti-cancer therapy.

[0136] In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of fibrotic diseases in which T cell exhaustion or T cell senescence is observed.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of macular diseases in which T cell exhaustion or T cell senescence is observed.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of muscular degenerative diseases in which T cell exhaustion or T cell senescence is observed.In certain embodiments, the mitochondrial-replaced T cells provided herein, for example, the mitochondrial-replaced T cells generated according to the method described in Section 6.1 or the Examples below, are suitable for use in the treatment of neurodegenerative diseases in which T cell exhaustion or T cell senescence is observed.

[0137] In certain embodiments, treatment with mitochondrial-replaced T 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.

[0138] In some embodiments, e.g., in the methods described in Sections 6.2.1, 6.2.2, and 6.2.3, the mitochondrial-replaced T cells for use as a cell-based therapy or the like are autologous or allogeneic to the subject receiving the mitochondrial-replaced T cells. In some embodiments, e.g., in the methods described in Sections 6.2.1, 6.2.2, and 6.2.3, the mitochondrial-replaced T cells for use as a cell-based therapy are autologous to the subject receiving the mitochondrial-replaced T cells. In some embodiments, e.g., in the methods described in Sections 6.2.1, 6.2.2, and 6.2.3, the mitochondrial-replaced T cells for use as a cell-based therapy are allogeneic to the subject receiving the mitochondrial-replaced T cells. 6.2.1 Methods of Treating Age-Related Diseases

[0139] In one aspect, provided herein is a method for treating or ameliorating symptoms of age-related disease, comprising administering to a subject an effective amount of a composition comprising mitochondrial-replaced T cells generated according to the method described in section 6.1 and a pharma- ceutically acceptable carrier. In some embodiments of the methods provided herein, the age-related disease is selected from the group consisting of autoimmune disease or cancer. In a specific embodiment, the subject is a human.

[0140] In certain embodiments, the method for improving symptoms of age-related diseases (e.g., cancer or autoimmune diseases) includes one, two or more, or all of the following: (1) reducing the severity, progression, spread, and / or frequency of occurrence of one or more symptoms; (2) eliminating one or more symptoms and / or underlying causes; (3) preventing the occurrence of one or more symptoms and / or their underlying causes; and (4) improving or correcting damage. In one embodiment, the method for improving symptoms of age-related diseases (e.g., cancer or autoimmune diseases) includes reducing the severity of the symptoms. In one embodiment, the method for improving symptoms of age-related diseases (e.g., cancer or autoimmune diseases) includes reducing the progression of the symptoms. In another embodiment, the method for improving symptoms of age-related diseases (e.g., cancer or autoimmune diseases) includes reducing the spread of the symptoms. In another embodiment, the method for improving symptoms of age-related diseases (e.g., cancer or autoimmune diseases) reduces the frequency of occurrence of the symptoms. In another embodiment, the method for ameliorating a symptom of an age-related disease (e.g., cancer or autoimmune disease) comprises the elimination of the symptom. In another embodiment, the method for ameliorating a symptom of an age-related disease (e.g., cancer or autoimmune disease) comprises the prevention of the onset of a symptom of an age-related disease (e.g., cancer or autoimmune disease). In another embodiment, the method for ameliorating a symptom of an age-related disease (e.g., cancer or autoimmune disease) comprises the amelioration of damage caused by the age-related disease (e.g., cancer or autoimmune disease). In another embodiment, the method for ameliorating a symptom of an age-related disease (e.g., cancer or autoimmune disease) comprises the correction of damage caused by the age-related disease (e.g., cancer or autoimmune disease).

[0141] In a specific embodiment, provided herein is a method for treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising mitochondrial-replaced T cells generated according to the methods described herein (e.g., in Section 6.1 or in the Examples) and a pharma- ceutically acceptable carrier. In another specific embodiment, provided herein is a method for ameliorating symptoms of cancer in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising mitochondrial-replaced T cells generated according to the methods described herein (e.g., in Section 6.1 or in the Examples) and a pharma-ceutically acceptable carrier. In certain embodiments, the exhausted T cells and the mitochondrial-replaced T cells comprise an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In certain embodiments, the exhausted T cells and the mitochondrial-replaced T cells are genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In other embodiments, the mitochondrial-replaced T cells are genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0142] In another specific embodiment, provided herein is a method for treating the accumulation and persistence of senescent cells induced by anti-cancer therapy in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising mitochondrial-replaced T cells generated according to the methods described herein (e.g., in Section 6.1 or the Examples) and a pharmaceutical acceptable carrier. In another specific embodiment, provided herein is a method for reversing the symptoms of senescent cells induced by anti-cancer therapy in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising mitochondrial-replaced T cells generated according to the methods described herein (e.g., in Section 6.1 or the Examples) and a pharmaceutical acceptable carrier. 6.2.2 Methods of Treating Chronic Infections

[0143] T cell exhaustion has also been reported in various human chronic viral infections, such as human immunodeficiency virus (HIV), hepatitis B (HBV), and hepatitis C (HCV), as well as non-viral chronic infections, such as malaria and Mycobacterium tuberculosis. Thus, in one aspect, provided herein is a method for reversing symptoms of a chronic infection in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising mitochondrial-replaced T cells generated according to the methods described herein (e.g., in Section 6.1 or the Examples) and a pharmaceutically acceptable carrier.

[0144] In certain embodiments, the chronic infection is a viral infection. Non-limiting examples of viral infections suitable for treatment with mitochondrial-replaced T cells as described herein include human immunodeficiency virus (HIV) infection, hepatitis B (HBV) infection, hepatitis C (HCV) infection, cytomegalovirus infection, and SARS-CoV-2 infection. In one embodiment, the chronic viral infection is HIV. In one embodiment, the chronic viral infection is HBV infection. In another embodiment, the chronic viral infection is HCV infection. In another embodiment, the chronic viral infection is cytomegalovirus infection. In one embodiment, the chronic viral infection is SARS-CoV-2 infection. In other embodiments, the chronic infection is a non-viral infection. For example, in certain embodiments, the chronic infection is a bacterial or fungal infection.

[0145] In certain embodiments, a method for reversing a symptom of a chronic infection includes one, two, or more, or all of the following: (1) reducing the severity, progression, spread, and / or frequency of occurrence of one or more symptoms; (2) eliminating one or more symptoms and / or underlying causes; (3) preventing the occurrence of one or more symptoms and / or their underlying causes; and (4) improving or correcting damage. In one embodiment, a method for reversing a symptom of a chronic infection includes reducing the severity of the symptom. In one embodiment, a method for reversing a symptom of a chronic infection includes reducing the progression of the symptom. In another embodiment, a method for reversing a symptom of a chronic infection includes reducing the spread of the symptom. In another embodiment, a method for reversing a symptom of a chronic infection reduces the frequency of occurrence of the symptom. In another embodiment, a method for reversing a symptom of a chronic infection includes eliminating the symptom. In another embodiment, a method for reversing a symptom of a chronic infection includes preventing the occurrence of a symptom of a chronic infection. In another embodiment, a method for reversing a symptom of a chronic infection includes improving damage caused by a chronic infection. In another embodiment, the method for reversing symptoms of a chronic infection comprises correcting damage caused by the chronic infection. 6.2.3 Methods of Treating Mitochondrial Diseases or Disorders

[0146] In another 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 a composition comprising mitochondrial-replaced T cells generated according to the methods described herein (e.g., in Section 6.1 or the Examples) and a pharmaceutical acceptable carrier.

[0147] 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.

[0148] Thus, in some embodiments, the mitochondrial-replaced T cells are Treg cells, and the mitochondrial-replaced T cells are administered to a subject with mitochondrial complex III deficiency. In a specific embodiment, the subject is a human subject.

[0149] In certain embodiments, the method for improving symptoms of mitochondrial complex III deficiency includes one, two, three or more, or all of the following: (1) reducing the severity, progression, spread, and / or frequency of occurrence of one or more symptoms; (2) eliminating one or more symptoms and / or underlying causes; (3) preventing the occurrence of one or more symptoms and / or their underlying causes; and (4) improving or correcting the damage. In one embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the severity of the symptoms. In one embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the progression of the symptoms. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency includes reducing the spread of the symptoms. In another embodiment, the method for improving symptoms of mitochondrial complex III deficiency reduces the frequency of occurrence of the 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 reversing symptoms of mitochondrial complex III deficiency comprises ameliorating damage caused by mitochondrial complex III deficiency.In another embodiment, the method for reversing symptoms of mitochondrial complex III deficiency comprises correcting damage caused by mitochondrial complex III deficiency. 6.3 Biological Assays

[0150] In a specific embodiment, successful generation of mitochondrial-replaced T cells from exhausted T cells results in T cells with improved effector function compared to exhausted T cells without mitochondrial replacement. A variety of functional assays can be used to assess and evaluate the phenotype of mitochondrial-replaced T cells.

[0151] In some embodiments, mitochondria-replaced T cells have improved mitochondrial function compared to T 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.

[0152] Increased cell proliferation can also be an indicator of improved T cell function. An exemplary assay for measuring cell proliferation of T cells is a mixed lymphocyte reaction (MLR) assay. MLR assay generally involves combining a population of mitochondrial-replaced T cells, such as CD4+ T cells, with a different population of lymphocytes and measuring proliferation. In some embodiments, the mitochondrial-replaced T cells generated according to the methods provided herein have increased cell proliferation compared to exhausted T cells.

[0153] 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 effect of test item on this immune function to be investigated.Thus, in some embodiments, mitochondrial replacement T cells generated according to the method provided herein have enhanced CTL response compared to exhausted T cells.

[0154] Ca2+ signaling is crucial for 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 evaluating 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 T cells generated according to the methods provided herein have increased Ca2+ signaling compared to exhausted T cells.

[0155] Telomere length can also serve as an indicator of the function of mitochondrial replacement cell.Telomere length can be evaluated 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 T cell produced according to the method provided herein has reduced telomere shortening compared with exhausted T cell.

[0156] In some embodiments, mitochondrial replacement cells show reduced T cell exhaustion. FACS analysis for exhaustion markers (e.g., PD-1 / TIM3 / LAG3) can be used to assess T cell exhaustion. Thus, in some embodiments, mitochondrial replacement T cells produced according to the methods provided herein have reduced exhaustion compared to exhausted T cells.

[0157] As provided herein, in some embodiments, the T cells used to generate mitochondrial-replaced cells are senescent, and the mitochondrial-replaced cells exhibit reduced senescence. Thus, assessing 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 cell population doubling rates, 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-replaced T cells generated according to the methods provided herein have reduced senescence compared to senescent T cells. 6.4 Nucleotide and Protein Detection Assays

[0158] Techniques known to those of skill in the art or described herein (e.g., those in the Examples) can be used to assess the amount of nucleotides encoding XbaIR (e.g., PCR or qPCR), or the amount of XbaIR or fusion protein (e.g., immunoblot, ELISA). 7. Embodiment

[0159] The present invention provides the following non-limiting embodiments.

[0160] A1 1. A method for generating mitochondrial-replaced T cells from exhausted T cells, comprising: Incubating exhausted T cells having a reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondria-replaced T cells having at least a 1.1-fold reduction in expression of programmed cell death-1 (PD-1) relative to the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are generated. Including, The method, wherein the mitochondrial-replaced T cells have improved effector function compared to the exhausted T cells.

[0161] A2 1. A method for generating mitochondrial-replaced T cells from exhausted T cells, comprising: (a) electroporating exhausted T cells with a nucleic acid sequence comprising a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR to reduce endogenous mitochondrial DNA (mtDNA) copy number; (b) incubating the exhausted T cells having reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time to generate mitochondrial-replaced T cells having at least a 1.1-fold reduction in expression of PD-1 compared to the expression of PD-1 by the exhausted T cells from which the mitochondrial-replaced T cells were generated. wherein the mitochondrial-replaced T cells have improved effector function relative to the exhausted T cells.

[0162] A3 The method of embodiment A1 or A2, wherein the incubation of said exhausted T cells with said isolated exogenous mitochondria is performed in the presence of rapamycin.

[0163] A4 The method of embodiment A3, wherein the rapamycin is present at a concentration of 100 nM to 1000 nM.

[0164] A5 The method of any one of embodiments A1 to A4, wherein expression of PD-1 is reduced by at most 1.2 fold.

[0165] A6 The method of any one of embodiments A1 to A4, wherein expression of PD-1 is reduced by at most 1.25 fold.

[0166] A7 The method of any one of embodiments A1 to A4, wherein expression of PD-1 is reduced by at most 1.5 fold.

[0167] A8 The method of any one of embodiments A1 to A4, wherein expression of PD-1 is reduced by at most two-fold.

[0168] A9 The method of any one of embodiments A1 to A4, wherein expression of PD-1 is reduced by at most 5-fold.

[0169] A10 The method of any one of embodiments A1 to A4, wherein expression of PD-1 is reduced by about 1.1 to about 1.5 fold.

[0170] A11 The method of any one of embodiments A1 to A10, wherein the expression of T-cell immunoglobulin and mucin domain-containing protein 3 (TIM3), lymphocyte activation gene 3 (LAG3), T-cell immunoglobulin and ITIM domain (TIGIT), TOX, or a combination thereof is reduced.

[0171] 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 about 80 μg per cell.

[0172] A13 The method of any one of embodiments A1 to A12, wherein the mitochondrial-replaced T cells comprise at least 20% of the exogenous mtDNA.

[0173] A14 The method of any one of embodiments A1 to A12, wherein the mitochondrial-replaced T cells comprise at least 20% exogenous mtDNA and no more than 80% exogenous mtDNA as measured by TaqMan single nucleotide polymorphism (SNP) assay.

[0174] A15 The method of any one of embodiments A1 to A14, wherein the period of time sufficient for the generation of mitochondrial-replaced T cells is at least approximately 24 hours.

[0175] A16 The method of any one of embodiments A1 to A14, wherein the period of time sufficient for the generation of mitochondrial-replaced T cells is at least 36 hours.

[0176] A17 The method of any one of embodiments A1 to A14, wherein the period of time sufficient for the generation of mitochondrial-replaced T cells is at least 48 hours.

[0177] A18 The method of any one of embodiments A1 to A14, wherein the period of time sufficient for the generation of mitochondria-replaced T cells is from about 24 hours to about 72 hours.

[0178] A19 The method of any one of embodiments A1 to A18, wherein said improved effector function comprises increased proliferation, increased cytotoxicity, increased secretion of cytokines, or a combination thereof.

[0179] A20 The method of any one of embodiments A1 to A19, wherein the exhausted T cells comprise an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0180] A21 The method of any one of embodiments A1 to A19, wherein the exhausted T cells are genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

[0181] A22 A mitochondrial-replaced T cell produced by the method of any one of embodiments A1 to A19.

[0182] A23 A mitochondrial-replaced T cell produced by the method of embodiment A20 or A21.

[0183] A24 A composition comprising an effective amount of the mitochondrial-replaced T cells of embodiment A22 and a pharma- ceutically acceptable carrier.

[0184] A25 A method for reversing symptoms of a chronic viral infection in a subject in need thereof, comprising administering to the subject a composition of embodiment A24.

[0185] A26 The method of embodiment A25, wherein the chronic viral infection is a human immunodeficiency virus (HIV) infection, a hepatitis B virus (HBV) infection, a cytomegalovirus infection (CMV), and a severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection.

[0186] A27 A composition comprising an effective amount of the mitochondrial-replaced T cells of embodiment A23 and a pharma- ceutically acceptable carrier.

[0187] A28 A method for treating cancer in a subject in need thereof, comprising administering to the subject a composition of embodiment A24 or A27.

[0188] A29 A method for ameliorating the symptoms of cancer in a subject in need thereof, comprising administering to the subject a composition of embodiment A24 or A27.

[0189] A30 A method for treating a disease or condition associated with, accompanied by, or caused by T cell exhaustion in a subject in need thereof, comprising administering to the subject a composition according to embodiment A24 or A27, wherein the disease or condition is (a) Cancer; (b) viral infection; (c) bacterial infections; (d) obesity or metabolic disorder; (e) Alcoholism; (f) hyperactivity; (g) undue mental stress; (h) hypoxia; (i) injury; (j) Aging; (k) age-related immunological dysfunction; (l) fibrotic diseases; (m) macular disease; (n) a muscular degenerative disease; or (o) Neurodegenerative diseases That is, the method.

[0190] A31 A method for ameliorating the symptoms of a disease or condition associated with, accompanied by, or caused by T cell exhaustion in a subject in need thereof, comprising administering to the subject a composition according to embodiment A24 or A27, wherein the disease or condition is (a) Cancer; (b) viral infection; (c) bacterial infections; (d) obesity or metabolic disorder; (e) Alcoholism; (f) hyperactivity; (g) undue mental stress; (h) hypoxia; (i) injury; (j) Aging; (k) age-related immunological dysfunction; (l) fibrotic diseases; (m) macular disease; (n) a muscular degenerative disease; or (o) Neurodegenerative diseases That is, the method.

[0191] A32 A method for treating a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, comprising administering to the subject a composition according to embodiment A24 or A27, wherein the disease or condition is (a) CD8+ T cell dysfunction; (b) CD4+ T cell dysfunction; (c) impaired T cell priming; (d) memory T cell dysfunction; (e) effector B cell dysfunction; (f) impaired B cell priming; (g) memory B cell dysfunction; (h) congenital lymphoid cell dysfunction; (i) a congenital T-cell dysfunction; or (j) Congenital B cell dysfunction That is, the method.

[0192] A33 The method of any one of embodiments A25, A26 or A28 to A32, wherein the subject is a human. EXAMPLES

[0193] 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 Reducing Immune Cell Exhaustion Using Mitochondrial Replacement

[0194] The following examples demonstrate that mitochondrial replacement can be used on exhausted T cells to generate Mir T cells, thereby reducing phenotypic markers of exhausted T cells. 8.1.1 Materials and Methods

[0195] Isolation and cell culture of primary human T lymphocytes: Peripheral blood was donated voluntarily by healthy individuals who volunteered to participate in the recruitment process under approval by the ethical committee. Heparinized venous blood was obtained from the median cubital vein according to standard procedures (NP-EN0507, NIPRO, Osaka, Japan). Human peripheral blood mononuclear cells (PBMCs) were isolated from human peripheral blood using density gradient centrifugation with 1.077 g / ml Percoll (GE Healthcare Life Sciences, Buckinghamshire, England). Cells were cultured on cell culture plates activated with anti-CD3 and anti-CD28 antibodies (Miltenyi Biotec) in TexMACS medium (Miltenyi Biotec) supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin (Thermo Fisher Scientific incorporated), 20 μM IL-7, and 10 μM IL-15. The cells were incubated at 37° C. in a humidified 5% CO 2 incubator.

[0196] Generation of CAR T cells: CAR T cells were generated from peripheral blood T cells drawn from healthy volunteers by transferring a recombinant vector carrying a ligand for ephedrine type B receptor 4 (EPHB4), which is expressed in rhabdomyosarcoma (see Kubo, H., et al. (2021). "Development of non-viral, ligand-dependent, EPHB4-specific chimeric antigen receptor T cells for treatment of rhabdomyosarcoma." Mol Ther Oncolytics 20: 646-658).

[0197] Induction of CAR T cell exhaustion: CAR T cells expressing the ligand for EPHB4 were co-cultivated with rhabdomyosarcoma cells, Rh30 cells, which express EPHB4, without any cytokine signaling. After 3 days of co-culture, CAR T cells significantly expressed PD-1, a marker of exhausted T cells. The presence of CAR T, persistent antigen stimulation, and the absence of supportive signals (CD3, CD28, IL-7, and IL-15) mimicked the microenvironment of a solid tumor.

[0198] Mitochondria isolation and transfer into human T cells: Mitochondria were isolated from DsRed-Mt EMCs by differential centrifugation as previously described. 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 Bio-Rad protein assay kit (Bio-Rad Laboratories, incorporated, Richmond, CA, USA). Three days after the exhaustion CAR-T generation protocol, cells were electroporated with XbaIR fused with MTS to reduce mtDNA copy number. After gene transfer, cells were re-plated on CD3 / CD28-coated dishes in complete T cell growth medium. Mitochondrial transfer was performed by co-incubating isolated mitochondria with CAR-T-derived ρ(-) cells followed by centrifugation (1,500×g for 5 min at room temperature).

[0199] Direct sequencing of mitochondrial DNA D-loop sequence: Total DNA was extracted from cells using NucleoSpin Tissue (MACHEREY-NAGEL GmbH & Co.KG). The extracted DNA was used as a template to amplify mitochondrial DNA D-loop sequence using GoTaq® Green Master Mix (Promega KK.) according to the GoTaq® Green Master Mix Protocol. The primer sequences were forward primer: 5'-ctctgttctttcatggggaagc-3' (SEQ ID NO: 3) and reverse primer: 5'-cataaactgtggggggtgtct-3' (SEQ ID NO: 4). After amplification, the PCR product was electrophoresed on a 1% agarose gel, and the 1,134 bp band was purified using NucleoSpin Gel and PCR Clean-up kit (MACHEREY-NAGEL GmbH & Co.KG). The extracted PCR products were sequenced using the forward primer used for PCR amplification and an Applied Biosystems 3730xl DNA analyzer (Thermo Fisher Scientific Inc.).

[0200] Heteroplasmy analysis for mtDNA using TaqMan single nucleotide polymorphism (SNP) assay: To determine the heteroplasmy ratio, wild-type (NHDF) allele-specific TaqMan probe and mutant (YG cell) allele-specific TaqMan probe for TaqMan SNP assay were designed. Extracted DNA (10 ng) was used for quantitative PCR 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) using TaqMan Universal PCR Master Mix kit (Thermo Fisher Scientific Incorporated) on CFX connect real-time system (Bio-Rad Laboratories, Incorporated). The primer sequences were 5'-TTACTGCCAGCCACCATGAA-3' (SEQ ID NO: 5) as forward primer and 5'-TTGATGTGGATTGGGTTTTTATGT-3' (SEQ ID NO: 6) as reverse primer. The probe for wild-type (NHDF) was FAM-ACAGGTGGT T AAGTATT-MGB (SEQ ID NO: 7), and the probe for the mutation (YG cells) was VIC-CAGGTGGT C The mtDNA D-loop fragment was AAGTAT-MGB (SEQ ID NO: 8). Calibration curves were generated using known copy numbers of plasmids containing the amplified mtDNA D-loop fragment for either the wild-type or mutant sequences. TaqMan SNP validation demonstrated that YG cells could be distinguished from NHDF cells based on the mtDNA sequence (Figure 2B).

[0201] Analysis of PD-1 expression in CAR-T cells: CAR-T cells (5 × 10 6 cells) to human rhabdomyosarcoma Rh-30 cells (1 × 10 7 The cultured cells were washed with PBS (300 × g for 5 min) and then diluted with 1 × 10 5The cells were suspended in 200 μl of MACS buffer (Miltenyi Biotec) and 5 μl each of FITC anti-human CD279 (PD-1) antibody (329904, BioLegend, Inc.) and PE anti-human CD3 antibody (300408, BioLegend, Inc.), and 1 μl of 7-AAD (559925, Becton, Dickinson and Company) were added. The mixture was incubated at 37° C. for 30 minutes. After washing with MACS buffer, the mixture was suspended in 500 μl of MACS buffer and analyzed for PD-1 expression on the cell surface using a Cell Sorter MA900 (Sony). The expression levels of PD-1 and CD3 were analyzed using FlowJo software (Becton, Dickinson and Company).

[0202] Measurement of mean fluorescence intensity using FlowJo software: Mean fluorescence intensity (MFI) was measured using the Geometric Mean Fluorescence Intensity method, a statistical function in FlowJo software (version 10.6). Briefly, select the FITC group in the workspace and click on the Statistics function button. Then select Geometric Mean, select the FITC parameters in the dialog and click on the Add button. The MFI value is then displayed in the workspace. 8.1.2 Results

[0203] Rh30 human rhabdomyosarcoma cells (2 × 10 5 EPHB4-specific chimeric antigen receptor CAR-T cells (1 × 10 7 (1000 pieces) were added to the same 6-well dish and co-incubated with Rh30 cells for 3 days. FACS analysis of the expression levels of the exhaustion marker PD-1 by CAR-T cells on days 0 and 3 showed that the percentage of CD3+ / PD1+ cells increased from 1.84% to 23.8% after co-culture (Figure 1). Thus, the co-culture generated exhausted CAR-T cells.

[0204] On day 3, we depleted endogenous mitochondrial DNA in exhausted CAR-T cells by electroporating them with XbaIR mRNA. As a negative control, exhausted CAR-T cells were electroporated without XbaIR mRNA and subjected to the same electroporation, in which CAR-T cells were contacted with XbaIR mRNA. 12S rRNA, a surrogate marker for mtDNA content, was halved on day 3, thereby confirming that XbaIR caused effective mtDNA reduction (Figure 2A).

[0205] After electroporation, CAR-T cells were replated on CD3 / 28-coated dishes with IL-7 / IL-15 and cultured for 2 days (Figure 1). On day 5, normal human derived fibroblast (NHDF)-derived donor mitochondria were cocultured with electroporated CAR-T cells with or without XbaIR contact for an additional 2 days to generate Mir CAR-T cells or negative control exhausted CAR-T cells, respectively. FACS and TaqMan SNP assays were performed on day 7.

[0206] TaqMan SNP assay results measured on day 7 indicated that Mir CAR-T cells contained approximately 40% NHDF-derived donor mtDNA (Figure 2B). Furthermore, FACS analysis on day 7 showed that the percentage of CD3+ / PD-1+ cells was 0.045% for unstained cells (Figure 3A), 63.8% for exhausted CAR-T cells that had not undergone mitochondrial depletion (Figure 3B), and 66.8% for exhausted Mir CAR T cells (Figure 3C).

[0207] Analysis of PD-1 antigen expression 2 days after mitochondrial replacement revealed that PD-1 expression on exhausted Mir CAR-T cells was reduced by approximately 1.3-fold (i.e., 24%) compared to exhausted CAR-T cells not transfected by XbaIR electroporation (Figure 4A and Table 1). The fold change was calculated by dividing the PD-1 antigen count in exhausted CAR-T cells by the PD-1 antigen count in exhausted Mir CAR-T cells. The percent change was calculated as follows: [[(PD-1 antigen count in exhausted Mir CAR-T cells)-(PD-1 antigen count in exhausted CAR-T cells)]÷PD-1 antigen count in exhausted CAR-T cells]×100.

[0208] Table 1 [Table 1]

[0209] Furthermore, the bar graph of mean fluorescence intensity showed that PD-1 intensity was decreased in Mir CAR-T cells, indicating reduced expression of PD-1 antigen (Figure 4B).

[0210] Taken together, these results demonstrate that mitochondrial replacement in exhausted T cells can reduce the expression of PD-1, an exemplary phenotypic marker of exhaustion. Such Mir T cells with reduced expression of PD-1 have utility in treating various conditions, such as cancer and chronic viral infections.

[0211] 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 encompassed by the appended claims.

Claims

1. A method for producing mitochondria-replaced T cells from exhausted T cells, comprising: incubating exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time such that the expression of programmed cell death-1 (PD-1) is at most 1 / 1.1 of the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are produced, to generate mitochondria-replaced T cells; comprising: The method, wherein the mitochondria-replaced T cells have an improved effector function compared to the exhausted T cells.

2. A method for producing mitochondria-replaced T cells from exhausted T cells, comprising: (a) electroporating an exhausted T cell with a nucleic acid sequence comprising a nucleotide sequence encoding a fusion protein comprising a mitochondrial targeting sequence (MTS) and XbaIR to reduce the endogenous mitochondrial DNA (mtDNA) copy number; and (b) incubating the exhausted T cells with reduced endogenous mitochondrial DNA (mtDNA) copy number with isolated exogenous mitochondria for a sufficient period of time such that the expression of PD-1 is at most 1 / 1.1 of the expression of PD-1 by the exhausted T cells from which the mitochondria-replaced T cells are produced, to generate mitochondria-replaced T cells; The method, wherein the mitochondria-replaced T cells have an improved effector function compared to the exhausted T cells.

3. The method according to claim 1 or 2, wherein the incubation of the isolated exogenous mitochondria with the exhausted T cells is performed in the presence of rapamycin.

4. The method according to claim 3, wherein rapamycin is present at a concentration of 100 nM to 1000 nM.

5. The method according to claim 1 or 2, wherein the expression of PD-1 is reduced to at most 1 / 1.

2.

6. The method according to claim 1 or 2, wherein the expression of PD-1 is reduced to at most 1 / 1.

25.

7. The method according to claim 1 or 2, wherein the expression of PD-1 is reduced to at most 1 / 1.

5.

8. The method according to claim 1 or 2, wherein the expression of PD-1 is reduced to at most 1 / 2.

9. The method according to claim 1 or 2, wherein the expression of PD-1 is reduced to at most 1 / 5.

10. The method according to claim 1 or claim 2, wherein the expression of PD-1 is reduced to about 1 / 1.1 to about 1 / 1.

5.

11. The method according to claim 1 or claim 2, which reduces the expression of T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), lymphocyte activation gene 3 (LAG3), T cell immunoglobulin and ITIM domain (TIGIT), TOX, or a combination thereof.

12. The isolated exogenous mitochondria are about 20 μg to about 80 μg of protein per 1 × 10 ６ cells, the method according to claim 1 or claim 2.

13. The method according to claim 1 or claim 2, wherein the mitochondrial replacement T cells contain at least 20% exogenous mtDNA.

14. The method according to claim 1 or claim 2, wherein the mitochondrial replacement T cells contain at least 20% exogenous mtDNA and 80% or less exogenous mtDNA as measured by TaqMan single nucleotide polymorphism (SNP) assay.

15. The method according to claim 1 or claim 2, wherein the period sufficient for generating the mitochondrial replacement T cells is at least about 24 hours.

16. The method according to claim 1 or claim 2, wherein the period sufficient for generating the mitochondrial replacement T cells is at least 36 hours.

17. The method according to claim 1 or claim 2, wherein the period sufficient for generating the mitochondrial replacement T cells is at least 48 hours.

18. The method according to claim 1 or claim 2, wherein the period sufficient for generating the mitochondrial replacement T cells is about 24 hours to about 72 hours.

19. The method according to claim 1 or claim 2, wherein the improved effector function includes increased proliferation, increased cytotoxicity, increased secretion of cytokines, or a combination thereof.

20. The method according to claim 1 or claim 2, wherein the exhausted T cells contain an exogenous polynucleotide encoding a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

21. The method according to claim 1 or claim 2, wherein the exhausted T cells are genetically modified to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR).

22. Mitochondrial replacement T cells generated by the method according to claim 1 or claim 2.

23. Mitochondrial replacement T cells generated by the method according to claim 20.

24. A composition comprising an effective amount of the mitochondrial replacement T cells according to claim 22 and a pharmaceutically acceptable carrier.

25. The composition according to claim 24 for effecting improvement of symptoms of chronic viral infection in a subject in need thereof.

26. The composition according to claim 25, wherein the chronic viral infection is human immunodeficiency virus (HIV) infection, hepatitis B virus (HBV) infection, cytomegalovirus infection (CMV), and severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infection.

27. A composition comprising an effective amount of the mitochondrial replacement T cells according to claim 23 and a pharmaceutically acceptable carrier.

28. The composition according to claim 27 for effecting treatment of cancer in a subject in need thereof.

29. The composition according to claim 27 for effecting improvement of symptoms of cancer in a subject in need thereof.

30. A composition according to claim 27 for effecting treatment of a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) cancer; (b) viral infection; (c) bacterial infection; (d) obesity or metabolic disorder; (e) alcoholism; (f) overexercise; (g) excessive mental stress; (h) hypoxia; (i) injury; (j) aging; (k) age-related immunological dysfunction; (l) fibrotic disease; (m) macular disease; (n) muscle degenerative disease; or (o) neurodegenerative disease is a composition.

31. A composition according to claim 27 for effecting improvement of symptoms of a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) cancer; (b) viral infection; (c) bacterial infection; (d) obesity or metabolic disorder; (e) alcoholism; (f) overexercise; (g) excessive mental stress; (h) hypoxia; (i) injury; (j) aging; (k) age-related immunological dysfunction; (l) fibrotic disease; (m) macular disease; (n) muscle degenerative disease; or (o) neurodegenerative disease is a composition.

32. The composition according to claim 27 for use in treating a disease or condition associated with, or involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) CD8+ T cell dysfunction; (b) CD4+ T cell dysfunction; (c) dysfunction of T cell priming; (d) memory T cell dysfunction; (e) effector B cell dysfunction; (f) dysfunction of B cell priming; (g) memory B cell dysfunction; (h) innate lymphoid cell dysfunction; (i) innate T cell dysfunction; or (j) innate B cell dysfunction A composition.

33. The composition according to claim 25, wherein the subject is human.

34. The composition according to claim 26, wherein the subject is human.

35. The composition according to claim 28, wherein the subject is human.

36. The composition according to claim 32, wherein the subject is human.

37. Mitochondrial replacement T cells generated by the method according to claim 21.

38. The composition according to claim 24 for use in treating cancer in a subject in need thereof.

39. The composition according to claim 24 for use in ameliorating the symptoms of cancer in a subject in need thereof.

40. The composition according to claim 24 for use in treating a disease or condition associated with, or involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) cancer; (b) viral infection; (c) bacterial infection; (d) obesity or metabolic disorder; (e) alcoholism; (f) excessive exercise; (g) excessive mental stress; (h) hypoxia; (i) injury; (j) aging; (k) age-related immunological dysfunction; (l) fibrotic disease; (m) macular disease; (n) muscle degenerative disease; or (o) neurodegenerative disease A composition.

41. The composition according to claim 24 for use in ameliorating the symptoms of a disease or condition associated with, or involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) cancer; (b) viral infection; (c) bacterial infection; (d) obesity or metabolic disorder; (e) alcoholism; (f) excessive exercise; (g) excessive mental stress; (h) hypoxia; (i) Injury; (j) Aging; (k) Age-related immunological dysfunction; (l) Fibrosis disease; (m) Macular disease; (n) Muscle degeneration disease; or (o) Neurodegenerative disease A composition which is [

42. ] The composition according to claim 24 for treating a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) CD8+ T cell dysfunction; (b) CD4+ T cell dysfunction; (c) Dysfunction of T cell priming; (d) Memory T cell dysfunction; (e) Effector B cell dysfunction; (f) Dysfunction of B cell priming; (g) Memory B cell dysfunction; (h) Innate lymphoid cell dysfunction; (i) Innate T cell dysfunction; or (j) Innate B cell dysfunction A composition which is [

43. ] A composition comprising an effective amount of the mitochondrial replacement T cells according to claim 37 and a pharmaceutically acceptable carrier. [

44. ] The composition according to claim 43 for treating cancer in a subject in need thereof. [

45. ] The composition according to claim 43 for ameliorating the symptoms of cancer in a subject in need thereof. [

46. ] The composition according to claim 43 for treating a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) Cancer; (b) Viral infection; (c) Bacterial infection; (d) Obesity or metabolic disorder; (e) Alcoholism; (f) Excessive exercise; (g) Excessive mental stress; (h) Hypoxia; (i) Injury; (j) Aging; (k) Age-related immunological dysfunction; (l) Fibrosis disease; (m) Macular disease; (n) Muscle degeneration disease; or (o) Neurodegenerative disease A composition which is [

47. ] The composition according to claim 43 for ameliorating the symptoms of a disease or condition associated with, involving, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) Cancer; (b) Viral infection; (c) Bacterial infection; (d) Obesity or metabolic disorder; (e) Alcoholism; (f) Excessive exercise; (g) Excessive mental stress; (h) Hypoxia; (i) Injury; (j) Aging; (k) Age-related immunological dysfunction; (l) Fibrotic disease; (m) Macular disease; (n) Muscular degeneration disease; or (o) Neurodegenerative disease A composition which is

48. The composition according to claim 43 for use in treating a disease or condition associated with, involved in, or caused by T cell exhaustion in a subject in need thereof, wherein the disease or condition is (a) CD8+ T cell dysfunction; (b) CD4+ T cell dysfunction; (c) Dysfunction of T cell priming; (d) Memory T cell dysfunction; (e) Effector B cell dysfunction; (f) Dysfunction of B cell priming; (g) Memory B cell dysfunction; (h) Innate lymphoid cell dysfunction; (i) Innate T cell dysfunction; or (j) Innate B cell dysfunction A composition which is

Images